Abhishek Dutt Tripathi

Kianoush Khosravi-Darani
Suresh Kumar Srivastava Editors

Novel Food
Grade
Enzymes
Applications in Food Processing and
Preservation Industries
Novel Food Grade Enzymes
Abhishek Dutt Tripathi •
Kianoush Khosravi-Darani •
Suresh Kumar Srivastava
Editors

Novel Food Grade

Enzymes
Applications in Food Processing and
Preservation Industries
Editors
Abhishek Dutt Tripathi Kianoush Khosravi-Darani
Department of Dairy Science & Food Department of Food Technology Research
Technology National Nutrition and Food Technology
Banaras Hindu University Research Institute
Varanasi, Uttar Pradesh, India Tehran, Iran

Suresh Kumar Srivastava

School of Biochemical Engineering
Indian Institute of Technology BHU
Varanasi, India

ISBN 978-981-19-1287-0 ISBN 978-981-19-1288-7 (eBook)

https://doi.org/10.1007/978-981-19-1288-7

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Preface

Enzymes play a significant role in pharma, agriculture, and other allied industries. In
the modern era, there is significant application of enzymes in food processing
industries and our proposed book will give deeper insight into food applications of
enzymes. This book covers all the aspects of enzyme including its classification,
kinetics, microbial production, biosynthetic pathways, commodity-wise industrial
applications, and its downstream processing strategies. The broad focus of this book
will be on the application of various classes of enzymes in dairy, fruits and vegeta-
ble, cereals and oilseeds, meat and poultry, brewing and food packaging industries.
The first chapter of this book gives a brief introduction about enzymes classifications
and their kinetics. Chapters 2, 3, 5, 6, 7, and 8 provide information regarding enzyme
applications in different food commodities like fruits and vegetables, dairy, bever-
age, and meat processing industries. These chapters also focus on different food
grade enzyme biosynthesis mechanism in simpler and illustrative way. Besides that,
this book will also allow the readers to understand the biosynthesis, production,
optimization, and purification of certain less known enzymes such as CGTase and
naringinase. Chapters 9 and 12 of this book will be giving some novel information
related to application of enzymes specifically for improvement of flavor, color,
organoleptic attributes, shelf-life extension, and consumer acceptability of value-
added diversified food products. Chapters 10 and 11 also cover some of the very
interesting aspects of enzymes application in the food packaging and development of
biosensors which will be highly beneficial for those researchers and students who are
interested in learning the food safety issues and indulged in product quality improve-
ment. Enzymes play a significant role in the development of GM (Genetically
Modified) food production. Chapters 13 and 14 provide detailed information related
to enzymes application in GM foods. Chapter 15 highlights the various enzyme
infusion strategies adopted in the food processing industries that will enable the
students and entrepreneurs to understand the basic concept of enzyme activity and
stability after infusion in food. Future prospects of enzymes and their
nanotechnological applications have been discussed in Chapters 16 and 17 of this
book. The illustrated case studies related to certain novel enzymes will allow the
academicians and researchers to broaden their knowledge and will provide an
opportunity to carry out extensive research work pertaining to the applications of
such enzymes. The authors contributing to this book have already done extensive

v
vi Preface

research work in the proposed area and the contributed chapters will also be
highlighting some of the salient research work already published in the reputed
journals by internationally recognized authors in the proposed area. The flowchart
described in certain chapters will provide all the necessary information related to the
production, optimization, and characterization of food grade enzymes in simpler
manner. This book will not only cover the basic enzyme kinetics part but also
provide information related to the efficient enzyme recovery and purification pro-
cess. It also gives broad information on the factors affecting enzyme activity and
stability as well as involvement of enzyme in the development of functional food
combating chronic diseases and disorders.
As per our survey, only few books are available on the role of enzymes in food
processing although this is a prominent area of study and needs to be covered in
detail. The earlier books primarily focused on the general enzyme properties and its
application. However, no deep insight knowledge pertaining to the commodity-wise
application of food grade enzymes has been previously illustrated. We have tried to
cover all the aspects of food grade enzymes including its general properties, kinetics,
wide application in all the food processing and packaging industries. Although
numerous books on medical and therapy-based application of enzymes are available
in the market, no books on wider perspective of food grade enzymes applications are
available till now. The simpler language, clearly illustrated concepts, and broadly
discussed case studies related to various food grade enzymes will enable the readers
to strengthen their concept and knowledge. This book will be beneficial for the
faculty members, undergraduate, postgraduate, and PhD students of Microbiology,
Food Technology, Dairy Technology, Horticulture, Biotechnology, Biochemistry,
and other allied discipline of Life Science. This book will also be beneficial to those
authors who are indulged in the research activities related to the above-mentioned
subject areas such as food science, enzyme and fermentation technology, bioprocess
technology, and biochemical engineering. The involvement of foreign authors and
editors will provide deeper insight about the subject knowledge and will improve the
quality of book content making it readable and acceptable at global level. I hope the
readers will enjoy reading this book and will develop interest in the area of enzyme
technology.

Varanasi, Uttar Pradesh, India Abhishek Dutt Tripathi

Tehran, Iran Kianoush Khosravi-Darani
Varanasi, Uttar Pradesh, India Suresh Kumar Srivastava
Acknowledgments

I am grateful to my co-editors, Prof. Suresh Kumar Srivastava, School of Biochemi-

cal Engineering Indian Institute of Technology (IIT), Banaras Hindu University,
India, and Prof. Kianoush Khosravi-Darani, National Nutrition and Food Technol-
ogy Research Institute of Iran for providing the immense support in compilation of
this book. I am thankful to all my authors from India, Iran, and Nigeria who
contributed different chapters for this book as per their expertise. I am grateful to
Springer Nature group for selecting our book proposal and permitting us to complete
this task. I am thankful to external reviewers who approved the book proposal after
thorough evaluation. I pay my sincere gratitude to our Director and Esteemed Dean
for their constant encouragement. I am grateful to my Head of the Department Prof.
Dinesh Chandra Rai, Department of Dairy Science and Food Technology, Institute
of Agricultural Sciences, Banaras Hindu University, India, for his immense motiva-
tion and providing necessary facilities required for compilation of this book. I would
like to pay my sincere thanks to my parents Shri Vishnu Dutt Tripathi and Mrs.
Urmila Tripathi for their encouragement and support. I would like to pay my sincere
thanks to my beloved wife Mrs. Priyanaka Upadhyay and my kids Master Ojas and
Baby Prajul for their kind support and cooperation. Lastly, I am thankful to almighty
who blessed me with the completion of this task.

vii
Contents

1 Food Enzymes: General Properties and Kinetics . . . . . . . . . . . . . 1

S. M. Khade, S. K. Srivastava, L. H. Kamble, and J. Srivastava
2 Plants- and Animal-Derived Enzymes and Their Potential
Application in Food Processing and Preservation . . . . . . . . . . . . 17
Mahmoud Aminlari
3 Role of Enzymes in Fruit and Vegetable Processing Industries:
Effect on Quality, Processing Method, and Application . . . . . . . 65
Memthoi Devi Heirangkhongjam, Kanika Agarwal, Aparna Agarwal,
and Nidhi Jaiswal
4 Production of α, β, and γ-Cyclodextrin Gluconotransferase
(CGTase) and Their Applications in Food Industry . . . . . . . . . . 107
Rizul Gautam and Shailendra Kumar Arya
5 Enzyme in Milk and Milk Products: Role and Application . . . . . 139
Aparna Agarwal, Naman Kaur, Nidhi Jaiswal, Memthoi Devi
Heirangkhongjam, and Kanika Agarwal
6 Enzymes in Brewing and Wine Industries . . . . . . . . . . . . . . . . . . 165
S. Pati and D. P. Samantaray
7 Enzymes in Meat, Fish, and Poultry Products Processing
and Preservation-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Khadijeh Abhari and Hedayat Hosseini
8 Enzymes in Meat, Fish, and Poultry Product Processing
and Preservation-II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Sandesh Suresh Karkal, Anushma Venmarath,
Suresh Puthenveetil Velappan, and Tanaji G. Kudre
9 Enzymes in Functional Food Development . . . . . . . . . . . . . . . . . 217
Iran Alemzadeh, Asma Sadat Vaziri, Kianoush Khosravi-Darani,
and Pierre Monsan

ix
x Contents

10 Enzymes as Active Packaging System . . . . . . . . . . . . . . . . . . . . . 253

Amir Heydari and Negin Mahmoodi-Babolan
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen
Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Preethi Sudhakara, Jerrine Joseph, S Priyadharshini, Jemmy Chirsty,
Alex Anand, Davamani Christober, and Aruni Wilson Santhosh
Kumar
12 Enzymes in Flavor Development and Food Preservation . . . . . . . 317
Fataneh Hashempour-Baltork and Parastou Farshi
13 Enzymes from Genetically Modified Organisms and Their
Current Applications in Food Development and Food Chain . . . 357
Senthilkumar Muthusamy, Shilpa Ajit, Asha V. Nath,
J. Anupama Sekar, and T. S. Ramyaa Lakshmi
14 Current Applications of Enzymes in GM (Genetically Modified)
Food Development and Food Chain . . . . . . . . . . . . . . . . . . . . . . 383
Nafiseh Sadat Naghavi, Fatemeh Mahmoodsaleh,
and Masoumeh Moslemi
15 Enzyme Immobilization and Its Application Strategies in Food
Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Nafiseh Sadat Naghavi, Nazanin Sanei, and Martin Koller
16 Prospects and Challenges in Food-Grade Enzymes Industrial
Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Musliu Olushola Sunmonu and Mayowa Saheed Sanusi
17 Nanotechnology and Food Grade Enzymes . . . . . . . . . . . . . . . . . 455
Zahra Beig Mohammadi, Khadijeh Khoshtinat, Sanaz Ghasemi,
and Zahra Ahmadi
About the Editors

Abhishek Dutt Tripathi is an assistant professor in the Department of Dairy

Science and Food Technology, Institute of Agricultural Sciences, Banaras Hindu
University, India. He has published more than 75 research papers and articles in
journals of national and international repute. His field of specialization is food
chemistry, enzyme technology, fermentation technology, and food microbiology.
He has also published two books, two manuals, and many book chapters associated
with food and bioprocess technology. He has received several prestigious national
and international awards and is also serving as editor and reviewer with many
international and national journals.

Kianoush Khosravi-Darani (co-editor) is a professor at the National Nutrition and

Food Technology Research Institute of Iran. She has more than 20 years of teaching
and research experience. She has published more than 125 research papers in
journals of national and international repute. She is editor-in-chief of the journal
Applied Food Biotechnology indexed in web of science. She has received a number
of prestigious awards in the field of food biotechnology. She has done extensive
research work in the area of food science and technology, enzyme and bioprocess
technology, and biopolymers. She has published number of books in reputed
publication.

Suresh Kumar Srivastava is an institute professor at the School of Biochemical

Engineering, Indian Institute of Technology (BHU), India. He has more than
30 years of teaching and research experience. He has published more than
70 research papers in journals of national and international repute. He has received
number of prestigious awards from different government organizations and held
prestigious positions in board of studies at different Indian Universities and also
served as member of different administrative committees related to R&D. His area of
specialization in enzyme engineering, enzyme technology, food technology, and
biochemical engineering.

xi
Food Enzymes: General Properties
and Kinetics 1
S. M. Khade, S. K. Srivastava, L. H. Kamble, and J. Srivastava

Abstract

Enzymes are the biocatalysts having a catalytic power that speeds up a chemical
reaction without changing the equilibrium of the reaction. Almost all enzymes are
protein in nature and the biocatalytic power lies in the integrity of their structural
conformation. The enzymes are highly specific with their substrate molecules that
convert into the product by decreasing the activation energy, without getting
changed itself. However, the biocatalytic power of the enzymes also depends on
several physico-chemical parameters such as temperature, pH, salt
concentrations, etc. of the reaction. The enzyme catalysis can be quantitatively
revealed by the enzyme kinetics mechanism which measures the reaction rates
and the affinity of enzymes towards the substrates and inhibitors. The enzymes
can be isolated from the various biological factories such as plant, animal, or
microbial cells depending upon the type of enzyme. However, in order to produce
at large scale, the microbial sources are the best choice which can effectively
reduce the production and purification cost of enzymes. The enzymes are widely
used in various sectors such as agriculture, environmental, leather tanning, paper
and pulp, chemical and pharmaceutical, detergent, food and beverages, etc. In this
chapter, we are mainly focussing on the role of enzymes in the food industry.

S. M. Khade (*) · J. Srivastava

School of Engineering, Ajeenkya D Y Patil University, Pune, Maharashtra, India
S. K. Srivastava
School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University),
Varanasi, Uttar Pradesh, India
L. H. Kamble
School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra,
India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 1

Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_1
2 S. M. Khade et al.

Keywords

Enzyme · Biocatalyst · Substrate · Physico-chemical parameters · Enzyme

kinetics · Inhibitors

1.1 Introduction

An enzyme is a biomolecule which acts as a biocatalyst that increases the rate of a

chemical reaction without altering the equilibrium of the reaction. The enzyme
transforms a substrate into a product, without getting changed itself. Enzymes are
produced by the living cells. The term ‘enzyme’ was first named by Kuhne in 1878
for the catalytically active substances earlier called ferments. However, a German
biochemist Eduard Buchner in 1897 was the first to discover the enzyme zymase
from the yeast which is involved in the fermentation of sugar into alcohol. In 1907,
he was awarded the Nobel Prize for the biochemical investigations of non-cellular
alcoholic fermentation (Kohler, 1971). Most of the metabolic reactions are catalysed
by the enzymes in the living cell. All the enzymes are not present at all the time in the
cell, but are produced whenever the cell needed from the blueprint is present in
DNA. There are nearly 75,000 enzymes that are functional in the human body which
are divided into three types: metabolic enzymes (responsible for anabolic and
catabolic processes), digestive enzymes (mainly extracellular responsible for the
digestion of food), and food enzymes from raw foods (responsible for initiation of
food digestion) (Barnes-Svarney & Svarney, 2014).

General Properties:
1. Almost all enzymes are protein in nature except a small group of catalytic RNA
molecules. The catalytic activity of the enzyme relies on the structural integrity of
native protein conformation.
2. Enzymes are very specific to their substrates.
3. Enzymes possess huge catalytic power which increases the rate of a chemical
reaction by reducing the activation energy without altering the equilibrium
position of the reaction.
4. The biocatalytic power of the enzyme depends on the optimum physio-chemical
parameters of the reaction such as pH, temperature, and salt concentration.
5. Enzymes are highly regulated in nature.

Generally, the enzymes are classified as

(a) Simple enzymes: The enzymes which are entirely made up of the proteins
constituting the amino acids only, e.g. trypsin, pepsin, etc. (Bisswanger, 2017).
(b) Conjugated enzymes: The enzymes which contain the non-protein part called
cofactor along with the protein part, called apoenzyme. In these enzymes, the
apoenzyme part is generally biologically inactive, if the cofactor is removed.
The complete biologically active conjugated enzyme (apoenzyme and cofactor)
1 Food Enzymes: General Properties and Kinetics 3

is called holoenzyme. The cofactor may be inorganic or organic in nature that

linked either covalently or non-covalently to the apoenzyme. The inorganic
cofactors are simple metal ions, whereas the organic cofactors are complex
organic groups called coenzymes. Also, the organic cofactors which are firmly
attached to the apoenzyme are called prosthetic group. The conjugated
enzymes are alkaline phosphatase, horseradish peroxidase, etc. (McComb
et al., 2013).

1.2 Nomenclature and Classification

Most of the enzymes are named with the suffix –ase; however, some old names are
exceptions, e.g. trypsin, ptyalin, pepsin, chymotrypsin, renin, etc. Also, some of the
old names indicate the source rather than the action, e.g. papain from Papaya and
bromelain from Pineapple (Bromeliaceae family). Many enzymes are named based
on substrate and the chemical reaction they catalyse with the suffix –ase,
e.g. pyruvate kinase that helps in the synthesis of pyruvate from the phosphoenol-
pyruvate, whereas pyruvate dehydrogenase enzyme which converts pyruvate to
acetyl-CoA. To avoid the confusion arose due to the common names, an Interna-
tional Commission on enzymes was established for the systematic nomenclature of
enzymes.
The enzyme commission (EC) has set a regulation for the nomenclature of
enzyme. According to EC, each enzyme is classified and named based on the type
of chemical reaction, and for each enzyme, number with four parts is given, e.g. EC
1.7.3.3 (Uricase); EC 3.5.1.1 (Asparaginase). The first three numbers denote major
class, subclass, and sub-subclass, respectively, and the last number is a serial number
in the sub-subclass, which indicates the order in which each enzyme is added to the
list (Boyce & Tipton, 2001) (Table 1.1).
All the enzymes are classified into six classes based on the type of chemical
reaction they catalyse. The first integer in the EC number denotes the class of
enzymes.

Table 1.1 Common name and EC numbers of some enzyme

Common name of an enzyme EC number Reference
Hexokinase EC 2.7.1.1 Dai et al. (1995)
Lactate dehydrogenase EC 1.1.1.27 Vanderlinde (1985)
α-Amylase EC 3.2.1.1 Janeček (2009)
Pectinase EC 3.2.1.15 El Enshasy et al. (2018)
Lipase EC 3.1.1.3 Kouker and Jaeger (1987)
Invertases EC 3.2.1.26 Vu and Le (2008)
Xylanase EC 3.2.1.8 Huhtanen and Khalili (1992)
Alcohol dehydrogenase EC 1.1.1.1 Heinstra et al. (1989)
Maltase EC 3.2.1.20 Nawaz et al. (2015)
4 S. M. Khade et al.

1.2.1 EC 1 Oxidoreductase

The enzymes which catalyse the oxidation-reduction reaction are classified under
this class. The enzymes are involved in the transfer of electrons from one molecule
(oxidant) to another molecule (reductant) (Table 1.2).

Xoxi þ Yred ! Xred þ Yoxi

1.2.2 EC 2 Transferase

The enzymes which catalyse the transfer of groups from one molecule to another are
classified under transferase class. The groups involved in the transfer are phosphoryl
(–PO32), methyl (–CH3), carboxyl (–COOH), amino (–NH2), acyl (–RC¼O), and
carbonyl (–C¼O). The common names for these enzymes start with prefix trans with
exceptions of kinases, phosphorylases, etc. (Table 1.3).

XAþY!XþYA

Table 1.2 Some examples of enzymes under oxidoreductase class

Oxidoreductase Type of reaction Reference
Peroxidases Utilize H2O2 as an electron acceptor Gramss and
Rudeschko (1998)
Dehydrogenases Oxidize a substrate by reducing the electron acceptor Pumirat et al.
other than oxygen, e.g. NAD+ (2014)
Oxygenase Directly incorporate oxygen into the substrate Huang et al. (2008)
Oxidases Use molecular oxygen as an electron or hydrogen Kiess et al. (1998)
acceptor

Table 1.3 Some examples of enzymes under transferase class

Transferase Type of reaction Reference
Transaminases Transfer amino group from amino acids to keto acids Smit et al. (2009)
Kinases Transfer of phosphate group from ATP to substrate Martín et al.
(2010)
Methyltransferases Transfer of methyl group from methyl donor to the Shixian et al.
substrate (2006)
Acyltransferases Transfer of acyl group to the substrate Kuhajda et al.
(2011)
1 Food Enzymes: General Properties and Kinetics 5

1.2.3 EC 3 Hydrolases

These are the enzymes which catalyse the cleavage of a covalent bond by adding
water (Table 1.4).

X  Y þ H2 O ! X  H þ Y  OH

1.2.4 EC 4 Lyases

These are the enzymes which cleave C–C, C–O, C–N, C–S, and other bonds without
hydrolysis or oxidation, but by means of elimination, which results into the forma-
tion of double bond or a new ring or contrarily adding groups to double bonds
(Table 1.5).

X ¼ Y þ SP ! X  S þ Y  P

1.2.5 EC 5 Isomerases

The isomeric structures are created by means of intramolecular arrangements that

convert one isomer to another (Table 1.6).

XY!YX

Table 1.4 Some examples of enzymes under hydrolases class

Hydrolases Type of reaction Reference
Esterases Cleave esters into an acid and an alcohol Faulds et al. (2004)
Phosphatases Remove phosphate group from a substrate Morisseau et al. (2012)
Peptidases Cleave amide bonds present in the proteins Perrier et al. (2005)

Table 1.5 Some examples of enzymes under lyases class

Lyases Type of reaction Reference
Decarboxylases Removal of CO2 by elimination reactions van der Werf et al.
(1994)
Dehydratases Formation of double or triple bond by elimination of Shibata et al. (2010)
water
Ferrochelatases Addition of divalent metal cations to the tetrapyrrole Gazel and Yildiz
structure (2016)
6 S. M. Khade et al.

Table 1.6 Some examples of enzymes under isomerases class

Isomerases Type of reaction Reference
Racemases Interconversion of L and D stereoisomers Staudigl et al. (2014)
Tautomerases Interconversion of keto- and enol-groups Orita et al. (2001)
Mutases Intramolecular transfer of functional groups Rhimi et al. (2008)

Table 1.7 Some examples of enzymes under ligases class

Energy
Ligases Type of reaction source Reference
Carboxylases Catalyse the formation of C–C bond by ATP Portevin et al.
using CO2 (2005)
T4 DNA Catalyse the formation of ATP Wilson et al. (1997)
ligases phosphodiester bond
E. coli DNA Catalyse the formation of NAD Nandakumar et al.
ligases phosphodiester bond (2007)

1.2.6 EC 6 Ligases

These are the enzymes which join the two molecules by forming a chemical bond
like C–C, C–N, C–O, and C–S. The energy required for these is gained by the
cleavage of ATP or NAD. Ligases are also called synthetases, because of synthesis
of new molecule (Table 1.7).

X þ Y þ ATP ðNADÞ ! X  Y þ ADP ðNAMÞ

1.3 Role of Enzyme

The collision of an atom, ion, or molecule of one substance with an atom, ion, or
molecule of other results into a chemical reaction between the two substances. After
this collision, the intermediate product is produced, having higher chemical energy
than the chemical energies of both the reactants together. The transition state in the
reaction is achieved with the help of some of the energy entering the reaction apart
from the chemical energy of the reactants. The transition state is the stage with
highest free energy. The difference in free energy between the transition state and the
reactants is known as the Gibbs free energy of activation or only the activation
energy.
The enzyme plays an important role in lowering the activation energy of a
reaction which results in increase in the rate of reaction in both the directions.
However, the enzyme cannot alter the relative energies of the initial and final states.
In addition, the catalyst does not alter the position of equilibrium of a chemical
1 Food Enzymes: General Properties and Kinetics 7

Fig. 1.1 Free energy profile

of uncatalysed vs. catalysed
reactions. The standard free
energy of reaction (ΔG0) and
free energy of activation for
uncatalysed (ΔG#uncat ) and
catalysed (ΔG#cat) reactions are
shown here (Punekar, 2018)

reaction, but only increases the rate of a reaction by lowering the activation energy
(Fig. 1.1).
The biocatalysis of a chemical reaction begins with the binding of enzyme and the
substrate, the substrate to be catalysed. The substrate specifically binds at the
specialized region in the enzyme called active site with multiple weak
non-covalent interactions such as hydrophobic, hydrogen bond, ionic interaction,
or reversible covalent bonds. Upon binding of the enzyme and substrate, a large
amount of free energy is released, called binding energy, which is used to lower the
activation energy. The general catalysed reaction between enzyme and substrate may
be represented as

EþS
ES!EþP

where E is enzyme, S is substrate, the complex E–S is the enzyme-substrate

complex, and P is the product formed.
The binding of substrate at the active site of the enzyme has been proposed with
two models.

1.3.1 Lock-and-Key Model

This model assumes that the binding of substrate at the active site of enzyme is
highly compact, which may be due to the high degree of similarity between the shape
of the substrate and the geometry of the binding site, i.e. active site on the enzyme.
This binding of substrate and enzyme is just like a lock and a specific key (Fig. 1.2).
8 S. M. Khade et al.

S
Acve site P

E-S E
E

Fig. 1.2 Lock-and-key model of enzyme-catalysed reaction

Enzyme changes
S Slightly different shape slightly as P
acve site substrate binds

E E-S E

Fig. 1.3 Induced-fit model of enzyme-catalysed reactions

1.3.2 Induced-Fit Model

This model describes the behaviour of protein with some three-dimensional flexibil-
ity. Based on this model, the conformational change occurs in the enzyme upon
binding of the substrate which leads to complementary fit as soon as substrate is
bound. However, prior to substrate binding, the active site has slightly different
three-dimensional shape. After binding of the substrate, the enzyme-substrate com-
plex with transition state is formed which subsequently converts into product and
free enzyme (Fig. 1.3).

1.4 Enzyme Kinetics

Enzyme kinetics is the quantitative study of chemical reactions catalysed by the

enzymes. The kinetic study helps to determine the reaction rates and the affinity of
enzymes for the substrates and the inhibitors. Let us consider a chemical reaction
X + Y ! P, where X and Y are the reactants and P is the product obtained, the rate of
reaction can be determined by either the rate of disappearance of one of reactants or
the rate of formation of product. The rate of disappearance of X is Δ[X]/Δt, where
Δ represents change, [X] is the concentration of A in mol/L, and t denotes time.
Similarly, the disappearance of another reactant Y is Δ[Y]/Δt and the rate of
formation of product P is Δ[P]/Δt. So, from these, the rate of reaction can be
measured which is related by the stochiometric equation for the reaction.
1 Food Enzymes: General Properties and Kinetics 9

Δ½X Δ½Y Δ½P

Rate ¼  ¼ ¼
Δt Δt Δt

1.4.1 Kinetics of Enzyme-Catalysed Reactions

Leonor Michaelis and Maud Menten in 1913 proposed a model for the kinetics study
of enzyme-catalysed reaction of hydrolysis of disaccharide sugar and sucrose by
invertase enzyme into monosaccharide sugars, glucose, and fructose units.
Michaelis-Menten assessed the relationship between the reaction velocity and the
substrate concentration. The results of this disaccharide hydrolysis into the two
monosaccharides by the invertase are represented with the following reaction:

k2
E þ S
kk11 E  S ! E þ P

Here, k1 represents the rate constant for the formation of enzyme-substrate

complex, ES after the binding of substrate S with the enzyme E; the rate constant
k1 denotes the reverse reaction of dissociation of enzyme-substrate complex to the
free enzyme and the substrate; whereas the rate constant k2 represents the conversion
of ES complex to product P and the free enzyme.
As per Michaelis-Menten, the rate or velocity of enzyme-catalysed reaction
depends upon the substrate concentrations [S], when measured at different substrate
concentrations with keeping all the physio-chemical parameters constant throughout
the course of experiment. At the low concentration of substrate, the initial velocity
(V0) increases at a linear rate with the increase in the concentration of substrate to the
point beyond which the velocity does not increase further called maximum velocity
(Vmax) with increase in the substrate concentration (Fig. 1.4).

Vmax

0 Km [S]

Fig. 1.4 Kinetic study of substrate-saturation curve

10 S. M. Khade et al.

1.4.1.1 Lineweaver–Burk Plot

In Lineweaver–Burk plot or doubsle reciprocal plot, this graph was plotted in
between reciprocal of velocity against the reciprocal of substrate concentration as
in the following equation:

1 K 1 1
¼ m  þ ð1:1Þ
v V max ½S V max

In this plot, x and y intercepts represent K1m and V max

1
, respectively (Fig. 1.5).

1.4.1.2 Eadiae-Hofstee Plot

The errors in kinetic parameters in Double reciprocal plot were minimized by
Eadiae-Hofstee plot which was plotted in between the rate of reaction (v) and v/
[S] and represented by the following equation:

v
v ¼ K m  þ V max ð1:2Þ
½S

where Vmax is the y-intercept and VKmax

m
is the intercept on x-axis (Fig. 1.6).

1.4.1.3 Hanes-Woolf Plot

The kinetic parameters were determined by the advanced plot called Hanes-Woolf
plot. The graph was plotted between the substrate concentrations vs. the rate of
reaction ‘v’. The following equation was used for the plot:

Fig. 1.5 Lineweaver–Burk plot to determine kinetic parameters

1 Food Enzymes: General Properties and Kinetics 11

Vmax

Fig. 1.6 Eadiae-Hofstee plot to determine kinetic parameters

- Km

[S]

Fig. 1.7 Determination of kinetic parameters by Hanes-Woolf plot

½ S ½S K
¼ þ m ð1:3Þ
v V max V max
1
The straight line graph will yield V max as the slope, whereas VKmax
m
as the y-intercept
and Km as the x-intercept (Fig. 1.7).
12 S. M. Khade et al.

1.5 Applications of Enzymes in Food Industries

The enzymes play a vital role either in the food processing or additives in the food
industries. The major applications of food enzymes are in the baking industries,
dairy industries, starch processing, juice industries, brewing industries, etc.

1.5.1 Baking Industry

To produce a valuable high-quality bakery product, the baking industry utilizes

yeasts and enzymes since long ago. The starch containing foods can be processed
by the addition of amylolytic enzymes like amylases to improve the softness,
freshness, and shelf life qualities of food. The other enzymes, such as xylanases,
oxidoreductases, lipases, and proteases, are also used to improve the dough stability,
gluten strength, stability of the gas cells in the dough, and by reducing the protein
content in the flour, respectively (Li et al., 2012).

1.5.2 Dairy Industry

The enzymes employed for the processing of dairy products become essential in the
improvement of the quality and physiologically health benefits. The enzymes,
chymosin (coagulation), lipases (low-down fat level), and lysozymes (to avoid late
blowing), are used in the production of cheese (Kilcawley et al., 1998; Marseglia
et al., 2013). In addition, β-galactosidases and lactases enzymes are predominantly
employed in the hydrolysis of lactose sugar in milk into glucose and galactose to
avoid lactose intolerance.

1.5.3 Starch Industry

The α-amylase is the prominent enzyme used in the baking industry to act on the
starch present in the dough of the bread into the dextrins, which enhances the
fermentation process. The released extra sugar in the dough improves the taste and
textural properties of the bread (Souza, 2010).

1.5.4 Juice Industry

The enzymes, amylases and glucoamylases, are mainly used in the clarification of
cloudy juices. Pectinases are used in the hydrolysis of structural
heteropolysaccharide of fruit cell wall pectin, leading to increase in the juice
production. Naringinases are mainly employed to reduce the bitterness of the citrus
juices (Tapre & Jain, 2014).
1 Food Enzymes: General Properties and Kinetics 13

1.5.5 Brewing Industry

The α-amylase hydrolyses the starch into maltose and glucose units with reduced
viscosity. β-Glucanases hydrolyse glucans to reduce the viscosity and smooth wort
separation. Proteases are used to process the malt which allows favourable content
for yeast growth. Xylanases are used to enhance the 5-C pentose utilization in malt,
barley, and wheat with extraction and beer filtration (Li et al., 2012).

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Plants- and Animal-Derived Enzymes
and Their Potential Application in Food 2
Processing and Preservation

Mahmoud Aminlari

Abstract

Enzymes are biological catalysts which increase the rate of biochemical reactions
in living cells. It is important that enzymologist understands the specific action of
particular enzyme in a plant or animal tissue and applies these properties in vitro
and in a food product. Most enzymes can be used as processing aids and as
protection agents against microbial and deteriorative processes. Although the
advent of recombinant DNA technology and advances made in food applications
by microorganisms are more practical and economic, some of these enzymes are
sufficiently abundant in their natural sources to make them amenable to large-
scale production (for example, egg-white lysozyme and plant proteases). In this
chapter, several plant and animal enzymes, their occurrence, and potential
applications in food industry will be presented. Emphasis will be made on
enzyme working on carbohydrates, proteins, and lipids. A section is devoted to
miscellaneous enzymes used in food industry, such as phenylalanine ammonia
lyase of wheat seedling which metabolizes Phe, thereby rendering foods suitable
for PKU patients. In the final section of this chapter, examples of chemical
modification of enzymes to improve their properties will be discussed and
examples of the studies on modification of chicken egg-white lysozyme to
enhance its functional and antimicrobial activities, performed in the laboratory
of this author, will be presented.

M. Aminlari (*)
Department of Food Science and Technology, School of Agriculture, Shiraz University, Shiraz, Iran
Department of Biochemistry, School of Veterinary Medicine, Shiraz University, Shiraz, Iran
e-mail: aminlari@shirazu.ac.ir

# The Author(s), under exclusive license to Springer Nature Singapore Pte 17

Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_2
18 M. Aminlari

Keywords

Plant enzymes · Animal enzymes · Food application · Antimicrobial · Bioactive

peptides · Chemical modification

2.1 Introduction

Enzymes perform many biochemical functions in living cells. They participate in

multitude of biochemical reactions which are vital for cell well-being, growth, and
production of various biochemical products. As such, enzymes are indispensible in
any living system. Some enzymes are unique to particular cell type, while others are
present in many different types of cells. It is the responsibility of the food enzymol-
ogist to determine and adapt the functionality of a particular enzyme in a plant or
animal tissue to a functional property in food systems.
Enzymes from plants and animals have been utilized in the food industry for
millennia. The maturation of sugar to liquor by yeast is an earliest illustration of a
biotechnological cycle. Enzymes present in various raw materials have been used to
produce traditional foods such as cheese, yoghurt, and bread and fermented
beverages such as wine, beer, and vinegar for many thousands years (Poulsen &
Buchholz, 2003). As early as 1783, it was shown that pepsin gastric juice could
digest meat in vitro. The first industrial application of enzymes involved using crude
protease mixture from animal pancreas as laundry detergents in the beginning of
twentieth century.
In view of abundance of starting raw material the greater part of the first
intracellular enzymes came from yeast and skeletal muscle. Starting late 1940s,
significant improvements in the separation techniques such as chromatography
applicable to proteins resulted in preparation of large quantities of industrially
important enzymes (Aehle, 2007). With the advent of recombinant DNA technol-
ogy, it became possible to transfer and express the genes encoding the enzymes of
interest to selected host microbes for production at industrial scale. Today, molecular
biology and genetic engineering play major roles in both the discovery of novel
enzymes and the optimization of existing enzymes and are the basis for the produc-
tion of the majority of industrial enzymes.
The grade or degree of purity of enzyme preparation depends on its application.
For many industrial applications, partially purified enzyme preparations can be used,
provided they do not contain interfering contaminating activities.
They are less costly and, therefore, preferred to highly purified products. They are
commonly produced in large scale worldwide and are mostly obtained from animal
or plant sources with few purification steps. Examples include proteases and
enzymes working on carbohydrates. Enzyme destined for analytical purposes must
be pure and do not contain unknown by-products that can interfere with enzymatic
analyses, for example, glucose oxidase and restriction endonucleases. Finally, clini-
cally important enzyme which must be highly purified is usually sold in crystalline
form and must be devoid of any contaminating activity. A very famous example of
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 19

this group of enzymes is asparaginase which is used for treatment of childhood

leukemia (Chahardahcherik et al., 2020). The last two types of enzyme are produced
from microbes, plants or animals. These enzymes are produced in mg-g quantities
worldwide using sophisticated purification procedures.
Food applications of enzymes represent wide and highly diverse fields including
baking, dairy, fruit juice, vegetable processing, and meat. Several factors must be
taken into consideration when one attempts to employ an enzyme in foods; reaction
specificity, requirement for mild conditions, possibility of formation of an unaccept-
able by-product by a chemical process, and relative cost-benefit.
Uses of exogenous enzymes are used in the production of many
commodities (glucose, high-fructose corn syrup, invert sugar, and other sweeteners),
protein hydrolysates, and modified lipids; modification of components inside a food
matrix (such as brew adjustment, cheese making, meat tenderization, citrus juice
debittering, and crumb relaxing), process improvement (such as cheese maturing,
juice extraction, juice clarification, fruit and oil seed extraction, drink filtration,
quicker batter blending, baked product raising, and adjustment), process control
(for example, online biosensors), and food analysis. The enzymes are utilized to
acquire various advantages, like more proficient cycles, prompting decreased utili-
zation of raw materials, improved or consistent quality, substitution of synthetic
food-additives and evasion of potential destructive side effects in the food, and
enhancements of nutritional attributes.
There are some regulations in food industry which allow adding certain chemical
agents to foods to achieve particular goals, such as oxidizing and reducing agents
(cysteine, bisulfites, and potassium bromide) in baking industry and preservatives
such as benzoate and sorbates in different foods. However, in recent years, consumer
demand for “natural” foods has driven development of products without additives. In
order to meet this demand, much attention and interest have been directed toward
identification and application of naturally made compounds. In many cases, the
beneficial effect of enzymes to replace chemical agents can be demonstrated.
This chapter aims to present an overview of recent developments in identification
of novel plant and animal enzymes with potential application in the food industry.
The aim is to increase awareness of and stimulate interest in developing novel
enzyme technologies to meet the new and changing needs of the food industry.
The activities of many endogenous enzymes continue postharvest and postmortem,
and in most cases, contribute to the food quality deterioration, such as excessive
ripening of fruits by pectic enzymes, hydrolytic rancidity of milk lipids due to lipase
activation, and extra meat tenderization by calpain system. However, the aim of this
chapter is to exclusively address the application of exogenously added enzymes of
plant and animal origin for their beneficial effects. Because of space limitation, this
chapter focuses on enzymes working on major food constituents (i.e., carbohydrates,
proteins, and lipids). A few enzymes with specific action in foods will be briefly
described under the heading “miscellaneous enzymes”. A section is devoted to
production of bioactive peptides through use of plant and animal proteases. In the
final section of this chapter, examples of chemical modification of enzymes to
improve their properties will be discussed and some of the studies done on lysozyme
20 M. Aminlari

in the laboratory of this author will be presented. Table 2.1 summarizes the use and
suggested use of enzymes in food industry.

2.2 Enzyme Working on Carbohydrates

Most of the enzymes working on food carbohydrates are hydrolase, i.e., use water as
one of the substrates. These enzymes are commonly referred to as glycosyl
hydrolases or glycosidases.
They catalyze the hydrolysis of glycosidic bonds in complex sugars. They are
amazingly normal enzymes with roles in nature including degrading of complex
biomass like starch (amylase), cellulose (cellulase), hemicellulose, and
as antibacterial safeguard methodologies (e.g., lysozyme), in pathogenesis
components (e.g., viral neuraminidases), and in ordinary cell work (e.g., managing
mannosidases associated with N-connected glycoprotein biosynthesis). Along with
glycosyltransferases, glycosidases’ are the major synergist apparatus for the synthe-
sis and breakage of glycosidic bonds (Bourne & Henrissat, 2001). Glycosidases
hydrlyze α-1,4 or α-1,6 glycosidic bonds. Glycosidases can be classified either as
“retaing” or “invering” types, in light of the destiny of the anomeric configuration (α
or β) of the hydrolyzed glycosidic bond. One more broad differentiation among
glycosidases is whether they are “endo” or “exo” splitting. Exo-acting works for the
most part on the non-reducing terminal of the substrate, while endo-acting types
rondomly assault internal glycosidic bonds of the substrate. Naming glycosidases as
“α” and “β” (as in amylases and glucosidases) perceives the anomeric configuration
of the freed reducing group as being axial and equatorial, respectively (Whitaker,
1994). Specialty applications for various glycosidases continue to emerge.
Glycosidases account for about half of enzyme used as processing aids in the food
industry, primarily for the production of low molecular weight sweetener and
bulking or thickening agents (dextrins) from starch (such as amylases), and for
carbohydrate modification in baking and fruit products (such as cellulases and pectic
enzymes) (Parkin, 2017). Most of these enzymes are commercially available. A
summary of the types and classification of glycosidases of most importance in foods
is provided in Table 2.2.

2.2.1 Amylases

2.2.1.1 a-Amylases
The amylases are utilized to hydrolyze starch into lower molecular weight dextrins
and consequently “thin” starch suspensions. α-Amylase is an endo- splitting glycosi-
dase that hydrolyses α-1,4 glycoside bond. The initial product is dextrin and the final
main products are maltose or maltotriose. The α-1,4 bonds close to α-1,6 branches
are impervious to hydrolysis. Broad hydrolysis of amylopectins with α-amylases
produces “α-limit dextrins,” as cleavage stops at the α-1,6 bonds. Figure 2.1 shows
the impacts of various glycosidases on starch molecule (Wong & Robertson, 2007;
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 21

Table 2.1 Some uses and suggested uses of enzymes in food industries (from Whitaker, 1994)
Enzyme Food Purpose or action
Amylases Baked goods Increase in maltose content for food fermentation
Brewing Conversion of starch to maltose for fermentation, removal of
starch turbidity
Cereals Conversion of starch to dextrins and maltose, increase water
absorption
Confectionary Recovery of sugar from candy scraps
Fruit juices Removal of starch to increase sparkling properties
Jellies Removal of starch to increase sparkling properties
Pectin Aid in preparation of pectin from apple pomace
Syrup Conversion of starch to low molecular weight dextrins (corn
syrup)
Vegetables Hydrolysis of starch as in tenderization of peas
Cellulase Brewing Hydrolysis of complex carbohydrates cell walls
Coffee Hydrolysis of cellulose during drying of beans
Fruits Removal of graininess of pears, peeling of apricot, tomatoes
Invertase Artificial Conversion of sucrose to glucose and fructose
honey
Candy Manufacture of chocolate-coated soft-cream candies
Lactase Ice cream Prevention of crystallization which results in grainy, sandy
texture
Milk Stabilization of milk proteins in frozen milk by removal of
lactose. Hydrolysis of lactose, permitting use by adults deficient
in lactase in intestinal and infants with congenital lactase
deficiency
Tannase Brewing Removal of polyphenolic compounds
Pentosanase Milling Recovery of starch from wheat flour
Naranginase Citrus Debittering citrus juice by hydrolysis of the glucoside naringin
Pectic Chocolate, Hydrolytic activity during fermentation of cocoa
enzymes cocoa
Coffee Hydrolysis of gelatinous coating during fermentation of beans
Fruits Softening
Fruit juice Improving yield of press juice, prevention of cloudiness,
improving concentration process
Olive Extracting oil
Wines Clarification
Proteases Baked goods Softening action in dough, cut mixing time, increase
extensibility of dough, improvement in grain, texture, loaf
volume, liberate β-amylase
Brewing Body, flavor, and nutrient development during fermentation,
aid in liberation and clarification, chill-proofing
Cereals Modification of proteins to increase drying rate, improve
product handling characteristics, manufacture of misu and tofu
Cheese Casein coagulation, characteristic flavor during aging
Chocolate, Action on beans during fermentation
cocoa
(continued)
22 M. Aminlari

Table 2.1 (continued)

Enzyme Food Purpose or action
Eggs Improve drying properties
Meat, fish Tenderization, recovery of proteins from bones and trash fish,
liberation of oils
Milk In preparation of soy-milk
Protein Condiments such as soy sauce, specific diets, dehydrated soups,
hydrolysate gravy powder, processed meats, production of bioactive
peptides, aids in digestion of proteinous foods
Wines Clarification
Lipase Cheese Aging, ripening, and general favor characteristics
Oils Conversion of lipids to glycerol, fatty acids, and
monoglycerides
Milk Production of milk with slightly cured flavor for use in milk
chocolates

Table 2.2 Catalytic properties of some glycolytic enzymes important in food technology (from
Whitaker, 1994; Parkin, 2017)
Bond Product
Enzyme selectivity selectivity Products
α-Amylase (endo) α-1!4 glucose Retaining Initial major product dextins, final
α!α product maltose, maltotriose
β-Amylase (exo) α-1!4 glucose Inverting β-Maltose
α!β
Pullulanase (endo) α-1!6 glucose Retaining Acts on pullulan to give maltotriose and
α!α on starch to give linear dextrins
Glucoamylase α-1!4 Inverting β-Glucose
(α-1!6) α!β
glucose
Cyclomaltodextrin α-1!4 glucose Retaining Acts on cyclodextrins and linear dextrins
transferase α!α to give maltose and maltotriose
Cellulase β-1!4 glucose 1,4-β-dextrins, mixed 1,3–1,4-β-dextrins
Invertase β-1!2 fructose Retaining Glucose and fructose
β!β
β-Galactosidase β-1!4 Retaining Galactose and glucose
galactose β!β
β-Gluctosidase β-1!4, β-1 Retaining Aglycan and glucose
aglycan, β!β
glucose
Polygalactourinase α-1!4 Inverting Galactouronic acid
α!β
Xylanase α-1!4 xylose Retaining
β!β
Lysozyme α-1!4 Retaining
NAM-NAG α!α
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 23

Fig. 2.1 The action of

different glycosidases on
starch molecule. (From Wong
& Robertson, 2007; Saini
et al., 2017)

Saini et al., 2017). α-Amylase is an important enzyme in carbohydrate metabolism

of microorganisms, plants, and animals. The active site requires a substrate of no less
than three glucose units long. The normal final results of α-amylase activity are
α-limit dextrins and maltooligosaccharides of up to 12 glucose units. The random
action of this enzyme on starch results in rapid decrease in the viscosity of
starch solution (Fig. 2.2). The average molecular mass of α-amylase from different
sources is 50–70 kDa. α-Amylases requires Ca2+ for activity. Ca2+ is firmly bound
and serves to widen the pH optimum of the catalyst to between pH 6 and 10 as
displayed in Fig. 2.2. α-Amylase from different sources are stable at temperatures
in the range of 30–130  C (Pandey et al., 2000). There are many commercial
sources of α-amylases available, the majority of which are microbial, despite the
fact that malt (grain or wheat) amylases are also accessible. The α-amylase routinely
is purified from barley using different purification steps, including salt precipitation,
centrifugation, ion exchange, and affinity column chromatographic methods such as
CM Sepharose CL-6B column and cyclohexaamylose (CHA)-Sepharose affinity
column. The enzyme can be also expressed in yeast and then purified from yeast
extract as described above (MacGregor & Morgan, 1992).
24 M. Aminlari

Fig. 2.2 Effect of pH, calcium ion, and salts on α-amylases: (a) pancreatic, (b) Bacillus subtilis
α-amylases, (c) effect of different salts on α-amylase activity (A: No salt, B: 0.04 M NaCl, C:
0.04 M bromide, D: 0.04 M iodide, E: 1.0 M nitrate, F: 1. 0 M chlorate), effect of β-amylase (d) and
α-amylase (e) on viscosity and reducing sugar content during starch hydrolysis. (From Wong &
Robertson, 2007; Saini et al., 2017)

Food Application of α-Amylases

Starch Hydrolysis
Ordinary applications include preparation of starch syrup, dextrose, liquor, brewing,
and bakery. α-Amylase is utilized economically in the liquefaction of starch to create
dextrins which are further saccharified by glucoamylase to yield glucose feed stock
for corn syrup, fuel ethanol, or alcoholic beverages. Heat stable α-amylase in the
presence of calcium ion and a 105 degree centigrade is used to produce a 30-40%
starch slurry.
This yields a mixture of linear and branched dextrins (maltodextrins) with extent
of hydrolysis ranging from 8 to 15 DE (DE: dextrose equivalence). The product of
this reaction is used for the production of 15–40 DE maltodextrins (corn syrups, used
as thickening, bulking, and viscosity building), sweetener production, and a 95–98%
glucose syrup (95 DE) in which other enzymes such as glucoamylase (which is often
used as an immobilized enzyme column), with or without pullulanase, might also be
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 25

used. The glucose syrup can then be treated with an immobilized glucose isomerase
column to generate a high-fructose corn syrup of 42% fructose (52% glucose)
(Wong & Robertson, 2007; Van der Maarel et al., 2002).
In future, improving starch handling and change will zero in on broadening
optimum pH (to pH 4–5), heat stability and decreasing Ca2+ prerequisite of
α-amylase, thereby improving starch processing and transformation, and improving
the capacity to process crude starch by β-amylases. For all enzymes included,
improving heat stability is of significance as this will increase efficiency in handling
just as advancing a single-step process. Likewise, improvement of determinants of
selectivity is also important.

Baking and Baked Goods Industries

Over the course of bread making, proteins have consistently assumed a significant
role. Indeed, even the old Egyptians utilized proteins present endogenously in the
flour, in spite of the fact that they might not have known about the efffect.
The first application of enzymes in baked goods was supplementation of
α-amylase by addition of malt to correct the concentration of endogenous
α-amylase in the flour. Today, a whole range of enzymes is available for end users
of flour. These make it possible to correct suboptimal concentrations of endogenous
flour enzymes. Virtually all of the glycosidases presented in Table 2.2 have been
added for some benefit in baking applications. The most widely used enzyme in
baking industry in terms of amount and value is α-amylase. Initially, amylases were
believed to function primarily by mobilizing fermentable carbohydrate for yeast.
The essential impact of amylase supplementation is getting satisfactory gassing
power by degradation of damaged starch granules in the mixture, which helps malt-
ose production by endogenous β-amylase. It is presently perceived that amylases
added straightforwardly to the dough will decrease batter viscosity and further
develop portion volume, bread softness(antistaling), and color. The majority of
these impacts can be considered to be due to incomplete hydrolysis of starch during
baking as the starch gelatinizes. Decrease in viscosity advances volume and texture
by permitting the reactions engaged with mixture molding and baking to happen
quicker (mass transfer effect) (Christophersen et al., 1998).
The most broadly utilized α-amylase in baking industry is the alleged
α-amylase from fangus or Taka-amylase from Aspergillus oryzae. The most well-
known other options, malt amylases from grain or wheat malt, have higher protease
contamination and higher thermostability, and this makes them more inclined to
negative incidental effects when used too much. The second important goal for use
of amylases in baking is antistaling, i.e., improving the fresh keeping of baked
goods.
Staling is a highly complex phenomenon, but it is generally accepted that
retrogradation of amylopectin is the main contributor to bread staling (Morgan
et al., 1997). This happens during the first hour of cooling subsequent to baking,
the initial crumb structure is set by amylose gelation, which makes an organization in
which the gelatinized starch granules are implanted. Recrystallization of amylopec-
tin side chains leads to rigidification of the starch granules and to a general
26 M. Aminlari

reinforcing of the crumb structure, considered as an increment in crumb firmness. An

inter- and intragranular amylose recrystallization is additionally associated with the
staling process. Estimates of worth of lost baked goods due to staling in the United
States in 1990 was about US $1 billion. All the more as of late, the maltogenic types
of α-amylases as and β-amylase have been perceived as being far better antistaling
agents, since they will generally produce smaller maltooliogosaccharides (DP 7–9)
and larger dextrins (which are plasticizers) (Hebeda et al., 1991).

Brewery and Fermentations

One of the primary uses of enzymes in brewing industry is preparing the materials
for fermentation and whether the enzyme is endogenous in kernel or been added
from outer sources, a profound knowledge and examination of those enzymes is
required for better production and better overall quality. Fermenting is one of the
most established food processes done by man. Brewing is characterized as the course
of beer production where the sugars in starch are converted to ethanol through the
activity of yeast. These days, brewing is one of the lead food industries in many
countrie. Use of enzymes is one of the fundamental mainstays of brewing industry
(Gomaa, 2018).
The four most normal enzymes utilized in brewing are β-glucanase, protease,
α-amylase, and β-amylase. The shared factor in the beer production is the conver-
sion of starch in the grains to ethanol. In the first stage, enzymes hydrolyze starch
into fermentable sugars. In a subsequent interaction stage, these sugars are changed
over to ethanol and carbon dioxide by yeast. The customary sources of
enzymes utilized for the change of cereals into beer lager is malted grain, and malted
barely is a vital factor in brewing. Commercial enzymes can be utilized for additional
quality ascribes, for example, clarification, color, texture or flavor, and it is obliga-
tory to utilize the enzymes from outer source since the amylases endogenous to
malted grain are insufficient to use all of the fermentable starch because of low,
absence of heat stability, as well as presence of endogenous inhibitors in the grains
(Bamforth, 2009).
Thus, α- and β-amylases are added to maximize the availability of fermentable
carbohydrate. Auxiliary enzymes such as glucoamylase, pullulanase, glucanases,
xylanases, and cell wall hydrolyzing enzymes are added to complete the degradation
of starch to α- and β-limit dextrins that the thermally labile malt amylases cannot
achieve, to render limit dextrins fermentable and to hydrolyze glucans (similar to
cellulose, but with β-1,3 and β-1,4 linkages) and xylans. The remaining limit
dextrins provide body to the final product. Exogenous enzymes are added during
(or right after) the “mashing” step, which is conducted at moderate temperatures
(45–65  C), and they are destroyed during the subsequent boiling stage. A range of
exogenous enzymes, such as glucanases, acetolactate decarboxylase, and prolyl
endopeptidase, are available for enhancement of the existing brewing process
(Bamforth, 2009).
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 27

2.2.1.2 b-Amylases
β-Amylase is an exoglucanase that catalyzes the hydrolysis of 1,4-α-D-glucosidic
bonds in polysaccharides to separate progressively maltose units from the nonreduc-
ing end of α-1,4-glucans like starch and glycogen. Sweet potato contains a bountiful
β amylase which represents 5% of the total soluble proteins in the tuber; while only
little α-amylase is available. The activity of this endogenous catalyst brings about
production of maltose which renders a pleasant sweet flavor to potato (Thacker et al.,
1992).

Properties and Applications

There are several similarities and differences between β-amylase and α-amylase.
Both enzymes cleave α-1,4 glucosidic bonds in amylose and amylopectin.
β-Amylase cleaves successively maltose units from the nonreducing end. Both
have an average molecular weight of about 50 kDa. While α-amylase is widely
distributed in nature, β-amylase is only found in plants.
Seeds of higher plants and sweet potatoes are the essential source of β-amylase.
β-Amylase is liable for the pleasantness of aged natural product since it hydrolyses
starch into maltose during ripening of fruits. pH 4.0–5.5 is optimum for the hydro-
lytic action of β-amylase. The product in α-amylase activity is an oligosaccha-
ride with α configuration, and in β-amylase, it is β-maltose.
α-Amylase is an endo-splitting glycosidae, while β-amylase is exo-splitting.
α-Amylase does bypass the branch point (i.e., α-1,6 glucosidic bonds) and
β-amylase cannot bypass. Both enzymes cause reduction in viscosity as
demonstrated in Fig. 2.2d, e, α-amylase acts more rapidly than β-amylase (Das &
Kayastha, 2019).
The amino acid sequences of β-amylases from different plants, including sweet
potato, soybean, barley, wheat, maize, rye, cowpea, alfalfa, and several species of
bacteria, have been deduced from their respective gene sequences. The sequences
between plant and bacterial β-amylases show only 30% homology (Thalmann et al.,
2019).
β-Amylase has been prepared in a crystalline form from a variety of plant sources
including germinated barley, sweet potato, and soybean. Purification mostly
involves multisteps fractionation by acetone, ethanol, or ammonium sulfate precipi-
tation followed by cation/anion exchange chromatography and gel filtration and the
affinity chromatography using cyclodextrin Sepharose 6B column.
β-Amylase has been used for various research and industrial applications.
β-Amylase is employed by the starch industry in the production of high maltose
syrups. Compared with glucose syrups, maltose syrups have a higher viscosity and
lower hygroscopicity, less tendency to crystallize, and more resistance to browning.
These characteristics make them particularly suitable for use in the confectionery
and baking industry (Aiyer, 2005).
28 M. Aminlari

2.2.2 Pectic Enzymic

2.2.2.1 Introduction
Pectins or pectic substances are collective names for a group of closely associated
polysaccharides present in plant cell walls, where they contribute to complex
physiological processes like cell growth and cell differentiation and so determine
the integrity and rigidity of plant tissue. They also play an important role in the
defense mechanisms against plant pathogens and wounding. Commercial pectins are
polymers of α-D-galactopyranosyluronic acids with various contents of methyl ester
groups and are obtained from citrus peel and apple pomace. Pectins have a unique
ability to form spreadable gels in the presence of sugar and acid or in the presence of
calcium ions and are used primarily in these types of applications. By definition,
preparations in which more than half of the carboxyl groups are in the methyl ester
form are classified as high-methoxyl (HM) pectins, the remainder of the carboxyl
groups will be present as a mixture of free carboxyl group and their sodium salt.
Preparations in which less than half of the carboxyl groups are esterified with
methanol are called low-methoxyl (LM) pectins. The percentage of carboxyl groups
is the degree of esterification (DE) or degree of methylation (DM) (Benen et al.,
2003; Schols & Voragen, 2003; Yapo, 2011).
The pectin degrading enzymes or pectic enzymes are classified into three gen-
eral groups, namely polygalacturonase, pectate and pecyin lyases, and pectin methyl
esterase. The specific responses brought about by these three pectinase activities are
displayed in Fig. 2.3. The depolymerizing enzymes are hydrolases and lyases. The
methylesteraseases hydrolyze the methylesterase at O6 of a
galacturonicacid residues, though acetylesterases hydrolyze the acetylester at O2
or potentially O3 of a galacturonic acids. These enzymes are commonly found in
plants and microorganisms (particularly in fungi like A. niger) and have been
purified and widely studied (Benen et al., 2003).
Pectic enzymes are normally present in many vegetables and fruits (endoge-
nous enzymes), yet they are also added as processing aids as exogenous enzymes.
In higher plants, especially pectin methyl esterase and polygalacturonase, both endo-
and exo-asplitting are present. Pectin acetylesterase and pectate lyase have been
found in citrus products of plants. The endogenous enzymes are expected to assume
significant parts in plant improvement and during maturing. The cutting edge
genomic approach will empower us to more readily comprehend their functions in
plants in various isoforms and their role in plant improvements. Taking into account
that pectic enzymes alone record for around one fourth of the world’s food enzymes
production, one can securely conclude that the technological innovations and
applications designed to reduce economical costs and increase the productivity of
these enzymes will be of great value (Alkorta et al., 1998; Whitaker, 1990).

2.2.2.2 Polygalacturonases
Polygalacturonases hydrolyze the α-1,4-D-galacturonosidic linkage (Fig. 2.3).
Whereas endo-polygalacturonases hydrolyze the polymer substrate randomly, the
exo-polygalacturonases are confined to cleave off galacturonic acid monomers or
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 29

Fig. 2.3 Site of action and reaction mechanism of pectin-degrading enzyme. (From Whitaker,
1994)

digalacturonides from the nonreducing end. The enzymes, as found for the pectate
lyases, use both polygalacturonic acid and low to moderate degree of esterification of
galactouronic acids as their substrate (Benen et al., 2003). Polygalacturonases are
widely distributed in higher plants and are believed to contribute to fruit softening of
pears, peaches, and avocado.
These enzymes assume significant parts in, food technology, for example, vege-
table and fruit juice extraction, such as, apples, grape wine, strawberries, grapes,
raspberries, and so on, degumming of plant fiber, waste water treatment, oil extrac-
tion, coffee and tea aging, paper and pulp industry, and animal feed. To clarify fruit
juices, a combination of amylases and pectinases is utilized. The time of filtration is
decreased up to half. Tomato is a rich source of polygalactouronase. Brilliant
practices are utilized in the tomato processing to heat inactivate the enzymes momen-
tarily (“hot break” process) to prepare thick juice liked by the purchaser and the high-
consistency concentrated juice (tomato pase) utilized for sauces, soups, catsup, and
comparable items. At the point when tomatoes are utilized for color and flavor, and
consistency is obtained by different agents like starch, thiner, “cold break”, juice is
the beginning material for paste production. A holding time is applied
30 M. Aminlari

between smashing and heat treatment to guarantee breakdown of the pectins by

joined pectin methylesterasease/polygalacturonase activity (Benen et al., 2003). To
the food enzymologist, the most intriguing endogenous polygalacturonases are those
present in the parts of the plant that are utilized as or in food. The pernicious impact
of endogenous polygalacturonases, i.e., diminishing in consistency of the homoge-
nate during aging, has become progressively clear. To prevent the activity of these
enzymes, tomatoes are homogenized after heat pretreatment to inactive the enzymes
and yield the purported hot break paste (Javed et al., 2018).

2.2.2.3 Pectin Methylesteraseases

Pectin Methylesteraseases have been industrially utilized in the commercial sector
for wine and fruit juice industry since 1930. Apart from the presence of many genes
encoding putative pectin methylesteraseases, these enzymes have also been detected
in various plants by their activity, which confirms their wide distribution (Gupta
et al., 2015; Dixit et al., 2013). Pectin methyl esterases are of major importance for
the preparation of pectins for specific applications. These enzymes have been
identified in higher plants and vegetables. Within each species, multiple forms of
pectin methylesterases can be present. These enzymes, along with
polygalacturonases, are used in clarification of juices in fruit juice industry. The
break down of pectins additionally decreases the viscosity of juices and facilitates
their filtration and concentration. One more utilization of pectin methylesterases in
natural product juice industry includes liquefaction of natural product mashes. In
such an application, the pectinases are joined with cellulases and hemicellulases for
the total interruption of the cell walls. In this multitude of enzymatic applications,
degradation of pectin facilitate the process and increase juice yield. Likewise, the
favorable aroma of juices is increased and amount of waste material decreases.
Pectin methylesterasease from Datura stramonium in blend with polygalacturonase
expanded clearity of orange, apple, pomegranate, and pineapple juices by 2.9, 2.6,
2.3, and 3.6 fold, respectively (Dixit et al., 2013).

2.2.2.4 Pectate Lyases

Pectate lyases cleave the α-1,4-D-galacturonosidic linkage by β-elimination,
resulting in the formation of a Δ4,5-unsaturated newly formed nonreducing end as
is shown in Fig. 2.3b. Pectin lyases perform the same reaction. However, the latter
enzymes require the galacturonic acid residues adjacent to the scissile bond to be
methylesteraseified. Pectate lyases have been identified in plants (Uluisik &
Seymour, 2020). The first clear cut demonstration of plant pectate lyase activity
was reported in the plant zinnia eleganse (Zinnia elegans) (Semenova et al.,
2006; Domingo et al., 1998). Pectate lyases have not found many applications in
food industry because of their harsh reaction conditions (Semenova et al., 2006).
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 31

2.2.3 Cellulases

Cellulose is the most plentiful biopolymer on the planet. It is mostly created in higher
plants in which it shapes the inflexible skeleton of the plant. Cellulose is a homopol-
ymer comprising of up to 1000 β-1,4-linked-anhydroglucopyranoside units. Single
glucose polymers are packed onto one another to shape an exceptionally crystalline
fibrillar material in which the singular cellulose chains are held together by hydrogen
bonds. Cellulose microfibrils likewise contain some amorphous areas, the level of
which depends on the source. The most crystalline cellulose is produced by algae,
and the least crystalline by plants (Klemm et al., 2005).
Cellulases belongs to the group of β-glucan hydrolases that can break down
cellulose. Several microorganisms, including filamentous organisms, yeast, and
microbes existing in the digestive tract and colon of monogastric, can hydrolyze
cellulose to oligosaccharides and in the long run to glucose. Ruminants can
completely degrade cellulose in their rumen by the wide range of microorganisms
present. Cellulases have generally been classified into two specific groups
endoglucanases and exoglucanases, which act in the middle or at either ends of the
cellulose chain, respectively (Tenkanen et al., 2003). Cellulases are produced by
fungi, bacteria, protozoans, and animals. There are additionally a few reports on the
presence of cellulases in plants. Up to this point, just the endoglucanases (1,4-β-D-
glucan-4-glucanohydrolase) have been found in plants. This enzyme catalyzes
random cuts in cellulose chains, subsequently creating more shorter cello-oligomers,
which can be additionally degraded by exoglucanases. Endogenous plant
endoglucanases have been recommended to assume a part in plant development,
for example, in fruit maturing and leaf abscission, or in the rearranging of
polysaccharides in developing cells. The producion of microbial cellulase in trans-
genic plants, for example, sugarcane and corn, has been reported. These advances
can offer one potential utilization of plant biomass as biofuel harvests or remaining
horticultural waste as a sustainable fuel source (Hefferon, 2017).
Cellulases are not among the central enzymes used in the food industries. They
are, nevertheless, applied in treatment of grain-based beverages and food
assortments like beer and bread. Cellulases are used in rather immense totals in
animal feed (Zhang & Zhang, 2013). Cellulase has a wide scope of utilizations in
industrial biotechnology and is the second most utilized modern enzyme after
protease. Cellulases are utilized in the textile industry, in cleansers, pulp and paper
industry, further developing edibility of animal feeds, and food industry. In numer-
ous food applications, cellulases are not utilized alone, but are added to help the
activity of other enzymes. In collaboration with enzymes, such as pectinases and
hemicellulases, cellulases disrupt the underlying rigidity of the plant cell walls and
upgrade the extraction efficiency of the molecules of interest. Thus, they are utilized
in any process of handling plant-based materials. Cellulases are utilized in wine
and production of fruit juice to facilitate the maceration, and, the extraction of color
and flavor compounds, fermentable sugars and the extraction of juices. Macerating
enzymes mix containing cellulases are likewise utilized in fruit juice production.
Furthermore, the utilization of these enzymes offers benefits in the treatment of
32 M. Aminlari

waste of the fruit juice industry by further developing the extraction yield and the
general process efficiency. In baking industry, cellulase is utilized to degrade gums
in the dough, improving bread structure. Added enzymes are utilized progressively
for improving the processes and final quality of products. Obviously, the biggest
modern utilization of cellulases is outside the food and feed area but in material
handling of fibers made out of cellulose (Zhang & Zhang, 2013; Autio et al., 1996).
Cellulases are likewise utilized for the extraction of nutraceuticals from plants.
Nutraceuticals are compounds from normal sources with extra medical advantages,
other than a nutritious agent (Fernandes, 2018). Cellulases and hemicellulases can be
utilized for the separation of pectin from citrus fruits to separate the cell wall,
expanding the pectin yield. Cellulases can be utilized alone or in blend with other
cell wall degrading enzymes in all processes in which significant mixtures like juice,
oil, polysaccharides, protein, and so on are removed from the plant material
(Laurikainen et al., 1998).

2.2.3.1 b-Glucosidases
β-Glucosidases are enzymes with great practical significance to natural systems.
These are divided in various glycoside hydrolase families in light of their catalytic
mechanism and amino acid sequences. Most investigations completed on
β-glucosidases are centered around their applications rather than their endogenous
capacity in the their natural environment (Singh, 2016). In a large number of leafy
foods tissues, significant flavor compounds are glycosylated. β-Glucosidases cata-
lyze the hydrolysis of alkyl and aryl β-glycosides disaccharides and short-chain
oligosaccharides. β-Glucosidases have an extraordinary potential to be utilized in
different biotechnological processes from freeing flavours, fragrances, and isofla-
vone aglycons to the biosynthesis of oligosaccharides and alkylglycosides. The
utilization of glycosidases to deliver flavor compounds from glycosidic compounds
was at first analyzed in wines. β-Glucosidases are omnipresent, generally dispersed
in nature, and can be found in microorganisms, plants, and animals (Romero-Segura
et al., 2009; Zhu et al., 2017).
One more attribute of glucosidases critical to food processing and quality is the
presence of cyanogenic glycoside in many plants that can potentially produce
hydrogen cyanide during preparation or by eating. The cyanogenic glucoside
linamarin in cassava roots and leaves (widely used as food staple in tropical areas
of Africa, Asia, and South America), lima beans, bitter almonds, and flax seeds are
examples of these glycosides. There are roughly 25 cyanogenic glycosides referred
to, for example, linamarin in cassava and lima beans; dhurrin in sorghum; amygdalin
in almonds, peaches, and apricot pits (Hughes, 1999). The enzyme rhodanese is a
ubiquitous enzyme active in all living organisms from bacteria to man.
It is a multifunctional enzyme, yet appears to have important role in cyanide
detoxification. This enzyme is likewise broadly present in plants (Chaudhary &
Gupta, 2012). The primary source of this enzyme was reported to be the liver of
animals. Nonetheless, broad examinations on the distribution of rhodanese in our
research have exhibited that different organs, specifically the gastrointestinal system,
is more prominent source of rhodanese, which show that cyanide delivered from
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 33

cyanogenic glycosides is mainly detoxified in the GI prior to being absorbed (see

Aminlari & Vaseghi, 2006).

2.2.3.2 Xylanases
After cellulose, xylan is the most abundant polysaccharide in nature. Xylans are a
major hemicellulose component, and together with cellulose, they comprise the bulk
of the cell wall material in botanical products. Xylanases are β-retaining
glycosidases capable of hydrolyzing linear β-1,4-linked polymers of xylose (with
various substitutive groups such as arabinose). Multiple isoforms exist and these
enzymes can be endo- or exo-acting (endo-acting are more important in foods). The
xylanases are ubiquitous in nature and their presence is observed diversely in a wide
range of organisms (Biely, 2003). They are in plants (especially important in
cereals), bacteria, and fungi, and they typically range in molecular mass from
16 to 40 kDa. Xylanolytic enzymes also occurring in plants participate in the process
of cell wall extension, cell division, seed germination, and other morphological and
physiological events in plants.
During germination, endogeneous xylanases catalyze hydrolysis of these
polysaccharides to eliminate the actual boundary imposed by cell wall on the free
dispersion of starch and protein degrading enzymes through the germinating
grain. Enzymes responsible for breaking down hemicellulose polysaccharides are
likewise reported to be available in wheat grain and wheat flour (Kumar et al., 2017).
Xylanase enzymes are beneficial to food handling by depolymerizing water-
unextractable arabinoxylan pentosans, which have high water-holding capacity
This increases the viscosity and thickness of the dough, gluten strength, and final
bread volume. The mixed xylanases are especially significant in the preparation of
frozen dough. Endoxylanases are one of the hemicellulases that are utilized in fruit
and vegetable processing. Xylanases are likewise utilized in brewing to lower
viscosity of the wort in preparing, easeing partition/filtration steps, decreased haze
formation, and marginally further developed process yields (Kumar et al., 2017;
Bhardwaj et al., 2019).

2.2.3.3 Glucoamylase
Glucoamylase catalyzes the hydrolysis of α-(1,4), α,β-(1,1), α-(1,6), α-(1,3), and
α-(1,2) glycosidic connections between adjoining glucosyl residues, arranged by
their decreasing rates of hydrolydid. It starts hydrolysing from nonreducing end of
starch-based substrates to liberate glucose. Glucoamylas assume a significant func-
tion in food processing to saccharify starch in cereals and produce glucose syrups
from maltodextrins after the activity of α-amylase. Glucoamylas is produced by
certain eubacteria, some archaea, various yeasts, and numerous filamentous fungi.
Despite the fact that there are reports of animal and plant glucoamylas, these seem,
by all accounts, to be essentially various enzymes with analytic properties that
crossover those of genuine glucoamylas. Thus, and notwithstanding wide utilization
of this enzyme in food industry, this catalyst won’t be additionally discussed in this
section.
34 M. Aminlari

2.3 Enzymes Working on Proteins

Proteolytic enzymes, proteinases, or proteases are used interchangeably to refer to

enzymes that hydrolyze peptide bonds in proteins and peptides, according to the
following equation:

NH  CHR1  CO  NH  CHR2  CO  þ H2 O

! NH  HR1  COOH þ N þ H3  CHR2  CO

Proteases are found in all living life forms (creatures, plants, and
microorganisms). They are fundamental for separating of proteins to peptides and
amino acids. In the human digestive system, the proteolytic enzymes pepsin (stom-
ach) and several proteolytic enzymes in small intestine (trypsin, chymotrypsin,
carboxypeptidases, leucine aminopeptidases, tripeptidases, and dipeptidases) con-
vert the proteins of ingested food varieties to amino acids.
Proteases are by far the best-portrayed enzymes as far as their fundamental roles
in the human digestion and early commercialization (the originally normalized calf
rennet for cheddar making was promoted in 1874) is concerned (Parkin, 2017).
Peptidases breakdown food proteins in situ or are added exogenously to cause
protein change.

2.3.1 Specificity

The investigation of protease specificity provide information on active site structure

and functionality, protein-protein association, controlling intracellular and extracel-
lular pathways, and evolution of protease and substrate genes (Diamond, 2007).
Proteolytic enzymes are specific as far as being endoproteases or exoproteases. The
endoproteases, like trypsin, chymotrypsin, and chymosin, hydrolyze peptide bonds
inside of the polypeptide chains. The exoproteases follow up on peptide bonds at the
N-terminal (aminopeptidases) or the C-terminal (carboxypeptidases) of the protein.
A few proteases are said to have broad specificity, i.e., they can hydrolyze peptide
bonds regardless of the side chain group adjoining the scissile peptide bond (pepsin
and bacterial subtilisin from different Bacillus spp). Majority of proteolytic enzymes,
nonetheless, are totally specific for one or few amino acid residue at the scissile
peptide bond. The scissile bond of the peptide substrates is assigned as that
connecting residue R1 and R2 in above equation. For instance, trypsine attacks
peptides on the C-terminal side of lysine and arginine amino acid residues. The
residues for chymotrypsine and elastase are aromatic amino acids, in particular
phenylalanine, tyrosine, or tryptophan and small hydrophobic amino acids like
alanine, respectively (Parkin, 2017; Grahn et al., 1999). The protease binding sites
are chemical and spatial complements of the substrate amino acid. This specificity
for the substrate is due to the favorable binding interaction of the substrate amino
acid side chain with residues that form the binding site of the protease.
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 35

. . . . . . S3  S2  S1  S10  S20  S30 . . . . . .

‐‐‐‐‐‐‐NH  CHR  CO  NH  CHR  CO‐‐‐‐‐‐
. . . . . . P3  P2  P1  P10  P20  P30 . . . . . .
"

In the protease active site, S1, S10 , . . . refer to the amino acids that provide the
chemical and spatial environment that complements the substrate amino acids
referred to as P1, P10 , . . . . Arrow shows the scissile peptide bond. These sites
have been mapped and characterized for many proteases (Parkin, 2017; Vizovišek
et al., 2018).

2.3.2 Classification

There are four distinct kinds of proteases, specifically, the serine proteases, the
cysteine proteases, the aspartic proteases, and the metalloproteases, in light of the
active site group of the enzyme engaged with the nucleophilic attack at the carbonyl
carbon of the scissile peptide bond of the substrate. These are portrayed below
(Whitaker, 2003).

2.3.2.1 Serine Proteases

Serine proteases share the presence of the side chain of a serine residue in the active
site which function in their catalytic activity. Among the serine proteases, the most
notable are trypsin, chymotrypsin and elastase, and the nine proteases engaged with
blood coagulation and collagenase. The serine protease subtilisin is from B. subtilus.
Most enzymes in this group have molecular masses of 25–35 kDa, and they are
featured by a surface or internal cleft as substrate binding site. The pH ideal of these
compound is around 8 (Whitaker, 2003; Hedstrom, 2002; Di Cera, 2009).

2.3.2.2 Aspartic (Acid) Proteases

Aspartic proteases are portrayed by two profoundly conserved ASP residues as the
catalytic unit, with optimum pH close to 3–4. Most natural enzymes from
this group are pepsin, calf chymosin (additionally called “rennin” or “rennet,”
utilized in cheese making), cathepsin (engaged with after death meat tenderization),
and the chymosin-substitute peptidases from Mucor spp. These endopeptidases are
typically 34–40 kDa in mass. The main mechanism for hydrolysis includes a dyad of
conserves ASP residues acting as general acid/base (Whitaker, 2003; Ulusu et al.,
2016; Nair & Jayachandran, 2019).

2.3.2.3 Cysteine (Sulfhydryl) Proteases

Cysteine proteases are a large group of enzymes (more than 130 known) present in
animals, plants, and microorganisms. Most of these enzymes are among the papain
family, and others being chymopapain (various isoforms) and papain from tropical
36 M. Aminlari

tree, Carica papaya (papaya), actinidin from kiwi fruit, ficin from fig latex, bromelain
from pineapple, and lysosomal cathepsins from animal tissues. Among them, papain,
bromelain, and ficin address 5% of the worldwide sale of proteases (Illanes, 2008;
Verma et al., 2016).
The most studied cysteine protease is papain, which will be examined later in
light of its significant uses in the food industry for chill proofing of brew and meat
tenderization, and for synthesis of peptides and other organic compounds. An
interesting cysteine protease in muscle is calpain (numerous isoforms) that is
activated by Ca2+ and plays a part in after death tenderization of muscle. Commonly,
the enzymes of this group are 24–35 kDa in mass, are active at pH 6.0–7.5, and can
endure temperatures up to 60–80  C (due in some extent by three disulfide bonds)
(Whitaker, 2003; Otto & Schirmeister, 1997).

2.3.2.4 Metalloproteases
A large group of the metalloproteases are exopeptidases. These are the
carboxypeptidases A and B, glycyl-glycine dipeptidase, carnosinase (work on
β-alanyl-L-histidine and related compounds), amino-acyl-histidine hydrolase, and
cytosolic aminopeptidase (α-amino-acyl-peptide hydrolase). All require Zn2+ as
cofactor. Some of these enzymes, for instance, prolidase (where proline or hydroxy-
proline is the carboxyl terminal residue) and iminodipeptidase (in which proline
or hydroxyproline is the N-terminal residue) require Mn2+. Other metalloprotases are
endo-acting thermolysin (from Bacillus thermoproteolyticus) and the nreutal
endoprotease from Bacillus amyloliquefaciens (Tavano et al., 2018).

2.3.3 Application of Proteases in Food Industry

Commercial proteases are available at various levels of purity, and some contain
multiple proteolytic agents. Some of the important commercial applications of
proteolytic enzymes are shown in Table 2.3 and are described below (Whitaker,
2003).

2.3.3.1 Production of Protein Hydrolysates

Hydrolysis of food proteins utilizing proteases, like pepsin, trypsin, chymotrypsin,
papain, and thermolysin, change their functional properties, for example, nutritional,
flavor/sensory, textural, and physicochemical (solubility, foaming, emulsifying,
gelling) and decreased allergenicity. Broad hydrolysis by nonspecific proteases,
for example, papain, causes solubilization of even inadequately soluble proteins.
Such hydrolysates ordinarily contain low molecular weight peptides with two to four
amino acid residues. Several functional properties of proteins, like gelation,
foaming, and emulsifying properties, suffer from excessive hydrolysis. Typically,
a protein is treated by an endopeptidase in a batch process for a couple of hours, after
which the added enzyme is inactivated by heat treatment. This treatment brings
about fast reductions in normal molecular weight of proteins, whereas exopeptidases
are utilized to hydrolyze small oligopeptides to composite amino acids. Proteins
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 37

Table 2.3 Use of proteolytic enzymes in food processing

Food Purpose of action
Baked goods Softening action in doughs. Cut mixing time, increase extensibility of doughs.
Improve texture, elasticity, and loaf volume. Liberate β-amylase.
Brewing Body, flavor, and nutrient development during fermentation. Aid in filtration
and clarification. Chill-proofing.
Cereals Modify proteins to increase drying rate, improve product handling
characteristics. Production of miso and tofu.
Cheese Casein coagulation. Characteristic flavor development during aging.
Chocolate, Action on beans during fermentation.
cocoa
Egg, egg Improve drying properties
products
Feeds Waste product conversion to feeds. Digestive aids, particularly for pigs.
Fish Solubilization of fish protein concentrate. Recovery of oil and proteins from
inedible parts.
Legumes Hydrolyzed protein products. Removal of flavor. Plastein formation.
Meats Tenderization. Recovery of protein from bones.
Milk Coagulation in rennet puddings. Preparation of soybean milk.
Protein Condiments such as soy sauce and tamar sauce. Bouillon. Dehydrated soups.
hydrolysates Gravy powders. Processed meats. Special diets. Antinutrient factor removal.
Specific protein inhibitors of proteolytic enzymes and amylases. Phytate.
Gossypol. Nucleic acid.
Wines Clarification
In vivo Conversion of zymogens to enzymes. Fibrinogen to fibrin. Collagen
processing biosynthesis. Proinsulin to insulin. Macromolecular assembly.

(ordinarily meat, milk, fish, wheat, vegetable, vegetable, and yeast sources) might be
exposed to a pretreatment that renders them to some degree of denaturation as this
improves peptidase access and hydrolytic activity. Protein hydrolysates can improve
functional properties, for example, emulsifying capacity, foam capacity and stability,
antioxidative effect, water absorption capacity, and nutritional properties
(Chalamaiah et al., 2012; Thiansilakul et al., 2007; Sinha et al., 2007; Clemente,
2000). Protein hydrolysates constitute an alternative to intact proteins and elemental
formulas in the development of special formulations designed to provide nutritional
support to patients with different needs. The production of extensive protein
hydrolysates by sequential action of endopeptidases and exoproteases coupled
with the development of post-hydrolysis procedures is considered the most effective
way to obtain protein hydrolysates with defined characteristics (Rocha et al., 2009).
Protein levels in production of protein hydrolysates are often 8–10%, provided there
are no limitations on solubility, and the amount of enzyme added is generally 2%
on a protein basis, depending on purity. Protein:enzyme levels are sufficiently high
so that the enzyme reacts at nearly Vmax with limited autodigestion of the enzyme,
although product inhibition by accumulating peptides may attenuate reactivity. The
hydrolysates can be characterized by the degree of hydrolysis (DH), sequence of
produced peptides, functional properties such as solubility, emulsifying properties,
38 M. Aminlari

etc. DH can be easily evaluated with o-phthaldialdehyde (OPA) method which

determines the number of free amino groups (Mirzaei et al., 2016). Typically, a
DH value of 3–6% corresponding to peptides of 2–5 kDa is desired for
physicofunctional properties, while DH of 8% or more means 1–2 kDa peptides,
demonstrating optimal solubility for use in sports and clinical nutrition products.
More exhaustive DH (as high as 50–70%) yields small peptides and amino acids of
<1 kDa which are useful for infant and hypoallergenic foods and savory flavoring
ingredient preparations (soups, gravies, sauces). One problem associated with the
greater DH is the possibility of production of bitter peptides (small and hydropho-
bic), thereby necessitating to control this potential flavor defect (Chalamaiah et al.,
2012).
Proteases can likewise be utilized to detach remaining muscle protein from bones
of fish and land animals as protein hydrolysates, and this normally includes heating
at 55–65  C for 3–4 h. The final product blend from enzyme hydrolysis of proteins
might require posttreatment refining as well as separation to get deravatives appro-
priate for the planned application (Chalamaiah et al., 2012; Mizani et al., 2005).

2.3.3.2 Application of Proteolytic Enzymes in Dairy Industry

Milk is an entirely perishable food item and therefore there has been a strong
motivation to change it over to more stable items, which is facilitated by specific
properties of milk. Traditionally, milk has been safeguarded by fermentation, nor-
mally with addition of salt (cheddar, fermented milks, butter). More current
safeguarding strategies incorporate drying, pasteurization/sterilization, and freezing.
A few qualities of milk additionally render it entirely amiable to modification by
enzymes. The reality that milk is a fluid facilitate enzyme addition. Cheese
manufacturing is presumably the oldest, as yet the biggest, utilization of exogenous
enzyme in food processing.
Since the principal components of milk are proteins (3:5%), lipids (3:6%), and
lactose (4:8), the principle enzymes used in dairy technology are proteinases and
peptidases, lipases, and β-galactosidase (lactase). However, several oxidoreductases
have significant applications. In this section, we will focus on the proteases. Other
enzymes will be dealt with in other sections. Proteolytic enzymes are the most
widely used enzymes in dairy technology. Applications include cheese
manufacturing, modification of functional properties, production of protein
hydrolyzates, and for nutritional and other applications (Fox, 1993; Shah et al.,
2014).

Cheese Manufacturing
The main gastric enzyme of neonatal ruminants is chymosin or rennet. Chymosin
has low broad proteolytic action, but high milk-coagulating action. Calf chymosin
(rennet) and chymosin substitutes (see beneath) are added to milk to cause the
underlying milk-clotting reaction prompting cheese formation. The rennet coagula-
tion of milk is a two-stage process. The first (essential) stage includes specific
hydrolysis of the PHE105-MET106 bond of κ-casein and creation of “para-casein”
and TCA (trichloroacetic acid)-soluble peptides (glycomacropeptides), while the
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 39

next stage includes the Ca-induced gelation of para-casein at a temperature of

30–35  C. Proteolysis is essentially complete before the beginning of coagulation.
The remaining from k-casein, which is referred to as para-k-casein, stays in the
micelle, yet the hydrophilic peptide, called the caseino(glyco)macropeptide, diffuses
into the whey and thus the micelle-settling properties of k-casein are lost. Starter
cultur are added to milk at 40–45  C to cause a pH reduction to 5.8–6.5, whereupon
chymosin is added to start clotting. Because of ensuing steps of cheese manufactur,
some enzyme activity stays in the curd and contributes to cheese aging and flavor
improvement during aging. Proteases in the starter cultures additionally add to
support proteolysis and flavor improvement during aging.
The gene for calf chymosin has been cloned in chosen microbes, yeasts, and
molds. A recombinant chymosin from E. coli has been created. Microbial recombi-
nant chymosin contain no pepsin, though 5 to 50% of the milk clotting action of calf
rennets might be because of pepsin (Kawaguchi et al., 1987; El-Sohaimy et al.,
2010).
Expanding world production of cheddar (3% each year throughout recent years),
accompanying with a diminished stock calf neonatal, the stockpile of veal rennet has
been deficient for a long time, which has led to a quest for rennet substitutes. During
the last 40 years, the expanding utilization of an assortment of milk-clotting catalysts
has turned into a significant specialized pattern in cheese production. Although
numerous proteinases can coagulate milk, just few have been viewed as pretty
much adequate as rennets. These are cow, porcine, and chicken pepsins; rennet
(watery concentrates produced using the fourth stomach of calves, kids, sheep, goat,
and cow); microbial rennets made by Endothia parasitica, and Mucor species
(Mucor miebei or M. pusillus) and Cryphonectria parasitica, and plant proteases
(Shah et al., 2014; Kim et al., 2004a).
Chicken pepsin is the most unreasonable of pepsins and is utilized uniquely in
extraordinary conditions. Bovine pepsin gives commonly palatable outcomes
regarding cheese yield and quality; numerous commercial “calf rennets” contain a
significant extent of bovine pepsin. Although the proteolytic specificity of the three
normally utilized fungal rennets on αs1- and β-caseins is extensively different
from that of calf chymosin, they by and large yield acceptable cheese and were
broadly utilized in the United States before the presentation of microbial recombi-
nant chymosin (Nelson, 1977; Green, 1977).

Milk-Clotting Enzymes from Plants

Although several properties of microbial milk-clotting enzymes such as high pro-
teolytic activity and high resistance to heat denaturation made them suitable for
cheese manufacturing, plant protease have attracted considerable attention and
interest in cheese industry since they are easily available and their purification is
simple. As discussed above plant protease are classified into several groups
depending on the mechanism of catalysis in the hydrolytic step. Aspartic, serine
and cysteine protease are the three major milk clotting enzymes from plants (Shah
et al., 2014).
40 M. Aminlari

Aspartic protease are present in many plant species and have aspartic acid
residues in their active site. They participate in many activities in plants. Examples
include:

1. Cardosin from flowers of the artichoke thistle (Cynara cardunculus)

2. Cynarase from flowers of globe artichoke (Cynara scolymu)
3. A Cardosin A like enzyme from thistle (Cynara humilis)
4. An enzymatic extract from the thistle Silybum marianum flowers
5. Oryzasin from seeds of rice (Oryza sativa)
6. Enzymatic extract from flowers of moringa (Moringa oleifera)
7. Onopordosin from cotton thistle (Onopordum acanthium)
8. Cirsin JN703462 from flowers of common thistle (Cirisum vulgare)
9. Enzymatic extract from cell suspension of red star-thistle (Centaurea calcitrapa)

Serine proteases have in their active sites serine residues. Their main role in plants
is almost the same as the aspartic proteases, with some additional features. Serine
proteases are widespread in plants and belong to several taxonomic groups. They are
extracted, purified and characterized from several parts of plants, especially fruits.
Some of the enzymes in this group are (Di Cera, 2009):

1. Dubiumin from seeds of Solanum dubium (Solanum, also called bittersweet or

woody nightshade) (from Wikipedia, the free encyclopedia).
2. Cucumisin from Cucumis melo fruits (Muskmelon is a species of melon that has
been developed into many cultivated varieties. These include honeydew, canta-
loupe, and Persian melon) (from Wikipedia, the free encyclopedia).
3. Lettucin from leaves of lettuce, Lactuca sativa (from Wikipedia, the free
encyclopedia).
4. Religiosin from Ficus religiosa (Sacred fig is a species of fig native to the Indian
subcontinent and Indochina) (from Wikipedia, the free encyclopedia).
5. Streblin from stems latex of Streblus asper (a tree known by several common
names, including Siamese rough bush, khoi, serut, and toothbrush tree).

Cysteine proteases or thiol-proteases have a catalytic mechanism that involves a

cysteine group in their active site. Plant extracts, or their purified proteases, have
been extensively characterized in many studies, for their potential use as milk
coagulants. Following is a list of examples of cysteine proteases from plants
(Amal Ben Amira et al., 2017; Lo Piero et al., 2011):

1. Enzymatic extract from seeds of Albizia lebbeck (a species of Albizia, native to

Indomalaya, New Guinea, and Northern Australia and widely cultivated in
tropical and subtropical regions). It is often called lebbek tree siris, frywood,
koko, and woman’s tongue tree.
2. Enzymatic extract from seeds of sunflower (Helianthus annuus).
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 41

3. Ficin from branches latex of figs tree Ficus carica sylvestris and an enzyme from
stem latex of Sideroxylon obtusifolium (Sideroxylon is a genus of trees in the
family Sapotaceae. They are collectively known as bully trees).
4. Actinidin from kiwi (Actinidia chinensis) fruits.
5. Calotropain from latex of crown flower Calotropis gigantean.

Recently, we reported on application of actinidin to the production of cheese from

cow’s milk. Comparison of actinidin and chymosin indicated that the former could
be a potential alternative to the latter in application as milk coagulant (Alirezaei
et al., 2011).
In general, the major drawback of most plant rennets is the development of an
increased bitterness and the appearance of cheese texture defects during storage
and/or ripening. These defects are mainly due to excessive proteolytic activities and
low ratios of milk-clotting activity/proteolytic activity. For this reason, the evalua-
tion of enzyme activities and their comparison with those of commercial rennet
(chymosin) is an important first step in selecting a suitable plant rennet.
Taken together, the utilization of various sorts of plant proteases in cheese
indudrty impacts the hydrolysis level of the protein matrix of milk, prompting
differeneces in sensory properties of cheese. The vast majority of plant rennets
produce cheeses with harsh flavors because of too much proteolytic activity,
which restricts their industrial application. Hence, the determination of a proper
plant coagulant and the control of various gelation properties are vital to acquire a
superior quality of end product.

2.3.3.3 Application of Enzymes in Meat Industry

Meat and meat product utilization (beef, pork, lamb, goat, and poultry) has
expanded, especially in nonindustrial nations. The world utilization of meat products
will arrive at 40 kg per capita in 2020. The platability of meat is affected by a few
factors, among which meat tenderness is viewed as the main determinant for
consumers. The processes associated with the transformation of muscle to meat
are complex. The chemical and physical properties of muscle tissue and the related
connective tissue are determinant of meat quality. Of the relative multitude of
characteristics of meat quality, consumers rate tenderness as the most significant.
Tenderness is a quality attribute coming about because of the interaction of actomy-
osin of myofibrillar proteins, the foundation of connective tissue, sarcomere length,
and the bulk density of fat. Meat tendeerness likewise relies on the degree of
protrolysis of muscles (Arshad et al., 2016; Marques et al., 2010).
There are multiple ways of tenderizing meat, by chemical or physical means,
which basically decrease the amount of recognizable connective tissue without
causing broad breakdown of myofibrillar proteins. The myofibrillar breakdown
begins after initiation of the enzymatic system and involves the proteins tropomyo-
sin, troponin T, troponin I, C-protein, connectin, desmin, vinculin, dystrophin,
nebulin, and titin. The first change in meat tenderness is because of the activity
of complex endogenous calpain-calpastatin system, which acts in muscle tissue after
slaughter. In muscles, these proteolytic enzymes play a huge part in after death
42 M. Aminlari

proteolysis and meat tenderization. Cathepsins were the main enzymes utilized in
meat tenderization, after which calpain was applied owing to its action in changing
the Z-line thickness found in post-mortem, despite the fact that it was not at first
connected with meat tenderization. Calpains are calcium-dependent proteases that
break down myofibrillar proteins. Calpastastin, on the other hand, inactivates
calpains, decreases the myofibrillar degradation, and hence lessens the tenderness.
Calpastatin effect is finished after being inactivated by cooking. The amount of the
enzymes changes among species, which affect the degree of meat tenderness,
because of expanded or diminished proteolysis of myofibrillar proteins (Lian et al.,
2013).
Meat tenderness relies upon the kind of muscle, pre- and post-mortem variables,
and after death pH and temperature. The research for significant proteases with
unique specificity for industrial applications is consistently a continued challange.
Proteolytic enzymes from plant sources have gotten extraordinary attention for being
active over a wide range of temperatures and pH’s. Treatment by proteolytic
enzymes is one of the most well-known techniques for meat tenderization. The
utilization of exogenous proteases for meat tenderization is a rather modern strategy
to further develop meat quality. There are few exogenous proteolytic enzymes, plant
proteases (papain, bromelain, ficin, and actinidin), and proteases from Aspergillus
oryzae and Bacillus subtilis, which have been considered as generally regarded as
safe (GRAS) for use in the meat. Papain and other sulfhydryl endopeptidases
(bromelain and ficin) are applied to muscle or meats that don’t turn out to be
sufficiently tendered during post-mortem aging. These enzymes are powerful in
this application since they can hydrolyze collagen and elastin, connective tissue
proteins that cause toughness in meat. These proteolytic enzymes are blended in the
meat to break down the proteins in muscle and hydrolyze collagen and elastin, which
helps in meat tenderization. Papain and bromelain are the most regularly involved
plant enzymes for meat tenderization. As meat tenderizers, proteolytic enzymes are
the most ideal for breakdown of collagen and elastin in connective tissue at some-
what low pH and low temperature. The structure of myosin and actin fibers is
impacted by the plant proteases. The tenderness of meat is deteremined by enzyme
activity assessment, myofibrillar fragmentation index, hydroxyproline estimation,
and examining by electron microscopy. The ideal meat tenderizer would be a
proteolytic enzyme with specificity for collagen and elastin in connective tissue, at
the generally low pH of meat, which would act either at the low temperature at which
meat is stored or at the high temperature accomplished during cooking (Arshad et al.,
2016; Lian et al., 2013).

Papain
Papain is a nonspecific thiol protease and the significant protein constituent of the
latex in the tropical plant Carica papaya. It is an important plant protease. The latex is
obtained by scoring, and afterward permitting it to dry on the fruit, and a crude
material is resulted. In the food industry, papain is regularly utilized for meat
tenderization, production of protein hydrolysate, and the clarifying of juice and
beer. Papain is also used in the baking and dairy industries (in cheese production),
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 43

and for the extraction of flavor and color compounds from plants. Papain is
prepared by decersing contaminants and further extraction. The enzyme has high
thermal and pressure resistances, requiring exceptional process conditions for suffi-
cient inactivation (to accomplish 95% inactivation of papain at 900 MPa and 80  C,
22 min of processing is required). The physiological job of papain in plants is to
protect them from insects. Papain tends to over tenderize the meat surface, making it
“soft”. Papain is utilized at a portion of 300 units/kg to release free amino acids in
dry fermented frankfurters. The three-dimentional structure for papain is reported.
Wide range enzymatic activity has been shown by papain in the pH range 5.9–7.5
and the temperatures of 70–90  C with optimum of 65–75  C. Under these
conditions, papain has superb activity in hydrolysis of myofibrillar proteins and
moderate impact on hydrolysis of collagen (Istrati, 2008; Gokoglu et al., 2017).
Papain can be utilized in the fruit juice and beer clarification. In beer, an
imperfection alluded to as chill-haze might be brought about by the combina-
tion (complexing) of tannins and proteins. Papain has for some time been utilized
to hydrolyze protein and limit cloudiness, despite the fact that bromelain and ficin
just as other bacterial and fungal proteases may now be utilized for this reason. The
endopeptidase is added after aging and preceding final filtering. Papain is finally
inactivated by pasturization, and unnecessary activity of papain might cause loss of
foam stability. Controlled or evaluated proteolysis in brew is important, since some
remaining protein is important to preserve specific quality attributes (Mosafa et al.,
2013).

Bromelain
Bromelain (or bromelin) enzymes are present in huge amounts in fruits, leaves, root,
and stems of the Bromeliacea family, of which pineapple (Ananas comosus) is the
best known. The juice of various pieces of the plant contains bromelain in soluble
form. Its high proteolytic action has made a wide interest in various applications,
principally in tenderization, food, detergents, and the textile industry. Bromelain
is commercially accessible in powdered form. It is assessed that 95% of the
utilized proteases in the United States are derived from plant proteases like papain
and bromelain, though microbial tenderizers are not utilized broadly. Reverse
micellar extraction is utilized for isolation and purification of bromelain from
pineapple. There is high bromelain recovery and purification in this strategy. This
enzyme, as different proteases, breakdown myofibrillar proteins and collagen, caus-
ing overtenderization of meat. Bromelain can be utilized in beef at 10 mg/100 g meat
and ideal tenderization happens after 24 h at 4  C. Following this time span, the
enzyme can be inactivated by heating at 70 . pH range for enzymatic activity for
bromelain is 4–7 and optimum pH is 5–6. The range of temperature for activity is at
50–80  C and optimum is 65–75  C. Under these conditions, bromelain has moder-
ate hydrolysis activity of myofibrillar proteins and good effect on hydrolysis of
collagen (Arshad et al., 2016; Gokoglu et al., 2017).
44 M. Aminlari

Ficin
Due to the problems raised by the use of animal or microbial recombinant proteases,
the use of ficin is becoming increasingly popular. Ficin is an outstanding example of
proteases from plants. This enzyme can be applied for protein hydrolysis, the
production of bioactive peptides, meat tenderization, milk coagulations in cheese
making, or peptide synthesis. Ficin does have huge potential and brilliant prospect in
the near future. Ficin is a sulfhydryl or cysteine protease commonly obtained from
Ficus carica (fig tree) that enhances the solubility of muscle proteins (Englund et al.,
1968; Morellon-Sterling et al., 2020). Ficin has a molecular weight of 44.5 kDa and
shows maximal activity at pH 5.0–9.0 and optimum pH is 7.0. The enzyme is fully
active at 45–75  C with optimum temperature of 60–70  C. Under these conditions,
ficin has moderate activity in hydrolysis of myofibrillar proteins and excellent effect
on hydrolysis of collagen. These properties make ficins a beneficial class of plant
proteases for use. We used ficin to tenderize beef used for manufacture of sausages.
Results indicated that solubility of meat proteins increased and SDS-PAGE results
showed the disappearance of several protein bands in ficin-treated meat. Ficin-
tenderized meat substantially improved water-holding capacity and emulsion stabil-
ity. The results of this study indicated that some quality attributes of meat products
can be improved by enzymatic modification of protein sources in the manufacture of
meat products (Ramezani et al., 2003).

Actinidin
Actinidin is a novel sulfhydryl protease extracted from gooseberry or the kiwi fruit.
Actinidin is the predominant enzyme in kiwifruit and can play a role in aiding the
digestive process. It has a molecular weight of 32 kDa. It is used commercially in
meat industry to tenderize meat. The ability of pre-rigor infusion of kiwifruit juice to
improve the tenderness of lamb was investigated. It was shown that actinidin in kiwi
fruit juice enhances proteolytic activity, resulting from the infused kiwifruit juice in
carcasses, and is associated with significant degradation of the myofibrillar proteins,
appearance of new peptides, and activation of m-calpain during post mortem aging.
Thus, kiwifruit juice is powerful and easily prepared meat tenderizer, which could
contribute efficiently and effectively to the meat tenderization process (Boland,
2013; Morton et al., 2009).
Actinidin has many applications in the food industry because of its advantages
over other plant proteases such as papain and ficin. Actinidin shows mild tenderizing
activity even at high concentrations, preventing surface mushiness. It has a relatively
low inactivation temperature (60  C), which makes it easier to control the tenderiza-
tion process without overcooking. A group in our lab studied the effect of actinidin
on beef for use in manufacture of sausage. Actinidin from kiwi fruit was partially
purified by precipitation with ammonium sulfate, followed by DEAE-Sephadex
column chromatography. The effect of purified actinidin on the protein solubility
(nitrogen solubility index [NSI]), water-holding capacity (WHC), texture, and
SDS-PAGE pattern of beef was studied and the quality attributes of a sausage
product were evaluated. Actinidin significantly increased NSI and WHC of beef;
the highest NSI and WHC (approximately 20% and 8% increase, respectively) was
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 45

observed when beef was incubated with 0.9 unit enzyme/g beef. Texture analysis
indicated increased tenderization (10% decrease in shear force) when slices of beef
were treated with actinidin at 37  C for 2 h. SDS-PAGE results indicated appearance
of several low molecular weight bands (<10 kDa) after treating beef with different
levels of actinidin for 30 or 60 min. Slight changes in protein band in the range of
100–120 kDa and 13–25 kDa were also observed. Use of actinidin-tenderized beef
significantly improved emulsion stability, texture, and organoleptic properties of the
sausage product (Lewis & Luh, 1988). We applied actinidin to tenderize camel meat
and beef and used the tenderized meats in the manufacture of emulsion type
sausages (Gheisari et al., 2008). The properties of sausages made from the meat of
both species were similar. Emulsion satiability, folding, texture, taste, and overall
quality of sausages produced from actinidin-tenderized meat were superior than
untreated samples (Aminlari et al., 2009). The actinidin has less tenderization
property as compared to other traditional plant proteases and still not approved as
GRAS by FDA (Lewis & Luh, 2007; Toohey et al., 2011).

Calpains
Endogenous proteolytic systems are responsible for modifying proteinases as well as
the meat tenderization. Abundant evidence has shown that calpains and calpastatin
(CAST) have the closest relationship with tenderness in livestock. They are involved
in a wide range of physiological processes including muscle growth and differentia-
tion, pathological conditions and postmortem meat aging (Lian et al., 2013). Meat
tenderness undergoes changes after slaughtering due to the activity of the endoge-
nous calpains and calpastatin. These calcium-dependent proteases degrade the
myofibrillar proteins. The calpain system consists of three members—m-calpain,
μ-calpain, and calpastatin, which is the calpain-specific endogenous inhibitor. Both
m-calpain and μ-calpain are cysteine proteases, and their proteolytic activity is
affected by oxidation, which can influence the quality of fresh meat. In the presence
of calcium, calpains autolyze, and this autolysis is indication of their proteolytic
activation during postmortem changes in muscles (Zhang et al., 2013). Calpain is an
important enzyme that is chiefly used for degradation of myofibrillar proteins. It also
aids in meat tenderizing and improves water holding capacity during postmortem
aging.

2.3.3.4 Conclusion
In meat, tenderness is the most important factor associated with meat palatability and
consumer satisfaction. Different plant proteases like papain, bromelain, actinidin,
and ficin have been used for tenderization of meat and meat products. These
enzymes are effective in this application because they can hydrolyze collagen and
elastin, connective tissue proteins that cause toughness in meat. Antemortem appli-
cation of enzyme is possible, as a fairly pure solution in saline can be injected
intravenously into animals 2–10 min before slaughter, sometimes after stunning; this
helps distribute the enzyme throughout the muscle tissues. Injection of inactivated
papain (disulfide form) obviates any discomfort among animals, since the enzyme
46 M. Aminlari

becomes activated by the reducing conditions that soon prevail postmortem (Arshad
et al., 2016).

2.3.4 Bioactive Peptides

Bioactive peptides have been defined as specific protein fragments that have a
positive impact on body functions or conditions and may influence health (Sánchez
& Vázquez, 2017). In recent years, peptides with known sequences have been
identified which have been shown to contain biological activities such as opiate-
like, mineral binding, immunomodulatory, antimicrobial, antioxidant,
antithrombotic, hypocholesterolemic, and antihypertensive actions. These bioactive
peptides benefit the human physiological system through the cardiovascular, ner-
vous, gastrointestinal, and immune systems (Choi et al., 2012). Most of the bioactive
peptides are inactive in the native protein sequences and become active only when
released when the parent proteins are hydrolyzed in vivo by the action of proteolytic
enzymes during digestion or in vitro by intentionally added proteases (Korhonen &
Pihlanto, 2006). The activity of these peptides is based on the inherent amino acid
composition and sequence. Bioactive peptides usually contain 3–20 amino acid
residues per molecule (Bhat et al., 2015), but in some cases may consist of more
than 20 amino acids. These peptides may be used as components of functional foods
or nutraceuticals because of their health-enhancing potential and safety profiles.
There is increasing commercial interest in the production of bioactive peptides
from various sources. Some of rich sources of bioactive peptides include milk and
egg, meat of various species of animals, fish, many plants including soy bean,
chickpeas, rice, and many other food-proteins and nonconventional protein sources
(Mazorra-Manzano et al., 2018; Sabbione et al., 2016).
Proteolytic enzymes have the ability to modify proteins through limited or
extensive cleavage, releasing free amino acids, peptides, or polypeptides with
physicochemical properties different from the original protein. Proteolytic enzymes
from proteolytic system of starters, proteases endogenous to food, or added enzymes
(e.g., rennet) differ in their specificity and therefore in their capacity to release
bioactive sequences (Korhonen & Pihlanto, 2006; Sabbione et al., 2016). Most
hydrolytic processes use one protease at a time; however, a combination of two
enzymes with different specificity has also been explored. Sometimes, proteins
partially hydrolyzed are further treated with pepsin and trypsin to simulate gastroin-
testinal digestion. Other proteases widely used for bioactive peptides’ production
include commercial preparations such as Alcalase, Neutrase, Flavourzyme,
Thermolysin derived from bacteria and fungi (Rui et al., 2012), and ficin (Shahidi
et al., 2018).
We have studied the production of peptide fragments produced from goat’s milk
whey proteins using trypsin and ficin and evaluated the bacterial growth inhibitory
activity of peptides. Goat’s milk whey proteins were subjected to enzymatic hydro-
lysis and peptides were purified by ultrafiltration followed by reverse-phase high-
performance liquid chromatography (RP-HPLC). Growth inhibitory activities of
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 47

hydrolysates ranged from 4.67% to 87.46% for E. coli and 3.03% to 98.63% for
B. cereus. Among all peptide fragments, permeate containing 3 kDa peptides
produced by trypsin showed maximum inhibition against Gram-positive and
Gram-negative bacteria. This fraction was further purified by HPLC. Fourteen
peptide fractions were collected and evaluated for their growth inhibitory activities.
Two fractions showed the highest growth inhibitory activities with MIC50’s of
383  8 and 492  10 μg/mL against E. coli and B. cereus, respectively. In a similar
study, we used trypsin and ficin to generate antibacterial peptides from goat milk
caseins. The peptide obtained by ficin with MW of <3 kDa showed the highest
antimicrobial activity and was selected for further purification by reversed-phase
high-performance liquid chromatography (RP-HPLC). Twenty-seven peptide
fractions were separated, and their antimicrobial activities were evaluated. The
results showed that one of the fractions (No. 14) possessed the highest activity
against Escherichia coli and Bacillus cereus (Esmaeilpour et al., 2016, 2017). The
authors of both papers suggested these novel antibacterial peptides can potentially
replace synthetic food preservatives in food industries.
In another research project, the yeasts Kluyveromyces marxianus and Saccharo-
myces cerevisiae protein hydrolysates were prepared by trypsin and chymotrypsin
hydrolysis and the peptides purified by reverse-phase high-performance liquid
chromatography (RP-HPLC). The antioxidant and ACE (angiotensin-converting
enzyme) inhibitory activities of the generated peptides were determined. From
K. marxianus two new peptides, LL-9, MW 1180 Da and VL-9, MW 1118 Da,
were identified. These peptides were sequenced and their functional properties
studied. Both peptides exhibited significant ACE inhibitory activity (IC50 of
22.88 mM for LL-9 and 15.20 mM, for VL-9). Molecular docking studies revealed
that the ACE inhibitory activities are due to interaction with the His513, His353,
Glu281 and Tyr520, Lys511, Gln281 pockets of ACE by LL-9 and VL-9, respec-
tively. In the case of S. cerevisiae, a fraction with molecular weight of <3 kDa
exhibited the highest activity. RP-HPLC resolved this fraction into five fractions,
one of which (fraction F3) with amino acid sequence of Tyr-Gly-Lys-Pro-Val-Ala-
Val-Pro-Ala-Arg (MW: 1057.45 Da) exhibited ACE inhibitory
(IC50 ¼ 0.42  0.02 mg/mL) and antioxidant activities (26.25  0.13 μM TE/μg
protein). Taken together, the results of these studies showed that K. marxianus and
S. cerevisiae proteins contain specific peptides in their sequences which can be
released by enzymatic hydrolysis.
These peptides have excellent bioactive properties that can potentially replace the
antioxidant and antihypertensive agents with chemical origin (Mirzaei et al., 2015,
2017).
The use of plant proteases in the production of bioactive peptides is still scarce.
Cysteine proteases such as papain, ficin, and bromelain are currently the most used
plant proteases. However, serine proteases such as zingibain, cucumisin, and
actinidin obtained from ginger rhizome, melon, and kiwifruit, respectively, are
three new emerging plant proteases which have been considered recently (Nafi
et al., 2013).
48 M. Aminlari

The search for novel specialized proteolytic enzymes with preference for specific
peptide bonds for the selective release of bioactive peptides requires further study in
order to improve process efficiency. The various plant proteases, the protein
substrates used, and the bioactive properties of peptides produced and their potential
to prevent or treat disorders such as hypertension, diabetes, obesity, and cancer have
been extensively reviewed (Mazorra-Manzano et al., 2018).

2.4 Enzymes Working on Lipids

2.4.1 Lipases

Lipases are key enzymes involved in fat digestion in vertebrates by converting

insoluble triacylglycerols into more soluble products, fatty acids, and
monoacylglycerols, which can easily be assimilated by the organisms’ intestinal
absorption. Most lipases have a basic pH optimum. Lipases act only at the oil–water
interface (Schmid & Verger, 1998).
While activity of endogenous lipases is often associated with acylglycerol hydro-
lysis and problems with lipid degradation and/or hydrolytic rancidity (or leading to
oxidative rancidity since liberated fatty acids tend to be more prone to oxidation),
exogenous lipases are used for beneficial purposes. Lipases for food processing are
obtained from edible forestomach tissue of calves, kids, or lambs, and animal
pancreatic tissues as purified edible tissue preparations or as aqueous extracts
(NAS, 1996). The porcine pancreatic enzyme is a glycoprotein composed of
450 amino acids with a calculated MW of ~50 kDa (Wong, 2003). Industrial lipases
are also produced by the controlled fermentation of Aspergillus niger var., Aspergil-
lus oryzae var., Candida rugosa, and Rhizomucor miehei as a powder or liquid
(Parkin, 2017).
Currently, commercial uses of lipases involve liberating flavoring (short-chain)
acids from lipids and rearranging fatty acyl groups along the glycerol backbone to
create highly valued and functional triacylglycerols from low-value lipids. Typical
applications in the food industry include: flavor generation in cheese. Using
pregastric lipases from goat, lamb, and calf, selective hydrolysis of short-chain
(C4–C8) fatty acids from triacylglycerols of milk fat can be achieved (Parkin,
2017). The lipase in papaya latex is selective for the sn-3-glycerol position, but
since papaya latex contains papain, it would not be suitable in cheese. Lipases are
also used for modification of lipids, manufacture of dairy products and confectionery
goods, and development of flavors in processed foods. These applications are
founded on selecting lipases with reaction selectivities required to yield the desired
products. Selectivity of lipases involves selectivity toward fatty acylgroup, ester
position along the sn-glycerol backbone, size of the glyceride (mono-, di-, or
triacylated), as well as interactions among these factors, which confer characteristic
stereoselectivity. The types of selectivity are exhibited by many of the commercially
relevant or promising lipases from over 100 characterized sources (Shahidi et al.,
2018).
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 49

Another major use of lipases is production of “structured lipids” in which the fatty
acyl groups are rearranged by lipases to yield a novel distribution along sn-glycerol
to create high-value lipids from low-value acylglycerol (Gunstone, 1999).
Lipases are used in baking industry and are added to bread doughs to supplement
endogenous cereal grain lipases and are added as dough improvers. This functional-
ity results in increased bread volume, more uniform crumb and air cell size, and
lesser tendency to stale, hydrolysis of lipids, thereby producing mono- and
diacylglycerolipids which function as emulsifying agents in the dough (Sonnet &
Gazzillo, 1991; Parkin, 2017).

2.4.2 Lipoxygenases

Lipoxygenase is a naturally existing and inexpensive enzyme widely distributed in

plant and animal cells and is abundantly found in legumes. Lipoxygenase is an
oxidoreductive enzyme catalyzing many oxidative reactions (Song et al., 2016). The
substrates of lipoxygenase in plants are mainly linoleic acid and linolenic acid.
Arachidonic acid is the primary substrate for lipoxygenase in animal cells.
Lipoxygenase catalyzes the dioxygenation of polyunsaturated fatty acids (PUFA),
converting them to diene hydroperoxy fatty acids which further decompose to
different chemicals and volatile compounds (Gigot et al., 2010). Lipoxygenase
utilizes atmospheric oxygen to react with PUFA and forms potentially useful and
valuable chemicals such as leukotrienes and lipoxins. The enzyme has many poten-
tial usages in the food industry; however, it is also associated with some unwanted
reactions, which include color change and nutrient deterioration (Parkin, 2017). The
flavor and aroma compounds produced by lipoxygenase activity on fatty acids affect
the food flavor. In one aspect, the aroma compounds might improve the desirability
of foods, and on the other hand, the oxidized compounds produced might give rise to
off-flavor of foods. By studying and understanding the properties and reaction
mechanism of LOX, food researchers could well control and utilize LOX as a natural
food additive (Shi et al., 2020). In the baking industry, it functions as an effective
baking conditioner and flour treatment agent. It is a potential substitute for potassium
bromate and benzoyl peroxide, which are commonly used as strengthening and
bleaching agents, respectively. Dough strengthening is probably through affecting
disulfide cross-links within the gluten. LOX has been widely used in the food and
beverage industries as a biocatalyst to produce aroma compounds at a low price and
large quantity (Parkin, 2017).
It appears that in the future, the enzyme will play a very important role in the food
industry. By better understanding the mechanism of action of the enzyme,
controlling the undesirable activities of the enzyme by inhibiting it allows food
producers to improve the quality of food.
50 M. Aminlari

2.4.3 Phospholipases

Mammal, plant, and microbial phospholipases are continuously being studied,

experimented, and some of them are even commercially available at industrial
scale for food industry. Phospholipases form a large class of enzymes with wide
diversity (Mansfeld, 2009). They are generally classified as acyl hydrolases and
phosphodiesterases. These enzymes are further divided into different groups
depending on the site of action within the phospholipids (PL) molecule. The acyl
hydrolases include the phospholipase A1 (specifically hydrolyzes 1-acyl ester bond
of PL) to release lyso-phospholipids (lyso-PL) and free fatty acids (FFA), phospho-
lipase A2 (catalyzes the hydrolysis of fatty acids at the sn-2 position of PL, releasing
lyso-PL and FFA), phospholipase B (which does not discriminate between the two
positional acyl ester bonds), and lysophospholipase A ½ (partially hydrolyzes PL)
(Parkin, 2017). The phosphodiesterases are represented by phospholipase C and D
(which cleave the phosphorus–oxygen bond between glycerol and phosphate, releas-
ing diacylglycerol and phosphate esters). Except phospholipases A2, most of these
phospholipases are not widely available at large quantities for industrial purposes.
The use of secreted PLA 2 from porcine pancreas or snake and bee venoms has a
very long tradition in food industry for modification of PL, such as egg yolk
production for emulsification in mayonnaise, sauces or salad dressings, baking
industry, or refinement of vegetable oils by degumming (Mansfeld, 2009).
The most representative examples of the use of phospholipases in food industry
can be found in the production of edible oils, dairy, and baking products or
emulsifying agents. Phospholipases are incorporated in processes such as the
degumming of vegetables oils during refinement for removing undesirable
compounds, the manufacture of cheese for yield increasing, or the production of
bread as bakery improvers for reducing the inclusion of emulsifying compounds
(Ramrakhiani & Chand, 2011). Lipases and phospholipases offer the opportunity to
generate compounds with technological effects and emulsifier characteristics in the
bread making, with the main final advantage of allowing the reduction or replace-
ment of added emulsifiers in bakery products. On the other hand, superior
emulsifying properties of released lysolipids can be obtained, leading to improved
dough rheological properties (Zhao et al., 2010; Casado et al., 2012).

2.5 Miscellaneous Enzymes

2.5.1 Catalase

The enzyme catalase is known to catalyze the breakdown of hydrogen peroxide into
oxygen and water. It is an oxidoreductase enzyme as it plays a crucial role in
quenching the reactive oxygen species (ROS), i.e., hydrogen peroxide, often pro-
duced as a by-product of aerobic respiration. Hence, it acts as an antioxidant and
protects the cell against oxidative stress (Kaushal et al., 2018).
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 51

Catalase is widely distributed in nature. It is found in all aerobic microorganisms

and in all plant and animal cells. The catalase activity of mammalian cells varies. It is
highest in liver and kidney and low in connective tissues. Catalase has been used in
the food processing industry to determine the adequacy of blanching (70–105  C) of
vegetables and fruits to destroy microorganisms and enzymes to preserve color,
texture, flavor, taste, and aroma of the products when stored frozen up to 2 years.
Early in the application of blanching for stabilization of frozen foods, catalase was
used especially for peas (24–26), the best on the market (Williams et al., 1986; Wong
& Whitaker, 2003). In the dairy industry, catalase activity is a sensitive and easily
detected indicator of contamination of milk by neutrophil granucytes. Also, the use
of catalase has proven to be a very cost-efficient and also a comparatively environ-
mental friendly method of getting rid of excess hydrogen peroxide from milk
samples for cheese production. H2O2 is used in the cold pasteurization process of
milk for cheese manufacture. The residual H2O2 is converted to water and molecular
oxygen by catalase, thereby avoiding its toxicity. Catalase enzymes are typically
obtained from bovine livers or microbial sources (Kilcawley et al., 1998).

2.5.2 Amino Oxidases

Biogenic amines and polyamines are present in variety of foods, such as fish, meat,
cheese, vegetables, and wines. These are organic bases with aliphatic, aromatic, and
heterocyclic structures. They are produced by decarboxylation of amino acid
substrates catalyzed by enzymes from contaminating bacteria. The most common
biogenic amines found in foods are histamine, tyramine, cadaverine,
2-phenylethylamine, spermine, spermidine, putrescine, tryptamine, and agmatine
(Ruiz-Capillas & Herrero, 2019). The formation of biogenic amines in food can
result in consumers suffering allergic reactions, characterized by difficulty in breath-
ing, itching, rash, vomiting, fever, and hypertension. Several methods for prevention
of the production or removal of biogenic amine in food have been practiced,
primarily by limiting microbial growth through chilling and freezing. However,
for many socioeconomic and technological reasons, such approaches are not always
practical. Therefore, alternative measures to prevent biogenic amine formation in
foods or to reduce their levels once formed are needed. One approach might be
application of enzymes which metabolite these amines (Naila et al., 2010).
Amine oxidase catalyzes the reaction with mono-, di-, or polyamine as substrate
(Ito & Ma, 2003):

RCH2 NH2 þ O2 þ H2 O ! NH3 þ RCHO þ H2 O2

The activity of these enzymes has been detected in Arthrobactor, yeast Candida
boidinii, Asp. niger, pea seedling, bovine plasma, and bovine lung. In human and
animals, the enzyme plays an important role for the metabolism of biogenic
monoamines in the central nervous system and peripheral tissues. Pea seedling
amine oxidase, a copper containing diamine oxidase, catalyzes the oxidative
52 M. Aminlari

deamination of histamine to the corresponding aldehyde, i.e., imidazole acetalde-

hyde concomitant with the reduction of dioxygen to hydrogen peroxide. The anti-
histamine properties of this enzyme make it an attractive candidate in fish industry to
combat the allergic responses elicited by consumption of fish. However, the
observed variation in the efficacy of the enzyme has been attributed to the diversity
and distribution of the enzyme inhibitors present in the variety of fish species
(Ebrahimnejad et al., 2013; Stránská et al., 2007).

2.5.3 Phenylalanine Ammonia-Lyase

Phenylketonuria (PKU) is an autosomal recessive genetic disorder affecting on

average incidence of about 1–3 case in 10,000 Caucasian live births. It is caused
by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) which is
responsible for converting phenylalanine to tyrosine using molecular oxygen and
tetrahydrobioptrin as necessary cofactor to perform its catalysis (Al Hafid &
Christodoulou, 2015). PAH deficiency leads to hyperphenylalaninenemia, a dra-
matic increase in blood phenylalanine concentration (<120 to >1200 μM) with
concurrent appearance of phenylalanine, phenylpyruvate, and phenylacetone in
urine. High levels of accumulated phenylalanine in PKU patients are toxic to
human body if left untreated and are associated with an abnormal phenotype
presenting with growth failure, microcephaly, seizure, and mental retardation. Chil-
dren with severe PKU can have normal cognitive development when dietary treat-
ment is initiated in early infancy and the blood phenylalanine level is maintained at
near normal or normal levels (Ding et al., 2004). The established treatment for PKU
resides in a phenylalanine-restricted diet, which has known deficiencies of several
nutrients and unsatisfactory organoleptic properties, making long-term compliance a
major challenge (Levy, 1999).
Phenylalanine ammonia lyase (PAL) is an enzyme that catalyzes a reaction
converting L-phenylalanine to ammonia and trans-cinnamic acid (which is excreted
as hippurate in urine). PAL is found widely in plants with isoenzymes existing
within many different species. It has a molecular mass in the range of 270–330 kDa.
A large number of plant species were screened for PAL activity and it was found that
enzyme activity was highest in grain seedlings, with maximal enzyme activity in
7-day-old red spring wheat (Goldson et al., 2008; Camm & Towers, 1973). We have
studied the stability of wheat seedling PAL during storage at different temperatures
and found a first order kinetic for inactivation of PLA with half-life of 30 and 18 days
at 18 and 4  C, respectively. Activity of PAL increased while germination
occurred at 25  C up to 8 days. These results can be used to develop appropriate
strategies for storing PAL containing materials with retained PAL activities
(Aminlari et al., 2010).
PAL has recently been studied for possible therapeutic benefits in humans
afflicted with phenylketonuria. The use of phenylalanine ammonia-lyase (PAL) is
an attractive alternative to the dietary treatment of PKU and both pharmacologic and
physiologic proofs of principle have been established using recombinant PAL for
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 53

enzyme substitution therapy in a murine PKU model (Sarkissian et al., 1999). A

large amount of active recombinant PAL from parsley (Petroselinum crispum) has
been prepared and chemically modified to render PAL nonimmunogenic and stable
in the circulation (Kim et al., 2004b). Oral therapy with phenylalanine ammonia
lyase (PAL), naturally encapsulated in plant cells, may provide a potential alternative
treatment for patients with PKU (Goldson et al., 2008). One significant challenge in
application of PAL for PKU therapy is protecting PAL from proteolysis and/or
denaturation and against immunologic responses. Different physical and chemical
methods have been investigated to address this problem. These include PAL immo-
bilization in semipermeable, microcapsules, the entrapment of PAL in silk fibroin,
and the immobilization of recombinant PAL in expression bacteria such as
Escherichia coli or Lactobacillus lactis, and modification with PEGylation (Bell
et al., 2017; Ikeda et al., 2005).

2.5.4 Lysozyme

In recent years, consumer demand for ‘natural’ foods has driven development of
products without additives. In order to meet this demand, much attention and interest
have been directed towards identification and application of naturally made
compounds such as antimicrobial agents, in food and pharmaceuticals (Branen &
Davidson, 2004). Some naturally occurring proteins such as lactoperoxidase,
lactoferrin, and lysozyme have received much attention and are being considered
as potential antimicrobial agents to replace the currently used synthetic food
preservatives (Demain, 2009).
Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, is
an antimicrobial enzyme found in many different sources, from viruses to
vertebrates, and has been subjected to extensive scrutiny, both as a protein model
and a natural antimicrobial and pharmaceutical agent. It forms part of the innate
immune system. Lysozyme is abundant in secretions including tears, saliva, human
milk, and mucus. Chicken egg white has the highest content of lysozyme
(it constitutes 3.5% of the total egg white proteins), from which this enzyme is
purified and is commercially produced (Proctor & Cunningham, 1988). Lysozyme is
a glycoside hydrolase that catalyzes the hydrolysis of 1,4-beta-linkages between N-
acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan, which is
the major component of gram-positive bacterial cell wall. In general, lysozyme
shows in vitro antimicrobial activity against some Gram-positive bacteria such as
Staphylococcus aureus, Micrococcus luteus, Bacillus stearothermophilus, and Clos-
tridium tyrobutyricum, but little action against Gram-negative bacteria (Cunningham
et al., 1991). Gram-negative bacteria, including foodborne pathogens, resist lyso-
zyme due to steric hindrance posed by the outer LPS layer, hence lysozyme cannot
access the peptidoglycan (Ibrahim et al., 1994). Thus, modification of lysozyme,
which can broaden its antibacterial properties against both Gram-negative and
Gram-positive bacteria, can also increase the usefulness of lysozyme (Seo et al.,
2013).
54 M. Aminlari

Lysozyme has received considerable interest as a food preservative (Cegielska-

Radziejewska & Szablewski, 2013). In many countries, lysozyme is used as a
preservative in many types of foods (including vegetables, sea foods, soy bean
products, meat products, and semihard cheeses), and as a component of pharmaceu-
tical products. In the European Union, its use in specific products such as hard
cheeses and in wine making to control infection (E number E 1105 as a food
additive) is allowed (Losso et al., 2000). Proctor and Cunningham (1988) and
Cunningham et al. (1991) have reviewed the use of lysozyme as a food preservative.
During the last two decades, extensive researches in the authors’ laboratory and
others have been directed toward modification of lysozyme in order to improve its
antimicrobial properties. Several types of these modifications are summarized in
Table 2.4. The results of modifications of lysozyme using its conjugation with

Table 2.4 Modified lysozymes and their properties (from Aminlari et al., 2014)
Modified lysozymes Effect on functionality
Palmitic acid Enhanced antimicrobial against E. coli (WT-3301)
Short and middle chain Enhanced antimicrobial against G-positive bacteria
saturated fatty acids
Glucose–stearic acid Enhanced activity against E. coli and E. tarda (G8104)
monoester
Perillaldehyde Enhanced activity against E. coli K12 and S. aureus
Cinnamaldehyde Enhanced activity against E. coli and S. aureus
Glucosamine Improved solubility at different pHs and temperatures, increased
heat stability, emulsion activity and stability, and foam capacity
Caffeic acid–cinnamic acid Antimicrobial activity against E. coli (ATCC 8739), decreased
activity against S. aureus (ATCC 6538)
Dextran Enhanced activity against E. coli and S. aureus in cheese
Enhanced activity against E. coli and S. aureus in milk
Treatment of bacterial isolates from cows with mastitis
Preparation of a lysozyme–dextran nanogel
Increased heat stability, higher emulsifying property
Galactomannan Antibacterial emulsifier
Emulsifier, antibacterial against G-negative pathogen E. tarda
Chitosan Enhanced bactericidal action against E. coli K12
Lysozyme-composite film with activity against E. coli,
L. monocytogenes, and S. faecalis
Cellulose Preparing a textile with potential barrier to microbial invasion
Gum Arabic Enhanced activity against E. coli and S. aureus in mayonnaise
Xanthan gum Enhanced activity against E. coli and S. aureus
Dextran sulfate Enhanced activity against E. coli and S. aureus
Barley beta-glucan Enhanced activity against E. coli and S. aureus
Tragacanth Enhanced activity against E. coli, S. typhimyrium, B. cereus, and
S. aureus
Inulin Improved functional properties
Trypsin and ficin digestion Enhanced activity of peptides E. coli and B. cereus dextran-
of conjugated lysozyme
2 Plants- and Animal-Derived Enzymes and Their Potential Application in. . . 55

different small molecule and polysaccharides by application of a Maillard-based

reaction, as well as modifications using proteolytic enzymes, have revealed that
these types of modifications have not only increased the functional properties (such
as solubility and heat stability), but also extended the antimicrobial activity of
lysozyme (Aminlari et al., 2014). We have shown that it is possible to broaden the
antimicrobial effect of lysozyme by several types of modifications, which conse-
quently make these lysozyme derivatives excellent food preservatives. Table 2.3
summarizes the effect of different types of modifications on lysozyme.

2.6 Conclusions

Nature provides us with remarkably vast array of enzymes with extraordinary

catalytic properties. In their natural reservoirs, these so-called “endogenous”
enzymes perform essentially all biochemical reactions vital to the living organisms.
When these enzymes are removed from their natural habitat, they continue to
catalyze same reactions when they have access to their substrate. At this stage,
they are indeed “exogenous” enzyme. Food scientists have used these exogenous
enzymes as food additives for many years. In this chapter, the properties of several
enzymes from plant and animal sources were presented and their application or
potential application in food industry was discussed. Over the years, it has become
clear that despite advances in experimental mutational studies, a quantitative under-
standing of enzyme catalysis will not be possible without computer modeling
approaches (Frushicheva et al., 2014). With the advent of new technologies such
as information technology, rational molecular design, and DNA enzyme
(deoxyribozyme) technologies (Breaker, 1997; Woolcock, 2016), it is expected
that new doors for application of these technologies will open to food enzymologists
in the near future. The discussion is by no means complete, and with the advent of
knowledge of enzymology, the endeavor will continue. For those of us who
(as Arthur Korenberg, the discoverer of DNA polymerase once said) are in love
with enzymes, the challenge has just began.

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Role of Enzymes in Fruit and Vegetable
Processing Industries: Effect on Quality, 3
Processing Method, and Application

Memthoi Devi Heirangkhongjam, Kanika Agarwal, Aparna Agarwal,

and Nidhi Jaiswal

Abstract

The significance of enzymes and their application in food processing industry is

increasing rapidly. Different kinds of enzymes are extensively used based on their
effective application. In fruits and vegetables processing, several endogenous
enzymes and newly developed enzymes are used. Enzymes present in fruits and
vegetables play a huge role in determining the texture, colour, flavour, and taste
attributes of the processed products. The continued enzymatic activity in fruits
and vegetables affects the storage quality, shelf life, and palatability of the
product. Therefore, several processing methods such as grinding, crushing,
slicing, juices, or preservation are used to prolong the shelf life and reduce
wastage of fruits and vegetables. Hence, it is very important to control the
stability and activity of endogenous enzymes present in fruits and vegetables
during food processing. Apart from the conventional techniques of thermal
processing such as blanching, heating, ohmic, and microwave, new and highly
advanced processing techniques like high hydrostatic pressure (HHP), high-
pressure homogenization processing (HPHP), pulsed electric field (PEF), and
ultrasound processing have been introduced successfully. The major advantage of
these new techniques is the use of non-thermal technology, which helps in
retaining the sensory attributes and nutritional content of the product. These
non-thermal processing techniques are effective at ambient or sub-lethal
temperatures and minimize the adverse thermal effects on the nutritional content
and quality of fruits and vegetables. Nonetheless, multiple advantages are ren-
dered by these techniques in food processing industry over conventional thermal
techniques which affect not only the enzymes, but also the texture, taste, and
colour of the product compelled for further investigation and improvement.

M. D. Heirangkhongjam · K. Agarwal · A. Agarwal (*) · N. Jaiswal

Department of Food and Nutrition, Lady Irwin College, University of Delhi, New Delhi, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 65

Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_3
66 M. D. Heirangkhongjam et al.

Keywords

Enzymes · Fruit and vegetable processing · HPP · PEF · Shelf life extension

Abbreviations

CAGR Compound annual growth rate

HG Homogalacturonan
HHP High hydrostatic pressure
HPHP High-pressure homogenization processing
HTLT High temperature-long time
HTST High temperature-short time
OH Ohmic heating
PEF Pulsed electric field
PME Pectin methylesterase
POD Peroxidase
PPO Polyphenol oxidase
RG-II Rhamnogalacturonan-II

3.1 Introduction

Enzymes play a very critical role in the growth and maturation of fruits and
vegetables. They also make significant contributions during the post-harvest phase
in maintaining stability of raw food materials and improving their quality attributes
like aroma, colour, texture, flavour, and nutritional quality. Apart from these,
enzymes also have the ability to act as catalysts in transforming raw materials to
food products during processing. They are also found to have extensive applications
in food processing and production by enhancing the nutritional, safety, functional
qualities, and overall acceptability of ingredients and processed products. Moreover,
enzymes are also known for their substrate specificity, effectiveness in catalytic
reactions, and enhancement rate under controlled conditions of moisture, pH, and
temperature (Berg et al., 2010).
Due to rapid growth of population and fast changing lifestyle, the production and
demand of convenience and processed food products have also been gradually
increasing. As per the study of Markets and Markets (2020), the fruit and vegetable
processing enzyme global market is expected to touch around $41.39 billion by
2022 at 6.7% CAGR from 2016 to 2020. During this corresponding period, the Asia-
Pacific region enzyme market is projected to grow at the highest CAGR due to high
growth of the food and beverage industry in big emerging economies like India and
China. Therefore, productive usage of the existing enzymes and the development of
new enzymes have become very significant in meeting the ever-increasing demands
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 67

in food processing industries. Hence, rapid advancement in enzymology is given

prime focus in the food processing sector.
Currently, new enzymes based on their sources (fungi and bacteria) and types
(amylase, pectinase, xylanase, and cellulase) have been isolated, characterized, and
developed commercially for productive usage either in enhancing the desirable or
delaying the unfavourable characteristics of foods during processing. Nonetheless,
the importance and significance of endogenous enzymes in the present scenario of
food processing industries cannot be overlooked for their productive usage in
meeting the desired colour, texture, flavour, appearance, and stability of foods during
industrial processing.
In light of the importance of enzymes in food processing, the uses of enzymes and
their application in fruit and vegetable processing industries are indispensable in
order to increase the productivity and shelf life, reduce cost of production, and also
develop new products.

3.2 Fruit and Vegetable Composition

Knowing the composition of fruit and vegetable is the first step in determining the
benefits and drawbacks of them. Fruits and vegetables are mainly composed of
cellulose (7.20–43.60%), hemicelluloses (4.26–33.50%), pectin (1.50–13.40%),
lignin (15.30–69.40%), and starch (3–21%) (Dhillon et al., 2013). The primary
cell walls in the young plants comprise largely of cellulose and tend to thicken and
become higher in hemicelluloses and lignin as they grow (Albersheim et al., 1996).
Now, let’s discuss in detail the different types of composition in fruits and
vegetables.

3.2.1 Cellulose

Cellulose is one of the main components in fruits and vegetables ranging from 7.20%
to 43.60%. It is a linear polymer of D-glucose present in the primary cell wall. It
forms microfibrils, which give a stiff shape, structure, and tensile strength to the cell
wall. It also helps to form a resistance against the degradation of this polymer
(Szymańska-Chargot et al., 2017). These microfibrils are coated with hemicelluloses
and help in binding firmly to their surface (Carpita & Gibeaut, 1993). Cellulose is
known for its diverse applications in food processing industry. It is used as an
emulsifier, bulking agent, texturizer, and as a fat substitute (Grassino et al., 2016).
However, the application of celluloses during the extraction process needs extra
precaution in order to avoid breaking down the cellulose network in maceration
processes as it decreases the quantity of juice production.
68 M. D. Heirangkhongjam et al.

3.2.2 Hemicellulose

Hemicellulose is another major component in fruits and vegetables with a variable

range of 4.26–33.50%. It is a matrix polysaccharide often present along with
cellulose in the cell walls and comprises of xyloglucan, arabinoxylan, glucomannan,
and galactoglucomannan. Xyloglucan is the most abundant hemicellulose with
similar structure as D-glucose backbone in cellulose. Hemicelluloses are bound via
hydrogen bonds, forming a network of cross-linked microfibrils between pectin and
cellulose. Such interconnections play a major role in the integrity of the pectin cross-
linking network. Vincken et al. (1994) demonstrated that the key structure in the
apple cell wall is xyloglucan and it helps in breaking down cell-wall embedded in
cellulose if it gets hydrolysed. In apple cell wall matrix, xyloglucan accounts for
24% of the total amount of sugar, whereas the cellulose–xyloglucan complex
accounts for 57% approximately. In food processing industries, hemicelluloses are
primarily used as enhancing agents in viscosity, stabilizing, and gelling. Moreover, it
is also widely used in technical and pharmaceutical fields as coatings, films, gel
materials, and adhesives (Wang et al., 2010).

3.2.3 Pectin

Pectin is one of the least present components in fruits and vegetable ranging from
1.50% to 13.40%. It forms a class of complex polysaccharides commonly found in
the cell walls of higher plants. It provides structure and firmness to the plant tissue in
the primary cell wall and middle lamella component involved in intercellular
adhesion (Thakur et al., 1997). There are three pectic polysaccharides, namely,
homogalacturonan, rhamnogalacturonans, and substituted galacturonans isolated
from primary cell walls. Homogalacturonan (HG) is a homopolymer, a linear
chain of 1,4-linked α-D-galactosyluronic residues, known for its gel forming ability.
In HG, some of the carboxyl groups are methylesterified at the C-2 and C-3
positions. Similarly, the backbone of Rhamnogalacturonan-II (RG-II) is composed
of 1,4-linked D-galactosyluronic residues like HG, with a non-saccharide and an
octa-saccharide side chains attached to C-2 and two disaccharides attached to C-3 of
the backbone residues. RG-II is significantly used in winery and fruit juice industries
because of its exceptional quality of binding heavy metals, thereby reducing the
toxicity level in the final product. It also has immunomodulating activities (Grassin
& Coutel, 2009). Galarturonan fractions are generally separated from other high
molecular weight pectin fractions by degrading purified pectins specifically present
in the galacturonan backbone either enzymatically or chemically. In fact, as
explained above, the different types of pectins perform certain specific functions,
but pectins in general are largely used as gelling and thickening agent in dairy and
baking. Also, pectins are being widely used as a carrier of drug delivery system in
cosmetic and pharmaceutical industries (Kollarigowda, 2015).
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 69

3.2.4 Starch

Starch is another main component in fruits and vegetables which is known very
commonly. It is a polysaccharide composed of amylose and amylopectin. Amylose
has linear chains of about 840–22,000 units of α-D-glucopyranosyl residues linked
by 1!4-α-D-glucan, whereas amylopectin is usually comprised of highly branched
α-1!6 and α-1!4 glucosidic linkages. Starch is a form of carbohydrate or energy
reserve mostly found in storage organs, seeds, unripe fruits, and vegetables. The
most important quality of starch is its water holding capacity which depends on the
specific shape and size of each granule. For example, a potato granule can hold
approx. 5–100 μm water/glucose unit (Jobling, 2004). Moreover, each granule
contains ‘blockets’ of amylopectin which are crystalline and amorphous in nature.
They are responsible for absorption of water, swelling, lose crystallinity, and seep
amylose. However, the swelling capacity tends to be lower with higher amylose
content and thus results in reduction in gel strength (Li & Yeh, 2001). Therefore,
because of its inconsistent natural properties due to the vagaries of weather and
agricultural conditions, most of the suppliers constantly try to make uniform starches
as functional ingredients. In food processing industry, the usage of starch is mani-
fold. It acts as an innate natural ingredient with different added functionalities like an
emulsifier, stabilizer, water binder, thickener, and gelling agent.

3.2.5 Lignin

Lignin is one of the major components present in fruits and vegetables ranging from
15.30% to 69.40%. It represents a class of natural aromatic polymers of
4-hydroxyphenylpropanoids units connected by ether and carbon-carbon linkages.
Lignins are generally considered a part of dietary fiber. They are mainly present in
cereals, fruit, and vegetables, in which wheat bran has the richest source of lignin.
Lignins have efficient antioxidant properties, mainly scavenging superoxide and
hydroxyl radicals. It also inhibits the activity of xanthine oxidase, glucose-6-phos-
phate dehydrogenase, and non-enzymatic and enzymatic lipid peroxidation
(Lu et al., 1998). In addition to this, its concentration and composition influence
the adsorption capacity of cell walls. Therefore, due to these properties, lignins are
considered as efficient adsorbers of hydrophobic heterocyclic aromatic amines
(Funk et al., 2006). Although lignin has many applications in other different
industries, in food industry it is mainly used as an additive, provides roughage to
foods, and as sequestering agents.
70 M. D. Heirangkhongjam et al.

3.3 Enzymes Used in Industrial Processing of Fruits

and Vegetables

As discussed, the role of enzymes in the overall natural growth, maturation, and
ripening of fruits and vegetables is very critical. This very importance of enzymes
remains the same or even more in the industrial processing of fruit and vegetables.
Another important positive aspect for enzymes is their ability in keeping the quality
of fresh fruits and vegetables post-harvest and during storage conditions, which
ultimately plays a very pivotal role in the industrial food processing.
In industrial processing, the ripening process with the application of enzymes is a
major step, wherein several changes like alterations in the cell wall, middle lamellae,
and membrane occur resulting in softening of tissues. Naturally, most of the
enzymes in fruits and vegetable tissues are important for the maintenance of metab-
olism; however, they are also associated with undesirable effects on colour, flavour,
odour, texture, and nutritional value. For example, in some vegetables, flavour and
odour development is affected by lipoxygenase, lipase, and peroxidase (Fleuri et al.,
2016). Furthermore, phenol oxidases result in discolouration and unfavourable
effects on the taste and nutritional quality of fruits and vegetables. Also, fruits and
vegetables containing pectic substances and α-amylases have major effects on their
textural integrity (Berg et al., 2010; Fleuri et al., 2016).
With the advancement in enzymology, different types of new enzymes have been
developed and are being used in improving the quality of products, development of
new products, and in processing aids such as peeling, extraction of juice, and
clarification, thereby increasing the efficiency in processing operation. For example,
amylases, cellulases, and pectinases facilitate maceration, liquefaction, and
clarification in processing of fruit juice, and hence they are cost-effective and
increase the yield. Moreover, different enzyme extracts from plant tissues, bacteria,
yeast, and fungi are applied in fruit and vegetable processing industries for the same
purpose (Leadlay, 1993). In Table 3.1, some of the important microorganism origin
enzymes used in industrial processing and its application are summarized. In
Table 3.2, different types of enzymes, product type, and its application in fruit and
vegetable processing are highlighted.
Enzymes are classified broadly into four types: Pectinases, Cellulase, Xylanase,
and Amylase. In the proceeding paragraphs, we will be discussing in brief the
different types of enzymes in fruit and vegetable processing industry.

3.3.1 Pectinases

Those enzymes whose primary role is to break down pectin, a structural

heteropolysaccharide found in primary cell walls of fruits and vegetables, cereals,
and fibers, are known as pectinases (Singh et al., 2003). The pectin substances are of
high molecular weight acidic heteropolysaccharide primarily composed of α-(1–4)-
linked D-galacturonic acid residues (Kavuthodi & Sebastian, 2018). Pectic acids are
water-soluble substances having variable degrees of methyl ester groups and for
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 71

Table 3.1 Enzymes derived from microorganisms and their application in fruits and vegetable
processing
Enzyme Microorganism Action Application
Pectinase Aspergillus spp., Pectin hydrolysis Degradation of pectins,
Penicillium funiculosum increases the overall juice
production, fruit juice
clarification
Pectinesterase Aspergillus spp. Remove methyl Increase firmness of
groups from vegetables and also used
galactose units of with pectinase
pectin depectinisation
technology
Protopectinase Kluyveromyces fragilis, Catalyse pectin Clarification and
Galactomyces reesei, solubilization reduction of viscosity in
Trichosporon fragilis, fruit juices
Bacillus subtilis
Hemicellulase Aspergillus spp., Bacillus Hemicellulose Helps extraction of fruit
subtilis, Trichoderma hydrolysis juices, vegetable oils, and
reesei aromatic compounds,
acts on hydrolysis of
soluble pectin and cell
wall components with
pectinases, lowers
viscosity and texture
α-Amylase Aspergillus spp., Bacillus Random Hydrolysing starch to
spp., Microbacterium hydrolyses α-1,4 reduced viscosity,
imperiale bounds to rupture liquefying adjunct, helps
starch and produce in sugar production, for
maltose softness and increases
volume of fruit juices and
vegetables
Glucoamylase Aspergillus niger, Hydrolyse dextrin Fruit juice extraction and
Rhizopus spp. from starch in also used for corn syrup
glucose and glucose production
Glucose Streptomyces rubiginosus, Conversion of Helps in high-fructose
isomerise Streptomyces lividans, glucose to fructose corn syrup production
Actinplanes (beverage sweetener)
missouriensis, Bacillus
coagulans
Cellulase Trichoderma spp., Hydrolyses Liquefaction of fruit in
Aspergillus niger cellulose juice production
Note: Adapted from “Enzymes in food and beverage processing”, Fleuri, L. F., Delgado, C. H. O.,
Novelli, P. K., Pivetta, M. R., Do Prado, D. Z., & Simon, J. W., 2015, p. 257, London, New York:
CRC Press, Taylor & Francis Group

neutralization, which form gels with the addition of sugars and acids under
favourable conditions (Guo et al., 2014). Pectic substances are classified into
protopectins, pectic acids, and pectin which are partially soluble in water (Uneojo
& Pastore, 2007). These substances are generally degraded by the enzyme
pectinases. They are further classified into different sub-types, namely,
72 M. D. Heirangkhongjam et al.

Table 3.2 Enzymes used in fruit and vegetable processing

Product
Name of fruit type Enzyme Application
Apple (Pyrus malus Juice Amylase Decrease starch concentration to
L.) improve durability
Banana (Musa Juice Pectinase Decrease turbidity and viscosity
sapientum L.)
Blueberry (Vaccinium Juice Pectinase Improve juice yield and anthocyanin
myrtillus L.) level
Citrus (Citrus sinensis Juice Pectinase and Decrease the turbidity of juice
L. Osbeck) cellulase
Cherry (Prunus avium Juice Pectinase and Decrease turbidity and increase
L.) protease stability
Cloudy ginkgo Juice Amylase and Reduce hydrolysis time to improve
(Ginkgo biloba L.) protease stability
Grape (Vitis vinifera Juice Pectinase Decrease turbidity and soluble solids
L.)
Lemon (Citrus Soft Pectinase Clarification of peel extract to
sinensis L. Osbeck) drink produce soft drinks
punch
Pineapple (Ananas Juice Pectinase and Improve soluble solids and aromas
comosus L.) cellulase
Pomegranate (Punica Juice Pectinase Improve concentration of
granatum L.) antioxidants and decrease turbidity
Carrot (Daucus carota Juice Pectinase Improved nutritional properties as
L.) the content of polyphenols and
flavonoids
Date syrup (Phoenix Syrup Cellulase and Reduce turbidity and increase the
dactylifera L.) pectinases extraction of soluble solids
High-fructose corn Syrup Amylase Conversion of glucose to fructose
syrup (HFCS—Zea
mays L.)
Olive oil (Olea Oil Pectinase, Oil extraction from olive residue and
europaea L.) cellulase, and improve soluble solids on oil
hemicellulase extraction
Sunflower oil Oil Pectinase Improve oil yield
(Helianthus annuus
L.)
Note: Adapted from “Enzymes in food and beverage processing”, Fleuri, L. F., Delgado, C. H. O.,
Novelli, P. K., Pivetta, M. R., Do Prado, D. Z., & Simon, J. W., 2015, p. 258, London, New York:
CRC Press, Taylor & Francis Group

methylesterases (based on mechanical action), polygalacturonases, and lyases (based

on their mode of action).
Then, polygalacturonases are further sub-classified into endo-polygalacturonases
(E.C. 3.2.1.15) and exo-polygalacturonases (E.C. 3.2.1.67). Similarly, lyases are
also sub-classified into three types, namely, pectin methylesterases (E.C. 3.1.1.11),
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 73

pectatelyases (E.C. 4.2.2.9 and E.C. 4.2.2.2), or pectin lyases (E.C. 4.2.2.10)
(Kc et al., 2020).
As per a recent study, it is reported that pectinases from microbial origin account
for about 25% of global industrial enzymes market which is projected to reach USD
6.3 billion by 2021 (Oumer, 2017; Oumer & Abate, 2018). This clearly highlights
the significance of microorganism origin enzyme as a reliable source of industrial
enzyme production. Pectinases or pectinolytic enzymes are also naturally produced
by many other organisms like insects, bacteria, nematodes, and protozoans (Khairnar
et al., 2009). Some of the other commonly used microorganisms for extensive
production of pectinases are Aspergillus spp., Bacillus spp., Erwinia spp., and
Penicillium spp. (Oumer, 2017).
In food processing industries, especially in fruits and vegetables processing,
pectinases have multiple usage. They are mainly used during processing of citrus
juice. Moreover, pectinases are also generally recommended for use with the com-
bination of other enzymes such as cellulases and hemicellulases. Such combinations
of enzymes are generally used in facilitating the process of maceration, liquefaction,
and clarification. It ultimately helps in increasing the extraction yield and enhancing
the concentration of acids, colourants, and flavourings (Oumer & Abate, 2018).
Apart from these, pectinases are also widely used in wine clarification, concentra-
tion, and fermentation of tea, cocoa, and coffee. Further, these enzymes are also used
regularly in pickling, preparation of jam and jellies, syrups, starches, and vegetable
oil extraction (Kubra et al., 2018).

3.3.2 Cellulases

Cellulases are enzymes that promote the process of hydrolysis of “cellulose”, a

fibrous, tough and water-insoluble polysaccharide and a homopolymer comprising
of several glucose units joined by β-1,4 linkages. They act synergistically as a
biocatalyst to release the sugar molecules, which helps in converting into ethanol
and organic acids (Fleuri et al., 2016). Cellulases are generally produced as a multi-
enzyme system comprising of glucosidase (β-D-glucoside gluco-hydrolase,
E.C. 3.2.1.21), β-1,4-endoglucanase (1,4-β-D-glucan 4-glucan hydrolase,
E.C. 3.2.1.4), and cellobiohydrolase or exoglucanase (1,4-β-D-glucan
cellobiohydrolase, E.C. 3.2.1.91) (Jecu, 2000). Cellulases are produced by bacteria
and fungi (Sharma et al., 2017). However, for commercial preparation of cellulases,
different filamentous fungi such as Trichoderma reesei (Megazyme) and Aspergillus
niger (Cellulocast from Novozyme) are used. Some other common cellulases pro-
ducing fungi are Aspergillus, Chaetomium, Fusarium, Humicola, Neocallimastix,
Penicillium, Piromonas, Thermoascus, Trichoderma, etc. (Singhania et al., 2010;
Bansal et al., 2011).
In food processing industry, majorly in beverage industry, cellulase enzymes are
extensively used in the production of fruit juices and wine processing. It facilitates
the extraction of juice and maceration process by breaking the cellulose chains
present in the plant cells. It also helps in extracting pigments and flavouring
74 M. D. Heirangkhongjam et al.

substances present in the grape skin. Besides releasing the flavouring substances and
improving the aroma and flavour of beverages, it also breaks the unpleasant-tasting
compounds present in the fruits and vegetables (Juturu & Wu, 2013).

3.3.3 Hemicellulase and Xylanase

Hemicellulase is an enzyme complex that breaks down the backbone of xylan and
arabinose side chains and releases pentoses (xylose and arabinose) (Yang et al.,
2017). Similarly, xylanases (endo-1,4-β-D-xylanohydrolase; E.C. 3.2.1.8) are hydro-
lytic enzymes involved in depolymerization of xylan. They are usually present in
superior plants, such as cereals, grasses, and trees that present noncellulosic
polysaccharides, such as D-glucose, L-arabinose, D-xylose, D-mannose, D-galactose,
D-glucuronic, and D-galacturonic acid (Cunha & Gandini, 2010). These enzymes
also degrade hemicellulose polymers, including acetyl xylan esterase
(E.C. 3.1.1.72), α-arabinofuranosidase (α-L-arabinofuranosidase, E.C. 3.2.1.55),
arabinase (endo-α-L-arabinase, E.C. 3.2.1.99), endo-xylanase (endo-1,4–βxylanase,
E.C. 3.2.1.8), feruloyl esterase (E.C. 3.1.1.73), α-glucuronidase (α-glucosiduronase,
E.C. 3.2.1.139), and β-xylosidase (xylan-β-1,4-xylosidase, E.C. 3.2.1.37) (Juturu &
Wu, 2013). Polymers such as xylan and arabinoxylan are completely hydrolysed by
the synergistic action of several xylanolytic enzymes: endo-1,4-β-D-xylanases. The
hydrolysis of these polymers degrades the β-D-xylan linkages; β-D-xylosidases
release a xylose monomer from the cleavage of the non-reducing end of xylo-
oligosaccharides and xylobiose (Terrasan et al., 2010).
Currently, commercial xylanases are produced on a large scale in many countries,
such as United States, Japan, Finland, Germany, Ireland, Canada, and Denmark, by
fermentation processes using bacteria, yeast, and fungi (Polizeli et al., 2005).
Substantial increase in the production of xylanases has been observed after the
development of improved microbial strains and efficient fermentation techniques
and recovery systems. It has several applications in food industries, agriculture as
well as in human health.
Furthermore, during processing of beer, the cellular wall is generally hydrolysed
releasing long chains of arabinoxylans which in turn increase the viscosity of beer
rendering it “muddy” in appearance. Then, xylanases are added to hydrolyse
arabinoxylans in order to lower oligosaccharides, thereby reducing the viscosity
and consequently improving its appearance (Dervilly et al., 2002). Xylanases in
combination with amylases, cellulases, and pectinases provide multiple advantages
such as increase yield of juice, stabilization of fruit pulp, and increased recovery of
flavours and aromas, essential oils, mineral salts, vitamins, etc.

3.3.4 Amylases

Amylases are enzymes that act as catalysts in the hydrolysis of starch into sugars
such as glucose and maltose (Sundarram & Murthy, 2014). These enzymes are
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 75

extensively found in plant, animal, and microbial kingdoms. Amylases are classified
into exo-amylases and endo-amylases based on their action. Exo-amylases are
involved in hydrolysis of α-glucan into maltose and glucose, whereas endo-amylase
hydrolyses α-glucan-forming oligosaccharides. Starch which is a polysaccharide that
is an essential factor of structure, consistency, and texture in foods is hydrolysed by
β-amylase. Moreover, the non-reducing terminals in starch polysaccharides and
malto-oligosaccharides are also hydrolysed by β-amylase (Fleuri et al., 2016).
Such enzymes are responsible for the degradation of starch and its related polymers
to yield products characteristic of individual amylolytic enzymes.
Amylase enzymes have some common types, namely, amylolytic, α-amylase,
β-amylase, and glucoamylase. These enzymes are largely present in a wide range of
organisms, including plants, animals, and microorganisms. Aspergillus spp. is the
most extensively used fungi for the production of amylolytic enzymes. The major
sources of α-amylase enzymes are A. niger, Aspergillus oryzae, B. circulans, Bacil-
lus amyloliquefaciens, B. subtilis, B. licheniformis, and B. stearothermophilus.
β-Amylase enzymes are obtained from species like Bacillus spp., Pseudomonas
spp., and Streptomyces spp. However, out of these enzymes, the production of
α-amylases accounts for about 30% of enzymes in world market for their extensive
use in food industrial processes, such as in baking, brewing, fruit juices, syrups,
starch, etc. It is also widely used in the production of drugs and other pharmaceutical
products (van der Maarel et al., 2002).

3.4 Significance of Enzymes in Food Quality

In recent years, the significance of enzyme application in food processing industries

has been increasing. It is due to the diversified role played by them in increasing the
rate of biochemical processes. But, sometimes enzymes are also liable to
unfavourable biochemical and physiological changes in fruits and vegetables.
These changes alter the colour, texture, aroma, and flavour in them. Therefore, it is
important to understand and characterize the interrelationships between the quality
components and the associated enzymes. It also helps in determining the optimal
post-harvest handling procedures and processing techniques to provide high-quality
products to consumers.

3.4.1 Enzymatic Reaction on Colour of Fruits and Vegetables

The colour of a product is the primary assessment factor of quality more than any
other factor. So, maintaining the natural colour of food is very critical. The colour of
food products is affected during pre-harvest and post-harvest factors which are
coupled by enzymatic reactions. The main enzymes responsible for changes of
colour in fruits and vegetables are polyphenol oxidase (PPO), peroxidase (POD),
and β-glucosidase (Zabetakis et al., 2000). Among the various processing changes,
enzymatic browning causes major problem in food processing (Terefe et al., 2014).
76 M. D. Heirangkhongjam et al.

It is due to the oxidation of phenolic compounds to quinones via polymerization

reactions resulting in the production of dark colour compound called melanin
(Marshall et al., 2000). Either to inhibit or delay this particular phenomenon, the
fruits and vegetables are treated with anti-browning agents prior to further
processing. The most commonly used anti-browning agents are citric acid, ascorbic
acid, and calcium chloride. They can be used either singly or in combination with
other chemicals. Ascorbic acid and citric acid are extensively used as anti-browning
agents since they act as a reducing agent on the enzymatic reaction of PPO.
Similarly, calcium chloride (CaCl2), also known for PPO inhibitors, reduces enzy-
matic browning by acting on pH and as a chelating agent. However, combined
treatments were found to be more effective to prevent the browning effect.
Polyphenol oxidases (PPOs) are a group of copper containing enzymes, capable
of catalysing the oxidation of several phenols to o-quinones (Oliveira et al., 2011).
These o-quinones in turn react with molecules which undergo non-enzymatic sec-
ondary reactions, resulting in the formation of melanin, brown complex polymers,
and cross-linked polymers with protein functional groups (Rolff et al., 2011). PPOs
are abundantly present in various fruits and vegetables, such as, apple, peach,
banana, potato, mushroom, coffee bean, microorganisms, etc. (Eisenmenger &
Reyes-De-Corcuera, 2009). Conversion of phenolics substrates into o-quinones
occurs in two oxidation steps. In the first step, hydroxylation of ortho-position
adjacent to an existing hydroxyl group, “monophenolase” or “monophenol oxidase”,
is generally referred to as cresolase or hydroxylase activity. The second step is the
oxidation of o-dihydroxybenzenes to o-benzoquinones, “diphenolase activity” or
“diphenol oxidase”, referred to as oxidase or catecholase activity (Yoruk & Mar-
shall, 2003).
The oxidation of phenolics compounds by polyphenol oxidase is the main reason
for most of the enzymatic browning in foods occurred during harvesting, handling,
storage, and processing (Eisenmenger & Reyes-De-Corcuera, 2009). It was reported
that the PPO activity in apple, tomato, and tobacco is encoded by multiple genes and
regulates tissue damage (Kim et al., 2001). In another study, expression of PPO
response was observed in both damaged and non-damaged leaves (Constabel &
Ryan, 1998). The expression of PPO in peaches was observed for 48 h of storage and
it was found that there was a slight or gradual decrease in the PPO activity in intact
tissues. On the other hand, in damaged tissues, there was a rapid increase in PPO
activity (Tourino et al., 1993). In peaches, enzyme inhibitors such as ascorbic acid,
sodium metabisulfite, β-mercaptoethanol, and cysteine were found to be effective
against PPO activity (Belluzzo et al., 2009).
Furthermore, enzymatic browning can also be controlled by several processing
techniques, such as freezing and heating coupled with natural and synthetic
inhibitors (Marshall et al., 2000). However, many PPO inhibitors are associated
with off-flavours, food security, and sustainability. For example, sulphites as PPO
inhibitors are reported to cause allergies and thus seek natural alternatives (Loizzo
et al., 2012). 4-Hexylresorcinol is a natural compound; when used along with
ascorbic acid, cysteine, or kojic acid, it resulted in the reduction in PPO activity
and changes colour in mango puree and apple juice (İyidoǧan & Bayındırlı, 2004).
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 77

Kojic acid is another natural compound; it chelates metal ions such as Fe3+ and Cu2+
in addition to impounding of free radicals (Kim & Uyama, 2005).
Proteases are another group of enzymes that catalyse the hydrolysis of proteins
which are degraded to peptides and amino acids (Omaña-Molina et al., 2013). It
inhibits PPO activity through proteolysis or binding to specific sites. According to a
study report, it was found that the browning reaction was decreased in apple when
papain was used (Labuza et al. 1992). Also, when pineapple juice and high-pressure
technique were applied, the browning reaction was reduced in apple slices. Such
inhibitory effect is due to the presence of bromelin, sulfhydryl groups, citric, or malic
acid (Perera et al., 2010). Similarly, the prevention of browning effect was also
observed in peeled banana when pineapple juice was used (Chaisakdanugull et al.,
2007). However, in some studies, bromelin was found to be ineffective in preventing
browning of apple juice (Tochi et al., 2009).
Peroxidases (POD) are generally isolated from plants, animals, and
microorganisms. These are oxidase enzymes which use hydrogen peroxide as a
catalyst in the oxidation reaction of polyphenols, aminophenols, monophenols,
and diphenols (Fatibello-Filho & Vieira, 2002). They are heat-stable and hence
used as a parameter to increase the efficiency in bleaching (Aguero et al., 2008).
POD lead to undesirable changes in colour, flavour, texture, and nutritional values in
foods (Gonçalves et al., 2007). The browning of sugar cane juice is due to the
presence of enzymes, POD and PPO, which oxidize phenolics compounds (Qudsieh
et al., 2002). The enzymatic activities of POD and PPD enzymes were studied in
different cultivars of grapes. In these studies, the POD extracts showed similar
activity in both the soluble and bound fractions, and highest PPD activity was
observed in cultivar Ruby. PPD and POD activities in cultivars Benitaka and
Ruby decrease when juice extracts were treated with higher temperature and longer
duration. It was observed that enzyme inactivation was achieved at 85  C with
10 min exposure time, but the thermal treatments were not sufficient to inactivate the
enzymes completely. For example, the thermal treatment in jam, jellies, and juices
causes reduction in PPO and POD activities, but not sufficient to inactivate the
enzymes (Freitas et al., 2008). Similar activities were also reported in processing of
guavas (Zanatta et al., 2006).
In other studies, it was shown that apple peel from Fuji and Gala cultivars when
compared to its pulp had elevated enzymatic activity both for PPO and POD. When
heat treatment was done in the concentrated extracts of pulp and peel, there was a
decline in PPO enzyme activity and total inactivation was also achieved after 10 min
of heat treatment at 75  C. However, such case of total inactivation was not observed
for POD enzyme activity (Valderrama et al., 2001). Studies have shown that the
enzymatic activity of PPO in fresh broccoli was higher compared to bleached
broccoli. In case of POD, the activity was found to be lower in bleached broccoli,
indicating that bleaching was partially effective in denaturation of these enzymes
(Lopes & Clemente, 2002).
78 M. D. Heirangkhongjam et al.

3.4.2 Enzymatic Reaction on Texture of Fruits and Vegetables

Texture of fruits and vegetables is an important indicator in determining the quality

of the product. It is dependent on the cellular structure of cell walls. Any changes in
the composition and structure of cell wall polysaccharides result into changes in the
firmness (Alkorta et al., 1998). It influences the keeping quality such as storage time,
handling damages, and consumer acceptance of fruits and vegetables after
harvesting. Degradation in the textural quality may be due to several factors such
as growth of microorganisms, infestation by insects, rodents, and other environmen-
tal conditions.
The texture quality of fruits and vegetables is also affected by a number of
enzymes. Pectin which is an important cell wall polysaccharide helps in maintaining
the texture by avoiding the breakdown of cellular structure. The enzymatic effect on
the texture of fruits and vegetables happens when enzymes such as pectinases,
cellulases, and hemicellulases break the cell walls. So, the inactivation of these
enzymes during processing would help in preserving the texture of the food.
Generally, pectin is used as a gelling and texturizing agent. It also improves the
taste and appearance of processed foods like jams, jellies, and marmalades (Alkorta
et al., 1998). However, pectins also have some major disadvantage in food
processing, especially in juice processing. Soluble pectins in fruits are responsible
for cloudiness or haziness in different fruit juices. Hence, the texture of such final
juice product may or may not have positive feedback from consumers. But, these
days, cloudy juices from tropical fruits are generally accepted and their market is
growing. Nonetheless, clear fruit juices from apples, grapes, oranges, etc. are still the
preferred choice of the consumers as they have appealing texture. Therefore, the
enzymatic application on such hazy or cloudy juices becomes important for improv-
ing their texture and ultimately increasing consumer acceptability in the market. In
order to overcome this issue, clarification process is conducted for the degradation of
pectins by the application of enzyme pectinases (Alkorta et al., 1998). Different
types of pectinases are used in this process and some names are pectin lyase, pectin
esterase, and polygalacturonase (Kant et al., 2013). But, the pectinolytic enzymes
from A. niger are commonly used in fruit and vegetable processing to improve the
texture of the final products (Dinu et al., 2007).
Transglutaminases is another important enzyme which has been used extensively
for food texture and new product development in food processing industries. The
enzyme helps in the catalytic cross-linking reactions between proteins which transfer
the glutamine residue, namely, γ-carboxyamide of one protein to the ε-amino group
of lysine residue of the same protein or another protein (Nandakumar & Wakayama,
2015). This results in the improvement in food properties, such as firmness, elastic-
ity, viscosity, and water-binding capacity. The main microbial sources of enzymes
for improving the texture in food are Bacillus sp., Streptomyces sp., and Corynebac-
terium sp. (Zhang et al., 2012; Placido et al., 2008). Glutaminase is another enzyme
that helps in texture by improving the protein properties, thereby increasing its
solubility, gelation, emulsification, etc. (Nandakumar & Wakayama, 2015).
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 79

3.4.3 Enzymes in Flavour and Aroma Production

Taste and flavour are the two major attributes in determining the overall quality of
various fruits and vegetables. Taste is determined by the contents of sugar, tannins,
phenols, organic acids, and other compounds. Analysis of flavour compounds has
given us an inclusive knowledge on the chemical compounds responsible for flavour
sensations of fruits and vegetables. However, different enzymes have multiple
effects on these attributes of fruits and vegetables. Enzymes such as peroxidases
and lipoxygenases are responsible for off-flavour formation in fruits and vegetables
(Bhowmik & Dris, 2004). The enzyme peroxidases are responsible for deterioration
in colour, flavour, texture, and loss in nutritional qualities in raw or processed fruits
and vegetables. The off-flavour is often linked with the oxidation of phenolic
compounds and indigenous lipids. It was observed that enzyme inactivation was
achieved at 70  C with 15 min exposure time (Sessa & Anderson, 1981). Therefore,
inhibition of such enzymes during processing is a must in order to retain or improve
the aroma and flavour.
Another enzyme that has a major effect in flavour and aroma is lipogenase. This
enzyme produces free radicals and conjugated unsaturated hydroperoxy acids by
catalytic oxidation of polyunsaturated fatty acids. Then, these free radicals interact
with other constituents like proteins, vitamins, phenolics, etc. present in fruits and
vegetables which helps in enhancing the flavour and aroma of the product. However,
these aromatic compounds may produce off-flavours in Brassicaceae family.
According to the reports of Sheu and Chen (1991), increase in colour losses and
development of off-flavour were observed in broccoli and asparagus during storage
of non-blanched and under-blanched products.
Furthermore, the volatile compounds such as aldehydes, alcohols, ketones, esters,
lactones, etc. are related to flavour and aromatic characteristics in foods (Beaulieu &
Baldwin, 2002). For example, the compounds present in alcohols and aldehydes are
extensively used as food additives due to the aroma referred to as ‘green touch’, a
characteristic observed in freshly harvested fruits (Schwab et al., 2008). These
volatile compounds are synthesized using various substrates like amino acids, fatty
acids, and carotenoids (Goff & Klee, 2006). The main enzymes involved in the
synthesis of volatile compounds from fatty acids are lipoxygenase, alcohol dehydro-
genase, hydro peroxide lyase, and (3Z ): (2E)-enal isomerise (Schwab et al., 2008).
These enzymes are used in the extraction from fruits and vegetables such as banana,
soy, tomato, and olive where they subsequently react with fatty acids to produce
volatile compounds (Akacha & Gargouri, 2009).
In order to improve the taste and flavour in citrus fruits, enzymes limoninase and
naringinase can be enzymatically tailored by degrading the bitter taste compounds
such as limonin and naringin (Ribeiro et al., 2010). The formation of limonin can
also be prevented by using limonoate dehydrogenase, as it catalyses the oxidation of
its precursor lactone-A-ring to 17-dehydrolimonoate, a non-bitter derivative which
cannot be changed into limonin (Merino et al., 1997). Bitter compounds in citrus
fruits can be reduced by using adsorbing polymers, such as Amberlite XAD-16HP
and Dowex Optipore L285 resins. Besides acting as a debittering agent, Dowex
80 M. D. Heirangkhongjam et al.

Optipore L285 can also induce other modifications in juice processing like reduction
of total titratable acidity (TTA), increasing total soluble solids (TSS), the ratio of
TSS to TTA, pH, etc. (Kola et al., 2010).

3.5 Application of Enzymes in Fruit and Vegetable Processing

The significance of enzymes and its application in food processing industry is

increasing rapidly. Different kinds of enzymes are used extensively based on their
effective application. In fruits and vegetables processing as well, several endogenous
enzymes and newly developed enzymes are used because of the following
advantages:

• Low temperature requirements

• Low energy requirements during processing
• Increased product yield especially in juice processing. They make the juice stable
without the addition of additives
• Less by-product formation during processing
• Improved product quality (Grassin & Coutel, 2009)

In the following paragraphs, detailed analyses are discussed on the use of

enzymes in different fruit and vegetable processing techniques.

3.5.1 Fruit Firming

Texture is one of the important quality attributes of fruits and vegetables (Pan et al.,
2014; Grassin & Coutel, 2009; Guillemin et al., 2008; Jensen et al., 2004; Degraeve
et al., 2003). However, the texture of processed fruits and vegetables, especially the
softer ones, such as strawberry, raspberry, and tomatoes, is adversely affected by
thermal processing treatments like blanching, sterilization, freezing or pasteuriza-
tion, and other mechanical method which may result in softening (Grassin & Coutel,
2009; Guillemin et al., 2008; Degraeve et al., 2003).
The texture and structural integrity of fruits and vegetables depend on the
composition of cell walls present in them. These cell walls are composed of an
interlinked fibrous structure of cellulose embedded in a matrix of pectin,
hemicelluloses, and celluloses (Guillemin et al., 2008; Sila et al., 2008). The enzyme
pectin methylesterase (PME) bound to the cell wall is a pectin degrading enzyme
which results in demethoxylation of pectin (de-esterification of the methylated
carboxy groups of polygalacturonic pectin), releasing methanol and forming
carboxylated pectin. This carboxylic acid is said to interact with calcium, resulting
in firmness of fruits by strengthening the cell wall (strong pectate network with
added calcium) (Pan et al., 2014; Sila et al., 2008; Guillemin et al., 2008; Degraeve
et al., 2003). This process is known as chelation, and it helps to overcome the
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 81

adverse effects of thermal and mechanical processing treatments resulting in reduced

fruit damage and disintegration (Grassin & Coutel, 2009; Degraeve et al., 2003).
Studies were conducted as early as 1965 and 1972 to assess the role of PME in
increasing the firmness of canned tomatoes and potatoes, respectively. In both the
studies, heat treatments such as blanching in case of tomatoes and preheating of
potato slices in water were used to assess the role of PME with firmness (Bartolome
& Hoff, 1972; Hsu et al., 1965).
As per a study conducted on peaches, vacuum infiltration of blanched peaches
with PME of citrus fruit for 2 h along with calcium chloride increased the firmness of
peaches (Javeri et al., 1991). Another study also demonstrated that vacuum
impregnated PME (Aspergillus niger) along with calcium chloride increased the
firmness in pasteurized fruits, namely, apples, strawberries, and raspberries
(Degraeve et al., 2003). Similarly, a study demonstrated enzymatic firming of red
pepper by using exogenous pectinesterase (Rheozyme from Novozyme A/S) and
retention of texture even after freezing or heating (Jensen et al., 2004). Thus, many
fruits and vegetables may be processed by using PME and calcium, thereby retaining
the texture of the products (Grassin & Coutel, 2009).

3.5.2 De-Skinning (Enzymatic Peeling)

De-skinning means removal of peel/skin of fruits and vegetables. It was initially

done mostly by different methods such as manually, chemical, steam, mechanical, or
freeze method. But, these methods involved high labour cost, high water consump-
tion, and peeling losses (Toushik et al., 2017; Pretel et al., 2007a, 2010). Now,
considering their disadvantages, these methods have been replaced almost by enzy-
matic peeling in processing industry. In this method, the product is infused in
enzyme preparation of pectic substances such as pectinases, hemicellulases, and
cellulases (Berry et al., 1988; Bruemmer et al., 1978). Once infused, the vacuum is
applied which helps the enzymatic solution to enter the intercellular spaces (Pretel
et al., 2008). In the process, cellulase releases pectin from albedo, while pectinases
break down the cell wall by hydrolysis of polysaccharides (Ben-Shalom et al., 1986).
Nowadays, many commercial enzymatic preparations are available and pectolytic
preparation (mixture of pectinases, cellulase, and hemicellulase) is one of them. It is
obtained from Aspergillus species fungi. It has been observed that the enzymatic
solution easily diffuses when there are large intercellular spaces in albedo, resulting
into easy peeling (Baker & Bruemmer, 1989). However, many studies have found
that the efficiency of enzymatic peeling effect depends on the treatment methods
such as heat (scalding), vacuum infusion, temperature, and pH (Pagán et al., 2005;
Suutarinen et al., 2003; Toker & Bayιndιrlι, 2003; Rouhana & Mannheim, 1994).
Details of some recent studies are given in Table 3.3.
Furthermore, the role of enzymes in the peeling process of vegetables such as
potato, carrot, onion, and Swedish turnips was examined by Suutarinen et al. (2003).
It was observed that enzymes did help in peeling of carrot and onions; however,
potato and Swedish turnips could not be peeled properly due to the presence of cutin
82 M. D. Heirangkhongjam et al.

Table 3.3 Studies assessing role of enzymes in enzymatic peeling

Pre-
Fruit/ processing
Reference vegetable condition Enzymatic solution Result
Prakash Grapefruit Scalding, Polygalacturonase, Peeling improved
et al. 1–4 min cellulase and pectin
(2001) methylesterase, incubated
for 12 min
Suutarinen Potato, Cellulase, Difficulty in
et al. carrot, polygalacturonse removing peel of
(2003) Swedish potato and Swedish
turnip, turnips
onion
Liu et al. Mandarins Pectinase, Peeling efficiency
(2005) polygalacturonse, and improved
cellulase, 20 min
Pretel et al. Orange, Hot water Pectinase, Peeling improved
(2007b) Thomson, bath polygalacturonse, and
and Mollar (45  C) cellulase, 40 min
Pretel et al. Orange, Hot water Pectinase, Peeling improved
(2007a) Sangrina bath polygalacturonse, and
(45  C) cellulase, 30 min

or suberin in vegetable surface (Suutarinen et al., 2003). As per a study by Toker and
Bayindirli, it was observed that the enzymatic peeling was easier at relatively
moderate temperatures such as 44–47  C for nectarines, 41–46  C for peaches,
and 45  C for apricots with a pH ranging from 3 to 4.1 (Toker & Bayιndιrlι,
2003). The steps involved during enzymatic peeling are illustrated in Fig. 3.1.
The first step is selection of fruits or vegetables and washing them with water. At
times, hot water treatment or scalding is done prior to enzymatic peeling to improve
the peeling process and have a high quality end product (Pretel et al., 2008, 2010). A
study on grapefruit as early as 1974 demonstrated dipping the fruit in a water bath of
60  C for 30–35 min resulted in a good quality product (Bruemmer et al., 1978). The
advantage of scalding is that it reduces the viscosity of pectin as well as enhanced the
ability of the peel to absorb the enzyme solution, thereby helping in enzymatic
peeling process (Pretel et al., 2008) (Fig. 3.1).
Therefore, enzymatic peeling has wide range of applications in fruits and
vegetables such as grapefruit, orange, mandarin, apricot, peaches, potato, carrot,
and Swedish turnips (Suutarinen et al., 2003; Toker & Bayιndιrlι, 2003). This
method is a significant alternative in food processing industry because of its
advantages in improving the quality of products, reducing wastage, minimal water
use, reduction in contamination, and being cost-effective (Toker & Bayιndιrlι,
2003).
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 83

Fig. 3.1 Steps in enzymatic peeling. (Adapted from Pretel MT, Sánchez-Bel P, Egea I & Romojaro
F. (2010). Enzymatic peeling of citrus fruits. In Bayindirli (Ed.), Enzymatic processing of fruits and
vegetables: Chemistry and engineering applications (pp. 145–174). CRC Press, Taylor and Francis
Group)

3.5.3 Application of Enzymes in Fruit and Vegetable Juice

Processing

The natural liquid substance contained in fruits and vegetables is commonly known
as juice. It can be obtained or prepared directly by extraction, pressing, or diffusion
from fruits or vegetables and such juices are meant for direct consumption. They can
be broadly categorized as juices without pulp (clarified/cloudy) or juices with pulp
such as purees, nectars, and pulps (Ceci & Lozano, 2010; Cautela et al., 2010). In
juice processing, different enzymes are extensively used in fruit and vegetable for
higher yield, clarification, and improvement in filtration, resulting in higher quality
of juices (Fleuri et al., 2016). Some of the commonly used enzymes in fruit and
vegetable juice processing are pectinases, hemicellulases, and cellulases. In juice
processing of fruits and vegetables, multiple steps are involved and below are the
standard procedures. The steps involved in fruit juice processing are also illustrated
in Fig. 3.2.

3.5.3.1 Selection of Fruit, Followed by Washing and Peeling

Enzymatic preparations can be used for peeling of fruits such as oranges, lemon, or
vegetables such as pumpkin and beet (Toushik et al., 2017) as discussed in Sect.
3.4.2. This is followed by juice extraction.
84 M. D. Heirangkhongjam et al.

Fig. 3.2 Steps in juice processing. (Adapted from Fleuri, L. F., Delgado., C. H. O., Novelli, P. K.,
Pivetta, M. R., do Prado, D. Z., & Simon, J. W. (2016). Enzymes in food and beverage processing
(pp. 255–280). CRC Press, Taylor and Francis Group)

3.5.3.2 Juice Extraction

Juice can be extracted by a number of methods—centrifugation, diffusion, extrac-
tion, and ultrafiltration. Enzyme preparations help in breakdown of cell wall and
release the juice contained within the structure in fruits such as apples, grapes,
berries, pears, and citrus fruits (Ribeiro et al., 2010). Among apples, the activity of
endogenous enzymes is low; therefore, commercial pectinases from Aspergillus
species are added so they can be macerated before pressing (Aehle, 2007). The
extracted juice is pasteurised for microbial stability (Grassin & Coutel, 2009). A
study on processing of blackberry juice observed that application of pectinolytic
enzymes during pressing resulted in an increase in juice extraction as high as 81.73%
in three cultivars as compared to 53.79% in the control arm (Granada et al., 2001).
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 85

Another important aspect in juice processing is the discolouration of juices during

pasteurization due to presence of anthocyanins. This issue can be prevented by the
use of β-glucosidase which hydrolyses anthocyanins into anthocyanidins, thereby
minimizing discolouration due to its low colour intensity and solubility (Villena
et al., 2007; Palma-Fernandez et al., 2002).

3.5.3.3 Clarification and Fining

Turbidity in juices is due to scattering of light caused by insoluble substances such as
cell fragments that come from pulpy tissue (pectin from cell walls) in suspension. It
is usually undesirable except when present in citrus fruits (Ribeiro et al., 2010).
Pectin is the cause of turbidity in juices especially apple juice (Aehle, 2007). Another
reason for the haziness in extracted juices is due to presence of starch in unripe fruits
(Ceci & Lozano, 2010). At this stage, the role of enzymes becomes critical as
enzymes like pectinase help in reducing turbidity by attracting pectin and forming
clusters (aggregation of cloud particles). Then, these particles usually settle down
and can be easily removed by filtration or centrifugation (Uneojo & Pastore, 2007).
Apart from enzymes, flocculating or fining agents such as gelatin and bentonite may
be added to aid in clarification process (Ceci & Lozano, 2010). Similarly, citrus
juices, especially lemon juice, are also clarified by pectinolytic enzyme preparations
(Fleuri et al., 2016).
Apple juice contains about 15% of starch which results in turbidity, slow filtration
rate, and gelling after concentration. The addition of amylase along with pectinase
hydrolyses this starch and removes the haze or cloudiness (Ceci & Lozano, 2010;
Carrín, 2004). Sometimes, haziness occurs in juice, so adding of fining agents can
prevent post-bottling haziness, thereby optimizing fining and ultrafiltration process.
Similarly, araban present in certain fruits may be responsible for post-concentration
haze in juice, and to prevent such haze formation, arabanase is added to the juice
(Ceci & Lozano, 2010).

3.5.4 Role of Enzymes in Wine Processing

The role of enzymes in the production process of alcoholic beverages such as

brewing of beer and winemaking is very important (Toushik et al., 2017; Gómez-
Plaza et al., 2010; Aehle, 2007). Different enzymes, namely pectinases, amylases,
glucanases cellulases, glycosidases, lyzozymes, and ureases, are used. They help in
improving the quality and yield of the product (Toushik et al., 2017; Gómez-Plaza
et al., 2010; Aehle, 2007).

3.5.4.1 Enzymes Used in Processing of Wine

Wine is a type of alcoholic beverage made from fermentation of fresh grapes
(Bruchmann & Fauveau, 2009). Pulp and skin of the grapes account for about
75–85% and 6–7%, respectively. The cell wall of grapes is composed of
hemicelluloses, pectin, and structural proteins like chitinase. Also, the cellulose
and pectin present in cell walls provide rigidity to the grapes, thus hindering juice
86 M. D. Heirangkhongjam et al.

Fig. 3.3 Steps in wine-

making. (Adapted from
Fleuri, L. F., Delgado.,
C. H. O., Novelli, P. K.,
Pivetta, M. R., do Prado,
D. Z., & Simon, J. W. (2016).
Enzymes in food and beverage
processing (pp. 255–280).
CRC Press, Taylor and
Francis Group)

extraction, clarification, and filtration (Claus & Mojsov, 2018). Moreover, the skin
and cell walls of grapes contain several essential compounds such as anthocyanins
and tannins. These compounds are responsible for colour and structure of wine
(Gómez-Plaza et al., 2010; Bruchmann & Fauveau, 2009).
The natural enzymes present in grapes and those obtained from yeast, fungi, and
bacteria collectively play an important role in wine making process (Gómez-Plaza
et al., 2010). Usually, the manufacturers extend the action of endogenous enzymes
by adding exogenous enzymes during production (Gómez-Plaza et al., 2010). The
commonly used commercial enzymes include pectinases, glucanases, glycosidases,
lysozymes, and ureases (Gómez-Plaza et al., 2010). The application of these exoge-
nous enzymes and yeast strains during processing increases the rate of production,
yield, and quality of the wine (Toushik et al., 2017).
The standard steps in wine processing are illustrated in Fig. 3.3.
These steps can be broadly categorized in three phases, namely pre-fermentation,
fermentation, and post-fermentation.

Pre-fermentation
This phase involves crushing the fruit and extraction of juice. In red wine prepara-
tion, the skin is not separated, while it is separated in white wine. During the
processing of red wine, pulp, skin, and seeds of grapes are kept together after
crushing and during fermentation in order to extract colour and flavour
(Byarugaba-Bazirake, 2008). The colour of red wine is due to the presence of
anthocyanins and tannins in grape skin and seed (Gómez-Plaza et al., 2010). In
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 87

this phase, exogenous enzymes derived from Aspergillus species such as pectinases,
cellulases, and hemicellulases (xylanases and galactanases) are employed, which
help in expediting the extraction of juice and colour control (Espejo, 2020; Toushik
et al., 2017; El Darra et al., 2016; Kelebek et al., 2007; Bautista-Ortίn et al., 2005).
These macerating enzymes also modify the stability, taste, structure, and mouth feel
of red wine (Gao et al., 2019).
Furthermore, maceration of the cell walls by pectinases results in the liberation of
liquid and other phenolic compounds (Espejo, 2020; Gao et al., 2019). It has been
observed in different studies that wines prepared by enzyme treatment tend to have
phenolic and tannin content, as well as colour intensity (Romero-Cascales et al.,
2008; Revilla & Gonzalez-San Jose, 2003). Sometimes, maceration is accompanied
by pressing (wine press extract must or wine from crushed grapes), resulting in better
yield (Gómez-Plaza et al., 2010). Another important attribute in wine making is its
aroma; reductases such as glycosidases and polyphenol reductase help in aroma
extraction and polyphenol reduction, respectively (Espejo, 2020). Glycosidases
improve the aroma of wines by releasing aromatic compounds from their
non-aromatic precursors or non-odouriferous sugar compounds (Zhu et al., 2014).
As per a study, treatment of Albarino wine with pectinolytic preparations and
glycosidase resulted in an improved aroma (Armada et al., 2010). However, in
industrial processing, usually exoglycosidases and β-glucosidase from Aspergillus
niger are used (Gómez-Plaza et al., 2010).

Fermentation
In this phase, yeast is added to the must in red wine to initiate fermentation
(Byarugaba-Bazirake, 2008). During the process, the sugars present in the juice
are converted into alcohol and release carbon dioxide. In this period, pectin plays an
important role by preventing the diffusion of intercellular components such as
phenolic and aroma compounds into the must (Claus & Mojsov, 2018; Bruchmann
& Fauveau, 2009).

Post-fermentation
This phase involves different steps such as clarification, filtration, and microbial
stabilization with the help of different enzymes. Proteases are used at the time of
clarification, while pectinase and β glucanase are used during filtration and lysozyme
for microbial stabilization (Espejo, 2020). After pressing, the grape must is quite
turbid and is rich in solid particles (Armada & Falqué, 2007). Thus, clarification is an
important step to improve the quality of wine, especially in white wine (Gómez-
Plaza et al., 2010). Moreover, activated carbon, bentonite, and pectinase may be
added to aid in clarification or physical removal of suspended matter in the must
(Gómez-Plaza et al., 2010). Commercial enzymes that help in clarification are:

(a) Pectin lyase which results in destabilization of the cloud, thereby reducing
viscosity.
(b) Pectin methylesterase (PME) results in demethylation of pectin, aiding
polygalacturonase in hydrolysis of pectin and leading to cloud flocculation
88 M. D. Heirangkhongjam et al.

Table 3.4 Enzymes and their roles in wine processing

Step Enzyme Action
Clarification Pectinase Removal of suspended particles
Maceration Pectinases, Degradation of pectin
hemicellulase,
cellulase
Maturation Pectinases and Ageing results in mouth feel and is done at the end
glucanases of fermentation
Filtration Pectinases and Glucanases are applied to the must to shorten the
glucanases glucan chains preventing problems during
filtration
Addition to wine/ Urease Elimination of urea and preventing the formation
must of ethyl carbamate
Addition to wine/ Lysozyme Elimination of bacterial
must
Late phase of Beta glucosidase Increased aroma by splitting glucose residue from
fermentation (white odourless precursors
wine)
Stabilization/haze Proteases Stabilization of wine, reducing betonite demand
formation
Note: Adapted from Gómez-Plaza et al., 2010. Use of enzymes in wine production. In Bayindirli
(Ed.), Enzymatic processing in fruits and vegetables: Chemistry and engineering applications
(pp. 215–243). CRC Press, Taylor and Francis Group

and clarification of must (Gómez-Plaza et al., 2010; Bruchmann & Fauveau,

2009).

Furthermore, during ageing or maturation, cellulases and β-glucanases are used to

improve the mouthfeel (Espejo, 2020; Gómez-Plaza et al., 2010). Other than the
above mentioned advantages of the macerating enzymes, studies have also observed
that pectinases are found to improve haze condition, increase pressability of grapes
to reduce filtration time, and increase must volume (Sharma et al., 2017; Garg et al.,
2016). Table 3.4 illustrates different enzymes and their actions during wine
processing.
Other important enzymes used in commercial wine production are cinnamyl
esterase, cinnamyl decarboxylase, glucanase, urease, and lysozyme. The yeast-
derived enzymes, cinnamyl esterase and cinnamyl decarboxylase, may be present
in commercial pectin preparations as a side activity and can cause off-flavours due to
volatile vinyl phenols (Bruchmann & Fauveau, 2009). At times, grapes infected with
Botrytis cinerea result in formation of glucans. These glucans prevent the natural
sedimentation of cloud particles and cause filtration problems as well. Thus,
glucanase enzyme is added to prevent the development of such glucans during
wine making process (Gómez-Plaza et al., 2010). Urease is another enzyme that is
added occasionally during wine preparation. This enzyme hydrolyses urea into
ammonia and carbon dioxide and prevents the formation of ethyl carbamate in
wine which is carcinogenic at high concentration (Gómez-Plaza et al., 2010).
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 89

Lysozyme is used in winemaking to prevent or delay malolic fermentation and

sulphur dioxide is added to the must or wine (Gómez-Plaza et al., 2010).

3.6 Effect of Processing Methods on Enzymes and Vegetables

Enzymes present in fruits and vegetables play a huge role in determining the texture,
colour, flavour, and taste attributes of the processed products (Oey, 2010). The
continued enzymatic activity in fruits and vegetables affects the storage quality,
shelf life, and palatability of the product. Therefore, several processing methods such
as grinding, crushing, slicing, juices, or preservation (pickling) are used to prolong
the shelf life and reduce wastage of fruits and vegetables (Oey, 2010). Hence, it is
very important to control the stability and activity of endogenous enzymes present in
fruits and vegetables during food processing (Oey, 2010).
Apart from the conventional techniques of processing such as blanching, heating,
or freezing, new and highly advanced processing techniques like high hydrostatic
pressure (HHP), pulse electric field (PEF), and ultrasound have been introduced
successfully. The major advantage of these new techniques is the use of non-thermal
technology, which helps in retaining the sensory attributes and nutritional content of
the product (Oey, 2010; Jaiswal & Sharma, 2016). In HHP technique, it employs
high pressure range of 100–600 MPa, resulting in enzyme and microbial inactivity
which may affect the shelf life of the products (Briones-Labarca et al., 2015). In PEF
technique, being a non-thermal processing, it uses a series of short and high voltage
pulses to inactivate microbes or enzymes (such as peroxidases and polyphenol
oxidases) in food (Segovia et al., 2015). However, in ultrasound processing tech-
nique, high frequency short pulses are used for inactivation (Jaiswal & Sharma,
2016). These non-thermal processing techniques are effective at ambient or
sub-lethal temperatures and minimize the adverse thermal effects on the nutritional
content and quality of fruits and vegetables (Tiwari et al., 2009).
However, these non-thermal processing techniques still have several drawbacks,
such as high equipment and processing cost, tedious to operate, hazardous, requires
stringent process control operations, etc. (Jaiswal & Sharma, 2016). Nonetheless,
multiple advantages are rendered by these techniques in food processing industry
over conventional thermal techniques, which affect not only the enzymes, but also
the texture, taste, and colour of the product compelled for further investigation and
improvement (Oey, 2010).

3.6.1 High-Hydrostatic Pressure (HHP) Processing

HHP, also known as High-Pressure Processing (HPP), is based on the Le Chatlier

principle, i.e. pressure is equally applied in all directions (Augusto et al., 2018;
Terefe et al., 2016; Keenan et al., 2012). As the name implied, this technique uses
very high pressure for application of uniform pressure to a product or food, leading
to the inactivation of certain microbes and enzymes in the food (Augusto et al.,
90 M. D. Heirangkhongjam et al.

2018). This technique can be applied in solid, semi solid liquid, or particulate food
products (Augusto et al., 2018).
The high pressure (HP) may increase the shelf life of fruits and vegetables,
especially when combined with temperature by resulting in enzyme activity or
inactivity and pressure-induced gel formation (Guerrero-Beltrán et al., 2005). In
HHP technique, enzyme inactivation may be affected by the type of food, pH,
temperature, and duration of treatment (de Castro Leite Júnior et al., 2017;
Guerrero-Beltrán et al., 2005).
The HHP unit consists of a pressure vessel, pressure generator, temperature
control, and pressure handling system. The HHP processing can be of two types—
batch (closed vessel system) or semi-continuous (number of closed vessels). In this
process, the hydrostatic pressure at a given point is transformed rapidly and uni-
formly in all directions. The intensity of HHP is determined by the process
parameters like pressure, treatment duration, and temperature. The entire HHP
processing cycle takes a few minutes and it can be performed at pressures as high
as 1400 MPa and temperatures between less than 0–150  C (Oey, 2010) (Augusto
et al., 2018; Oey, 2010). The maintenance of a product inside a vessel at a high-
pressure results in molecular changes affecting the enzymes (de Castro Leite Júnior
et al., 2017).
Several studies have been conducted to determine the effect of HHP (at times
combined with thermal processing) on enzyme activity in various fruits and
vegetables as shown in Table 3.5. Enzymes like PME, POD, and PPO are considered
baroresistant with POD and PME being the most and least resistant, respectively
(Augusto et al., 2018). Hence, some studies have found that PPO and POD are not
affected substantially by HHP (Augusto et al., 2018).

3.6.2 High-Pressure Homogenization Processing (HPHP)

HPHP, also called Ultra High-Pressure Homogenization, is a non-thermal

processing technique, especially used in fruit juice processing. In this method, the
fluid is pressurized with high or ultra-high pressure to flow through a narrow valve
and the shear stress distribution across the product helps to inactivate microbes and
enzymes present in the fluid (Augusto et al., 2018; de Castro Leite Júnior et al.,
2017). It is a technique which can be used only in fluids. HPH is a continuous
process and is often called dynamic high-pressure processing (Augusto et al., 2018;
de Castro Leite Júnior et al., 2017). The high shear and sudden drop in pressure,
turbulence, and caviation result in changes in processed foods (de Castro Leite
Júnior et al., 2017).
In HPH, factors such as enzyme structure, fluid, equipment, media, and process
parameters determine its effect on enzyme activity (Augusto et al., 2018). To
ascertain its efficacy, HPH was used to treat oranges and PME activity was measured
at five pressures (0–250 MPa) and three inlet temperatures (22, 35, 45  C). A
reduced activity of 70% can be achieved at 45  C and 250 MPa (Welti-Chanes
et al., 2009). Similarly, HPH has also been found to result in complete inactivity of
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 91

Table 3.5 Effect of HHP on enzyme activity

Fruits/ Enzyme stability after
Reference vegetables Enzyme Processing application of HHP processing
Andreou Tomato PME 800 MPa, 15 min, 70% reduction in enzyme
et al. juice 65  C activity
(2016) PG 500 MPa, 10 min, Complete inactivation of enzyme
55  C activity
Terefe Pear POD 600 MPa, 3 min, Enzyme activation by 23% at
et al. 40–100  C 40  C, while reduction by 92% at
(2016) 100  C
PPO 600 MPa, 3 min, Reduction in activity by 90%
100  C
PME 600 MPa, 3 min, Reduction in activity by 83%
100  C
Marszałek Strawberry POD 500 MPa, 15 min, Reduction of about half of
et al. puree 50  C enzyme activity
(2015) PPO 500 MPa, 15 min, Reduction of 72% of enzyme
50  C activity
Castro Green bell POD 100–200 MPa, 70% of reduction in enzyme
et al. pepper 10 and 20 min, activity
(2008) 18–20  C
Red bell POD 100–200 MPa, 40% of reduction in enzyme
pepper 10 and 20 min, activity
18–20  C
Green bell PPO 100–200 MPa, 50% reduction in enzyme
pepper 10 and 20 min, activity
18–20  C
Red bell PPO 100–200 MPa, No effect
pepper 10 and 20 min,
18–20  C

the enzyme pectate lyase at pressures more than 150 MPa (Calligaris et al., 2012).
Therefore, it is not recommended to use HPH independently and preferably; it needs
to be coupled with other processing methods for microbial or enzyme inactivation
(Augusto et al., 2018; de Castro Leite Júnior et al., 2017; Calligaris et al., 2012;
Welti-Chanes et al., 2009).

3.6.3 Ultrasound Processing (Ultrasonication)

In ultrasound processing technique, ultrasonic waves are employed with frequencies

above the hearing range of humans. The ultrasonic waves are of two types: high
frequency ultrasound (2–20 MHz) corresponding with low sound intensity
(0.1–1 W/cm2) and power ultrasound (20–100 kHz) with high sound intensity
(10–1000 W/cm2) (Feng et al., 2008). These techniques are used in processing
industry for improving the shelf life of fruits and vegetables by inactivating the
enzymes like PME, PPO, and POD present in the food (Bourke et al., 2010). But,
92 M. D. Heirangkhongjam et al.

when the technique, high power ultrasound with low frequency, is combined with
temperature/heat, the process is known as thermosonification or pressure
(manosonification) (Bourke et al., 2010). The combination of heat and ultrasound
helps to ensure product stability and inactivation of enzymes and microbes, thereby
resulting in retaining the quality and extending the shelf life of fruits and vegetables,
especially juices (Rojas et al., 2017; Saeeduddin et al., 2015). The inactivation of
microorganisms or enzymes in this technique may be due to physical factors such as
caviation or mechanical effects (Bourke et al., 2010; O’Donnell et al., 2010).
Ultrasonic processing in fruits and vegetables focuses on the inactivation of
endogenous enzymes which are more resistant to heat treatments (Feng et al.,
2008). Studies have It has been observed that ultrasonic processing either in combi-
nation with heat or pressure has minimal effect on quality of fruit juices such as
orange juice, guava, and strawberry juice (Bourke et al., 2010). Further, it has been
observed that high ultrasound and longer processing times may be required for
enzyme inactivation as the pulp complicates the inactivation process (Rojas et al.,
2017). Hence, ultrasound technique is usually combined with a thermal process to
increase the rate of inactivation of enzymes in juices (Chen et al., 2019; Rojas et al.,
2017; Anaya-Esparza et al., 2017; Abid et al., 2014). PME was also found to be
inactivated in lemon juice (Knorr et al., 2004) and mousambi (Siwach & Kumar,
2012) by using ultrasonic processing along with heat. Likewise, peroxidase enzyme
present in watercress was also found to be inactivated by the use of thermosonication
(Cruz et al., 2006).

3.6.4 Pulsed Electric Field (PEF)

In PEF processing technique, electrochemical effects and ohmic heating are respon-
sible for changing the structure and function of enzymes present in the food. At
times, large specific energy inputs are required to inactivate enzymes (Poojary et al.,
2017). Enzyme activity can be affected by a number of factors such as properties of
the enzyme, treatment parameters, processing conditions as well as the condition of
the medium (Poojary et al., 2017).
The PEF unit consists of a treatment chamber, pulse generator, a pulse monitoring
system, and a temperature monitor. It can be conducted in two ways—continuous or
batch processing. The unit also contains a fluid handling system for liquid foods in
continuous PEF processing (Min et al., 2007). The design of the treatment chamber
plays a key role in distribution of uniform temperature inside the PEF chamber (Oey,
2010). In PEF, the duration of processing takes substantially lesser time (micro to
milli seconds) as compared to other methods (Oey, 2010).
However, in PEF processing, the activity on enzyme may either limit its activa-
tion/inactivation or may not be affected at all (Poojary et al., 2017). In this process,
multiple factors such as food matrix, pH, enzyme dissolving medium, and certain
treatment conditions affect the enzyme treatment in fruits and vegetables (Poojary
et al., 2017; Oey, 2010). Two important parameters that affect the intensity of PEF
processing are electric field intensity and the total duration of treatment per energy
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 93

(Oey, 2010). A higher electric field and treatment duration may result in enzyme
inactivation, while a lower electric field and duration may result in enzyme activa-
tion (Poojary et al., 2017). Also, increasing pulse width lowers enzyme activity
(Poojary et al., 2017).
Furthermore, several studies have been conducted to assess the effect of PEF
processing on various food enzymes such as polyphenol oxidase, lipoxygenase,
peroxidase, and pectin methyl esterase (Poojary et al., 2017; Aguiló-Aguayo et al.,
2009; Noci et al., 2008; Espachs-Barroso et al., 2006) and are given in Table 3.6.
The findings from these studies indicate that a longer treatment duration, higher
electric field intensity, and pulse width result in enzyme inactivation.

3.6.5 Thermal Processing

In spite of advancement in alternative non-thermal treatments in food processing

industry, thermal treatments are still widely considered the most cost-effective and
simple method to ensure enzymatic and microbial inactivity in foods (Rawson et al.,
2011). However, these treatments have many drawbacks as well, such as slow in
conduction and convection of heat transfer, loses in nutritional and functional
properties, reduction in sensory attributes, etc. (Baysal & Icier, 2010; Gonzalez &
Barrett, 2010). In addition, food matrix, complexity of products, and the microor-
ganism also affect the efficacy of thermal processing (Chen et al., 2013). As a result,
optimization of thermal treatment becomes important to preserve the sensory
attributes, nutritional quality, and safety of the product.
The common thermal treatments (conventional and non-conventional) employed
during processing of fruits and vegetables are High temperature-long time (HTLT),
High temperature-short time (HTST), and Ohmic and Microwave heating (Lee et al.,
2015). Brief explanations of these treatments are discussed below.
High temperature-long time (HTLT) is a conventional thermal treatment. In this,
the processing of juice and beverages is done at temperature 80  C with >30 s
holding time (Miller & Silva, 2012). It inactivates or reduces the enzymatic activities
of polyphenoloxidase (PPO), pectin esterase (PE), peroxidase (POD), etc.
(Marszałek et al., 2017). During the treatment, it was observed that antioxidant
activity and PME enzymatic activity were reduced to 75% and 83% in mombin juice
and litchi beverage, respectively (Swami Hulle & Rao, 2016; de Carvalho et al.,
2015).
High temperature-short time (HTST) is another conventional thermal treatment
used in food processing. It is carried out at temperature 80  C with 30 s holding
time. Here, priority is given for destruction of microorganism than nutrient degrada-
tion (Achir et al., 2016). For example, HTST treatment reduces PME and PPO
activity by 95.3% and 90.9% in apple juice, respectively (Aguilar-Rosas et al.,
2013). In another study based on apricot nectar, complete inactivation of POD,
PPO, and PME was observed (Huang et al., 2013).
Ohmic heating (OH) is a common non-conventional thermal treatment. This
treatment is primarily used in fruit juices processing for its effectiveness. Fruit juices
94 M. D. Heirangkhongjam et al.

Table 3.6 PEF and effect on enzyme activity

Enzyme stability
Fruits/ after application
Reference vegetables Enzyme Processing of HP processing
Aguiló-Aguayo Tomato Lipoxygenase Electric field: An increase in
et al. (2009) juice 35 kV/cm for frequency or
1000, pulse width: pulse width
1–7 μs, frequency: reduced enzyme
50–250 Hz activity
Strawberry Polygalacturonase Electric field: Enzyme activity
juice 35 kV/cm for inactivated by
1000 μs, 26%
treatment time:
1700 μs,
frequency:
100 Hz, pulse
width: 4 μs
Strawberry PME Electric field: Enzyme
juice 35 kV/cm for inactivation
1000 μs,
treatment time:
1700 μs,
frequency: 100 Hz
Noci et al. Apple juice PPO Electric field: About 50%
(2008) 40 kV/cm, pulse reduction in
width: 1 μs, enzyme activity
treatment time:
6000 μs,
frequency: 15 Hz,
no. of pulses: 100
Espachs- Carrot PME Pulses of PME
Barroso et al. Banana 13.2–19.1 kV/cm inactivation—
(2006) Tomato for 40 μs, time: 45% banana,
1.6 ms, frequency: 83% carrot, 87%
Orange
0.5–5 Hz orange and
tomato. Enzyme
inactivity
increased with
increased
treatment time
and electric field
and pulses

that contain water and ionic salts in higher amount are found to be more effective
with this treatment (Miller & Silva, 2012). In other foods, this process has many
advantages like uniform and rapid heating which ultimately helps in nutritional and
sensory attributes of the processed products. As per the study by Somavat et al.
(2013), B. coagulans (ATCC 8038) in tomato juice when treated with OH at 60 Hz
and 10 kHz expedites the inactivation process as compared to conventional treatment
(Somavat et al., 2013). However, microwave heating is another common
3 Role of Enzymes in Fruit and Vegetable Processing Industries: Effect. . . 95

non-conventional treatment which is mainly used in urban areas. Even though it is a

thermal treatment, minimal thermal exposure is required for enzymatic inactivation
(Arjmandi et al., 2016).

3.7 Regulatory Aspects of Food Enzymes Used in Fruit

and Vegetable Processing

The regulation of enzymes for its application in food industry is very complex and
differs from county to country. The European Union (EU) classifies food enzymes as
processing aids of food additives. Majority of the enzyme preparations used in food
processing are categorized as processing aids by European Union since they have a
role in the technological aspect of food processing stage and not in the final product
(Aehle, 2007). The processing aid may not be labelled (Bruchmann & Fauveau,
2009). The enzyme lysozyme used in wine processing is considered as a food
additive (Bruchmann & Fauveau, 2009) since it has a role to play in the final product
and is regulated under Food Additives Directive (95/2). A vertical legislation of EU
on fruit juices (Council Directive 93/77/EEC) allows pectinolytic, proteolytic, and
amylolytic enzymes, while another regulation (Council Regulations 82/87/EEC)
allows only pectinolytic enzymes on wines (Aehle, 2007). The Organization
Internationale de la Vigne et du Vin (OIV) regulates the enzymes used in wine
processing in EU (Gómez-Plaza et al., 2010; Bruchmann & Fauveau, 2009; Aehle,
2007) and has recognized the important role of pectinases, hemicellulases, cellulose,
β-glucanase, and glycosidase. The European Commission Regulation 1493/1999
authorises the use of pectinases from Aspergillus niger, β-glucanase from
Trichoderma harzanium and urease from Lactobacillus fermentum. Pectinases are
assigned a GRAS status by United States Food and Drug Administration
(Bruchmann & Fauveau, 2009). In India, Food Safety and Standards Authority of
India (FSSAI) regulates the use and safety of food enzymes. As per the regulation
Food Safety and Standards (Food Products Standards and Food Additives) (Amend-
ment) Regulations, 2015, the food regulator had permitted the use of processing aids
in bread. The enzymes permitted for usage are glucose oxidase, lipase, and xylanase.
They are obtained from various microbial sources.
In some EU countries, namely Denmark, France, and United Kingdom, approval
is required for the use of any food enzymes (Aehle, 2007). However, in countries
like Poland, China, Japan, Australia, New Zealand, Canada, Brazil, and Mexico,
approval is needed for the use of enzymes produced traditionally, especially if they
are new enzymes (Aehle, 2007). On the other hand, countries such as Thailand,
Korea, and Taiwan require registration prior to their enzyme use, while USA
requires a GRAS (generally regarded as safe) assessment, GRAS notice, or Food
additive (Aehle, 2007). Even though countries have different set regulations, enzyme
preparations must comply with specifications recommended by Joint FAO/WHO
Expert Committee on Food Additives (JECFA) and by Food Chemical Codex (FCC)
for food enzymes (Bruchmann & Fauveau, 2009; Grassin & Coutel, 2009).
96 M. D. Heirangkhongjam et al.

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Production of a, b, and g-Cyclodextrin
Gluconotransferase (CGTase) and Their 4
Applications in Food Industry

Rizul Gautam and Shailendra Kumar Arya

Abstract

Cyclodextrins (CD) are cyclic oligosaccharides which can be produced by the

enzymatic degradation of starch and have the ability to form inclusion complex
(IC) with variety of molecules. Formation of inclusion complexes with
cyclodextrins (CDs) is known to enhance guest solubility in aqueous medium.
CD has α-(1–4) linkages forming a cone shaped structure with a hydrophilic outer
surface which makes them water-soluble. The rate of hydrolysis increases in the
order of α-CD < β-CD < γ-CD. Gamma cyclodextrins have been proved to retain
the maximum retention properties than the three cyclodextrins. Cyclodextrins are
also used to retain colours of food substances. Three common types of CDs are
α-CD, β-CD, and γ-CD with 6, 7, and 8 sugar-membered ring molecules. This
book chapter will discuss the preparation and properties of three types of CDs
with their applications in food industry.

Keywords
α-Cyclodextrin · β-Cyclodextrin · γ-Cyclodextrin · Food industry · Applications

4.1 Introduction

Cyclodextrins (CDs) are a family of cyclic oligosaccharides that show the ability to
form inclusion complex (IC) through variety of molecules (like Allyl isothiocyanate
(AITC)) (Del Valle, 2004). These molecules are produced through the action of

R. Gautam · S. K. Arya (*)

Department of Biotechnology, University Institute of Engineering Technology, Panjab University,
Chandigarh, India
e-mail: skarya@pu.ac.in

# The Author(s), under exclusive license to Springer Nature Singapore Pte 107
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_4
108 R. Gautam and S. K. Arya

Fig. 4.1 Basic structure of alpha, beta, and gamma cyclodextrins (Radi & Eissa, 2010; Das et al.,
2013; Astray et al., 2009)

cyclodextrin glycosyltransferase (CGTase) enzyme on starch or starch derivatives.

This enzymatic activity sometimes results in changing the properties of many
molecules like their stability, reactivity, bioavailability, and solubility (Larsen
et al., 1998). On the basis of number of D-glucose units attached to CDs, they are
classified as six-α-cyclodextrin, seven-β-cyclodextrin, and eight-γ-cyclodextrin
(Blackwood & Bucke, 2000; Singh et al., 2002). Cyclodextrins cannot be formed
if less than six glucopyranose units used because of steric hindrances (Qi &
Romberger, 1988). Cyclodextrin possesses a hydrophobic central and hydrophilic
outside cavity. The α-(1–4) linkages of CD help in the formation of a cone-shaped
structure that results in its hydrophilicity. The central cavity of the cone is hydro-
phobic in nature because of the presence of carbon and oxygen atoms of the glucose
residues. This arrangement of a hydrophilic exterior with a hydrophobic interior
design of CDs helps cyclodextrins to make inclusion complexes or compounds with
hydrophobic guest molecules. α-, β- and γ-CD are 6, 7, and 8 sugar-membered ring
molecules, respectively, and are the most common CD types (Del Valle, 2004), as
shown in Fig. 4.1 (Del Valle, 2004; Radi & Eissa, 2010; Das et al., 2013).
Cyclodextrins have number of applications in food industry due to their ability of
encapsulations of molecules and formation of inclusion complex with hydrophobic
compounds (Gawande et al., 1998). Cyclodextrins are preferred due to the following
reasons: they are made from renewable raw material such as starch or starch
derivatives. They are relatively cheap. They are toxic-free and beneficial for
human body. Applications of cyclodextrins in food industry are expressed in many
different ways, like for encapsulation of flavours, used as food preservative, used as
an eliminator of bitter tastes from food, used against oxidative degradations, heat-
and light-induced changes, and for modification of taste and odours of foods (Astray
et al., 2009).
Among all the three cyclodextrins, gamma cyclodextrin proves to be more
suitable to be used in the field of pharmaceutical and food industries because of its
properties like water solubility, size of internal cavity, etc. (Li et al., 2007). However,
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 109

the most marketed utilized CD is β-CD and to lesser extent α-CD. The production of
gamma CD is less due to high cost and low production associated with it. The
CGTases producing cyclodextrin of single type are of interest as it is difficult to
isolate the CD of interest from a complex mixture of CDs. Cyclodextrins are
hydrophobic molecules and their solubility increases with ethanol concentration.
The spectroscopic studies imply that the confirmation of cyclodextrins in solution is
similar to that in crystalline state; however, β-CD has a perfect regularity, whereas
alpha and gamma are slightly distorted (Astray et al., 2009).
Cyclodextrins are useful because of following reasons: They are made from
renewable material (starch). They are relatively cheap. Due to their biodegradability,
they do not produce any harmful effect to the environment. They are nontoxic for
human body (Duca & Boldescu, 2008). The stability of the cyclodextrins follows
α > β > γ. The cyclodextrins are as stable as sucrose in solid state and are resistant to
degradation and can be stored for several years (Kurkovsergey &
Loftssonthorsteinn, 2013). These are homogenous, crystalline, and
non-hygroscopic substances (Stella & Rajewski, 1997). The alpha cyclodextrin
has six glucopyranose rings, beta has seven, and gamma has eight glucopyranose
units (Astray et al., 2009). The rate of hydrolysis accelerates in the order of
α-CD < β-CD < γ-CD. Gamma cyclodextrins have been proved to retain the
maximum retention properties than the three cyclodextrins. Cyclodextrins are also
used to retain colours of food substances (Duca & Boldescu, 2008; Lopez-Nicolas
et al., 2007a, b). The hydrophobic interior of the cavity possesses some lewis base
characteristics due to the presence of high electron density.

4.2 History

First time cyclodextrin was discovered by Villiers in 1891 from starch using a strain
of Bacillus amylobacter (Das et al., 2013; Szejtli, 1990). The following table
represents the discoveries of cyclodextrins according to time passage and work
done on cyclodextrins in different fields (Del Valle, 2004; Dardeer, 2014).

Year of
working Work done on cyclodextrin References
1891 First time CDs produced from starch by Singh et al. (2002)
digesting it with Bacillus amylobacter
strain by scientist Villiers. Villiers also
produces cyclodextrin from impure culture
by using strain Bacillus macerans.
1903 Schardinger produces two crystalline Eastburn and Tao (1994)
products and named it as dextrin A and
dextrin B, but the strain from which they
are produced are not maintained.
1904 Schardinger discovered a new organism Eastburn and Tao (1994)
that has capability of producing ethyl
(continued)
110 R. Gautam and S. K. Arya

Year of
working Work done on cyclodextrin References
alcohol and acetone from sugar and starch-
containing plant materials.
1911 Schardinger produced large amount of Eastburn and Tao (1994)
cyclodextrin from strain Bacillus macerans
about 25–30% and named it α-CD and
β-CD.
1935 Schardinger discovered γ-CD. After that, Loftsson and Brewester (1996),
Freudenberg and Cramer explained the Matsuda and Arima (1999), Mabuchi
existence of large cyclodextrin molecules. and Ngoa (2001)
1942 Schardinger demonstrated the structures of Buschmann and Schollmeyer (2002)
β-CD and α-CD using X-ray
crystallography.
1948 γ-CD structure is determined by using Buschmann and Schollmeyer (2002)
X-ray crystallography. At that time, it is
observed that cyclodextrins can form
inclusion complexes.
1961 Some evidences show the existence of Qi and Romberger (1988), Hirose and
natural cyclodextrins like ξ, δ, ζ, and even Yamamoto (2001)
η-cyclodextrin.
1965 A method is developed in this year for Kainuma (1984)
isolation of larger homologues structures
of ζ-cyclodextrin and η-cyclodextrin,
method named fractionation method.
1970 In 1970, several numbers of Kainuma (1984)
microorganisms are considered as a source
for cyclodextrin glucanotransferases and
glucanotransferase was purified and named
during the exploration period. Applications
of cyclodextrins in different industries
started in this period.
1980 Development in biotechnology in the mid Crini (2014)
of 1980s, improved the purification and
production of cyclodextrins and led to
increases in their value for
commercial uses.
1982 In 1982, first book on cyclodextrins was Crini (2014)
published, written by Szejtli.
1992 In this period, Hedges R.A. published a Hedges (1992)
book on cyclodextrin under a title
Cyclodextrins and Their Applications in
Biotechnology.
2003 In this period, Tomasik P. published a Szejtli (2004a)
book on cyclodextrins, under a title
Cyclodextrins Chemical and Functional
Properties of Food Saccharides.
2004 In this year, review articles were published Szejtli (2004b)
on data related to cyclodextrin and its
(continued)
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 111

Year of
working Work done on cyclodextrin References
future aspects and also on application of
cyclodextrins.
2006 Helena Dodziuk published a book on Kainuma (1984)
Cyclodextrins and Their complexes:
Chemistry, Analytical Methods and
Applications.
2007 Investigation of cyclodextrin Kunamneni et al. (2007)
glucanotransferase production using
alginate-immobilized cells of alkalophilic
Bacillus sp. in an airlift reactor.
2010 Biochemical characterization of Zhaofeng et al. (2010)
α-cyclodextrin glycosyltransferase and its
extracellular expression from
Paenibacillus macerans have been
published by Zhaofeng Li et al.
2011 Encapsulation of ethylene gas into Ho et al. (2011)
α-cyclodextrin and characterization of the
inclusion complexes.
2012 Effects of cyclodextrins on the Liang et al. (2012)
antimicrobial activity of plant-derived
essential oil compounds.
2013 Optimization of reaction of γ-cyclodextrin Wang et al. (2013)
glycosyltransferase and isoamylase for
enhanced production of c-cyclodextrin.
2014 Cyclodextrins in capillary electrophoresis: Escuder-Gilabert et al. (2014)
Recent developments and new trends.
2015 Experimental setup and kinetic study of Maneechakr et al. (2015)
ultrasonic-assisted transesterification of
waste cooking oil over sulfonated carbon
catalyst derived from cyclodextrin.
2016 Use of cyclodextrins to recover catechin Lopez-Miranda et al. (2016)
and epicatechin from red grape pomace.

4.3 Properties of Cyclodextrins

CD have hydrophobic cavity and the ability to form reversible host–guest complexes
or termed as supramolecular complex via weak forces like, dipole–dipole
interactions, Van der Waals interactions, and hydrogen bonding with molecules
(Escuder-Gilabert et al., 2014). CDs have the ability to solubilize hydrophobic
materials and thus can entrap volatile components by forming inclusion complexes
with organic compounds, which results in enhancing their physical and chemical
properties. They have the property of selective recognition of a chiral compound and
enantio recognition of enantiomers of chiral compounds (Del Valle, 2004;
Steinbrunn & Wenz, 1996). Low toxicity and immunogenicity of CDs allow for
112 R. Gautam and S. K. Arya

their use as a transport system into the living systems. Also, CDs have the capacity to
form pseudorotaxanes and rotaxanes through number of linear species. Rotaxanes
are the compounds that consist linear and cyclic species and are linked together by
non-covalent interaction (Isnin & Kaifer, 1993; Buston et al., 2001). As inclusion
bodies, CDs help in increasing the stability, solubility, and bioavailability of
lipophilic organic molecules, improve the stability, increase the permeability of
water-insoluble compounds, and diminish the drug toxicity by manufacturing the
compound effective at lesser doses (Del Valle, 2004; Kamimura et al., 2014).

4.3.1 CGTase Synthesis

Cyclodextrin glucanotransferases (EC 2.4.1.19, CGTases) using starch and its

derivatives are used for the production of cyclic α-(1,4)-linked oligosaccharides
commonly known as cyclodextrins (CDs) via the cyclization reaction (Tonkova,
1998). CGTase is isolated from Bacillus sp. and degrade the starch from raw
substrate (like potato) to form cyclodextrin. It was reported that immobilization
increases the activity of CGTase enzyme, as when it was immobilized in mixed gel
beads, its activity increases. This fermentation process is carried out in a continuous
stirred tank reactor (CSTR) and a packed bed reactor (PBR) for commercial produc-
tion. Enzyme preparation is done according to production process required. Gener-
ally, CGTase is isolated from Bacillus sp. C26 and inoculated into medium and then
incubated at 37
C at 200 rpm for 48–50 h. Centrifugation is done for 15 min to
remove the cells and insoluble material at 4000  g and at 4
C. Precipitation of
enzyme is done by adding the cell-free supernatant with ammonium sulfate and
keeping them for overnight in controlled conditions at 4
C temperature and pH is
maintained at 7. Product purification is done by dialysing the solution obtained after
overnight incubation with molecular cut-off 8000 Da. Enzyme activity is monitored
by using phenolphthalein colourimetric method, and to measure the enzyme protein
concentration, folin ciocaltue method is used. It was reported that CGTases are an
extracellular enzyme produced by number of microorganisms, for example, bacteria
(mostly from Bacillus species) (Leemhuis et al., 2010).
B. circulans DF 9R was isolated from rotten potatoes (Ferrarotti et al., 1996).
Culture was then transferred to medium containing cassava starch, (NH4)2SO4,
phosphate buffer, MgSO4, and FeSO4. Culture was grown aerobically until the
absorbance at 620 nm becomes 0.3 and is then used as inoculum. The medium is
then transferred to 1000 mL Erlenmeyer flask containing medium. Then the mixture
is centrifuged and the supernatant obtained is used as a crude enzyme (Rosso et al.,
2002; Kunamneni et al., 2007). The microorganism producing β-CGTase is
alkalophilic Bacillus species (Bacillus sp. AK11) that has the capacity to grow in
high alkaline condition when cultivated in air lift reactor. Alkalophilic bacilli are
more trending and more commonly in use due to broad range specificities of pH and
temperature (Kunamneni et al., 2007; Park et al., 1999; Vassileva et al., 2005).
Microorganism is then transferred to growth medium containing starch, peptone,
KH2PO4, and MgSO4 and medium is adjusted to pH 8 using sodium carbonate and
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 113

maintained at a temperature of 37
C. Cells were immobilized using sodium alginate

method (Kunamneni et al., 2007; Higuti et al., 2004). The alginate beads were
loaded into the reactor containing the production medium and allowed to stay for
24 h to activate cells. The broth obtained was drained and further replaced with
production medium which contained different molar concentrations of the added
ingredients. The production medium is then constantly added so that there is
continuous mixing of the solution and overflow is regulated by peristaltic pump
and finally its activity can then be measured spectrometrically. It was observed that
with the increase in the air flow rate, the production of CGTase increased. The
concentration of carbon and nitrogen needs to be maintained for effective CGTase
production using immobilized beads (Kunamneni et al., 2007).
However, it was observed that the average volume productivity increased during
immobilization as compared to free cells (Kunamneni et al., 2007). The concentra-
tion of α-CD production is measured by observing the decrease in absorbance at
507 nm. The decrease in absorbance is due to formation of methyl orange α-CD
complex (Kunamneni et al., 2007; Higuti et al., 2004).
One unit of CGTase is defined as the amount of enzyme catalysing the production
of 1 μmol of β-CD per minute under the reaction conditions. The cyclising activity of
CGTase was tested by phenolphthalein method. In this test, the production of
β-cyclodextrin is measured on the basis of its ability to form inclusion complex
with this dye which is then measured by taking absorbance at 550 nm (Kunamneni
et al., 2007; Goel & Nene, 1995). Phenolphthalein is diluted in ethanol and the pH is
adjusted to 11 using Na2CO3. The activity assay consisted of substrate solution
containing 1% (w/v) maltodextrin in tris-HCl buffer at 50
C, taking water as a
blank.
The concentration of γ-CD was measured at 630 nm due to formation of inclusion
complex with bromcrescol green (Goel & Nene, 1995; Natalia et al., 2007) taking
water as a blank. In this case, the reaction mixture consists of 1% (w/v) soluble starch
in gly-NaOH buffer, pH 10, and enzyme solution diluted by buffer incubated at
40
C for 10 min. Then 0.1 N HCl was used to stop the reaction. Finally, the citric
acid–citrate sodium buffer (pH 4.2) were added to the reaction mixture, and then the
absorbance is taken at 630 nm.
Methods for CD-complex formation: The forces that are responsible for interac-
tion of these inclusion complexes include Van der Waals forces, hydrophobic
interactions and hydrogen bonds (as shown in Fig. 4.2). These inclusion complexes
change the chemical and physical properties of the molecule. The energy of Van der
Waals is proportional to molecular polarizability and molecular refraction. The
stability of the complex increases with the increase in the electron donor ability of
the substituents (Astray et al., 2009).
Cyclodextrins can also form complexes with the gases such as CH4, C2H6, C3H8,
Cl2, Xe, etc. Alpha cyclodextrin due to its smaller diameter (470–530 pm) can form
stable complexes with guest molecules having five carbon atoms or less. However,
ethylene gas is more suitable for formation of complexes with alpha CDs. The
encapsulation of ethylene into alpha cyclodextrin was done by dissolving alpha
cyclodextrin powder in water at 25
C. After the powder is completely dissolved,
114 R. Gautam and S. K. Arya

Fig. 4.2 Cyclodextrins structure and inclusion complex formation (Schmid, 1989; Gomes et al.,
2014; Garnero et al., 2010)

solution is then stirred for complete 5 min. Saturated alpha cyclodextrin obtained is
added to plastic container and ethylene gas was then pressurized at 0.2, 0.5, 1, and
1.5 Pa at 25
C. Afterwards, crystal precipitates formed are collected by vacuum
filtration and then analysed for ethylene gas concentrations and further characterized
for their physiochemical properties (Ho et al., 2011).
CDs have hydrophobic cavity. CDs have the ability to form host–guest
complexes via weak forces, like Van der waal interactions, dipole–dipole
interactions, and hydrogen bonding with molecules (Szejtli, 1988). CDs can solubi-
lize hydrophobic materials and thus entrap volatile components by forming inclusion
complex with organic compounds, resulting in enhancing their chemical and physi-
cal properties. CGTase production at large scale by using Bacillus sp. depends upon
the kinetic model of cyclodextrin-glucanotransferase (Burhan et al., 2005).
Advantages of CD to form IC are: higher thermal stability, high solubility, bioavail-
ability of hydrophobic guests, control volatility, masking off unpleasant odours, and
controlling release of drugs and flavours.
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 115

4.3.2 CGTase Sources

This table represents the various sources of CGTase from different spices, their mode
of production, and their optimum parameters like temperature and pH.

Optimum
Sources of CGTase Production temperature and pH References
Bacillus macerans Bacillus macerans Optimum Fujiwara et al.
IFO 3490 IFO 3490 mainly temperature is 55
C (1974), Kitahata
produces α-CGTase. and pH 5.2–5.7. et al. (1974),
Fermentation medium Rimphanitchayakit
is soluble starch, corn et al. (2005)
steep liquor.
Bacillus circulance E Bacillus circulance E Optimum Charoenlap et al.
192 192 produces mainly temperature 60
C (2004), Vassileva
β-CGTase. Agar- and pH 5.5–5.8. et al. (2005)
immobilized cells are
used for high
productivity of
CGTase. B. circulance
requires alkaline pH
for an efficient
CGTase formation.
Paenibacillus Main product is β-CD. Optimum Larsen et al. (1998)
temperature is 50
C
and pH is 6.0–8.0.
Klebsiella pneumonia The CGTase from It can be preferred at Gawande and
Klebsiella pneumonia. pH 6–8, temperatures Patkar (2001)
Main product is α-CD. of 40–50
C.
Bacillus CGTase from Bacillus Its optimum activity Rahman et al.
stearothermophillus stearothermophillus at 80
C temperature. (2004)
ET1 is a potential
antistaling enzyme
with cyclodextrin
(CD)-producing
activity. Main product
is α- and β-CD.
Bacillus lehensis Bacillus lehensis is Optimum Blanco et al. (2014)
isolated from cassava temperature is 55
C
starch wastewater. and pH 8.0.
Main product is
β-CD. Its CGTase
activity is 18.9 μm/L
Micrococcus Micrococcus variant Its optimum activity Kim et al. (1997)
M 849 produces α and temperature is
β-CD as main 55–65
C and pH is
products. 5.0–9.0.
Thermoanaerobacter Thermoanaerobacter Its optimum activity Starnes et al. (1992)
sp. sp. ATCC 53627 temperature is
mainly used for the 90–95
C.
(continued)
116 R. Gautam and S. K. Arya

Optimum
Sources of CGTase Production temperature and pH References
production of
β-CGTase.
Alkalophilic Bacillus Alkalophilic Bacillus Optimum Sian et al. (2005)
sp. sp. 38-2 mainly temperature is 45
C.
produces β-CD.
Bacillus CGTase is synthesized Growth medium is Andel-Naby et al.
amyloliquefaciens by maintained at pH 6.5 (2000)
B. amyloliquefaciens and microorganism is
by immobilized maintained at 4
C on
method in batch and potato dextrose agar
continuous cultures. slants.

4.3.3 Methods for Production of Inclusion Complexes

In this table, methods for CDs complexes or inclusion complexes formation have
been explained and the manufacturing of CDs.

Methods for
CD-complex
formations Process References
Precipitation Precipitation method is most widely used for Das et al. (2013),
method inclusion complex production. According to Sapkal et al. (2007)
Sapkal et al., by using co-precipitation method, a
poor aqueous solubility guest molecule is
combined with β-cyclodextrin to form inclusion
complex. In this method, drug and CD are
dissolved in water and solvent (like acetone) is
heated at 75
C. Stirring is done for 1 h at 75
C to
obtain concentrated, viscous, and translucent liquid
and then cooled at room temperature. Precipitates
are filtered or separated than dried and stored to get
solid inclusion complex.
Kneading method Conventional kneaders like low and high shear Das et al. (2013),
mixers are used to prepare CD paste, paste is Miller et al. (2007)
prepared with small amount of water in which
guest molecule is added (without a solvent) in a
small amount of ethanol, and then after grinding,
solvent is evaporated and powdered form
cyclodextrin-inclusion formed.
Spray drying In this method, first cyclodextrin is dissolved in Das et al. (2013),
solution that is already alkalinized with 25% Arias et al. (2000)
aqueous ammonia, then guest molecule is
dissolved in 96% ethanol alcohol solution in
100 mL, both solutions are then mixed, and finally
sonicated. The l solution obtained is then spray-
dried to get inclusion complexes; this is done at
(continued)
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 117

Methods for
CD-complex
formations Process References
equilibrium state which is attained by removing the
solvent by spray drying.
Neutralization The guest molecule is dissolved in basic or acidic Das et al. (2013),
method aqueous cyclodextrin solution. Cyclodextrin and Choi et al. (2001)
drug are dissolved separately in 0.1 N NaOH
solution, mixed and stirred for half an hour; pH is
maintained by 0.1 N HCl at 7.5; at this pH,
complexes precipitate and then residues are
separated and washed until free from chlorine and
then dried at 250
C for 24 h

4.3.4 Production Methods

(a) Batch process: Generally CDs are produced through batch process which is
quite simple and easy to control process. But there are several limitations of this
method, like long operation hours, high labour cost, and high enzyme concen-
tration requirement that leads to switching to other production method (Sakinah
et al., 2014; Lia et al., 2014).
(b) Enzymatic membrane reactor (EMR): EMR is basically a stirred tank reactor
combined with a separation membrane that recirculates the reaction mixture
through a membrane module placed outside the enzymatic reactor. As compared
to batch process, this is a continuous process method and is more practical and
economical. Also, reusability of the enzyme results in high yield productions of
cyclodextrins as compared to batch process (Lia et al., 2014). In this process,
product (CD) is separated from the enzyme and substrate through a semi-
permeable membrane, which is composed of an ultra-thin separation layer
widely used in enzyme separation. During the separation process, CDs penetrate
through the membrane and the enzymes get retained by the membrane within the
reaction reactor (Bodalo et al., 2001).
The working principle is such that the pressure difference across the membrane,
also called the transmembrane pressure (TMP), impels the product through the
membrane, while the unreacted substrate and free enzyme are recirculated to the
enzymatic reactor (Sakinah et al., 2009). Advantages of enzymatic membrane
reactor over batch reactor are its high production flow rates; reductions in cost,
energy, and waste products by the recycling practice; easy reactor operation and
control; and the high yields of pure material (Katzir, 1993). But the main
limitation of this method is fouling of membrane as the substrate gets deposited
onto the membrane surface and enzyme precipited within the membrane pores
(Hughes & Field, 2006; Balakrishnan & Dua, 2000; Yuan & Zydney, 1999;
Bansal et al., 2006). However, this problem can be overcome by employing a
high cross-flow velocity (CFV), but this condition requires large amount of
118 R. Gautam and S. K. Arya

energy and sometimes reduces the enzyme activity (Sakinah et al., 2014;
Gugliemi et al., 2007; Priyananda & Chen, 2006).

4.4 Alpha Cyclodextrin Production

α-Cyclodextrin containing six 1,4-linked D-glucose units makes it cyclic polysac-

charide, which differentiates it from other two, β (seven) and γ (eight)-cyclodextrins.
It is commercially produced from starch (used as a substrate) by an enzymatic
production process. α-Cyclodextrin has variety of application in food industry due
its small internal cavity and high resistance to its enzymatic hydrolysis.
α-Cyclodextrin is less important than other CDs because of its low production
yield and high cost. α-Cyclodextrin is generally used as a dietary fibre food.
α-Cyclodextrins are used in food industry due to following reasons: α-CDs has
both nutritional and technological properties (Lia et al., 2014). It acts as a carrier for
natural flavour, colours, and vitamins, and as a stabilizer of oil in water emulsions. It
is also used as a solubilizer of lipids.
A number of applications for α-cyclodextrins have been published in a variety of
papers. Some of the important applications are: α-cyclodextrin have been used in
bakery products: like biscuits, rolls, and cakes; snack food: like salty snacks, etc.;
dairy desserts: such as yoghurt and dessert mixes; beverages and powders: like
coffee whitener, vegetable and fruit juice, soy drinks, and soft drinks (diet); grain-
based foods: like pasta, maggie, macaronis, noodles, and instant rice dishes; and so
many other food items like chewing gum, sauces, fat, and oil products like mayon-
naise and dressing. So these all are the proposed applications of cyclodextrins in
food products by some users in particular papers (Lia et al., 2014).

4.4.1 Production

CDs’ production can be divided into four stages as follows:

1. Production of CGTase in the reaction medium from the cultivation of the

microorganism.
2. Extracting the enzyme through separation, concentration, and purification
method.
3. Converting pre-hydrolysed starch into cyclic and acyclic dextrin with the action
of enzyme.
4. Separation, purification, and crystallization of CDs (Katzir, 1993; Moriwaki
et al., 2014; Astray et al., 2009).

Cyclodextrins are produced from starch or their derivatives because of enzymatic

conversion that is catalysed by cyclodextrin glycosyltransferase (CGTase) (Rios
et al., 2004). In simple words, cyclodextrins are produced by cyclodextrin
glucosyltransferase (CGTase) through cyclization reaction of gelatinized starch
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 119

(Rakmai et al., 2015; Iyer et al., 2003). The result is a mixture of α-, β-, and
γ-cyclodextrins, containing trace amounts of cyclodextrins (Sakinah et al., 2014).

1. α-Cyclodextrin: It has small internal cavity and high resistance to enzymatic

hydrolysis, which make it suitable for application in the food industry. But
because of its low production yield and high price, its market share is currently
smaller than that of β-cyclodextrin. Thus, the production of α-cyclodextrin can be
improved by modifying the properties of CGTases.

4.4.2 Properties of a-Cyclodextrin

α-CD has the lowest hydrogen bond strengths between the 3-OH and 2-OH groups
around the wider rim. Diameter of internal cavity is 4.7–5.3 Å and the cavity volume
is about 66% or 41% of that of β- or γ-cyclodextrins, respectively (Li et al., 2007). It
typically forms inclusion complexes with benzene derivatives because of Van der
Waals interactions. α-Cyclodextrin has modest solubility in water which means it is
almost eight times greater than that of β-cyclodextrin, but 1.6 times lower than that of
γ-cyclodextrin at 25
C. Also, it is more resistant to hydrolysis in acid solutions. It
was observed that even after 3 h of incubation period in extremely acidic conditions
(pH ¼ 2.4) at 100
C, no breakdown of α-cyclodextrin is recognized (Jodai et al.,
1984; Saha & Zeikus, 1992). But α-cyclodextrin can’t be hydrolysed by human
salivary and pancreatic amylases for its commercial production (Kondo et al., 1990).
α-CGTases from Bacillus macerans or Paenibacillus macerans are most commonly
used. It is also effective at solubilizing free fatty acids (Qi & Zimmermann, 2005).

4.4.3 Enzymatic Production of a-Cyclodextrin

There are two types of processes that result in the production of cyclodextrins:

Non-solvent process: In this process, cyclodextrin mixture is produced without the

addition of any complexing agents and the mixture produced can be further
separated by chromatographic procedures.
Solvent process: This process requires an organic complexing agent that helps in
selecting one type of cyclodextrin and thus directs the enzymatic reaction to
produce the cyclodextrin of interest. This method is commonly used for the
production of α-cyclodextrin on industrial scale as shown below in the Fig. 4.3
(Flaschel et al., 1984; Rendleman, 1997).

4.4.4 Applications

Cyclodextrins are used as browning inhibitors in different fruit juices. The oxidation
of volatile precursors present in freshly squeezed pear juices can be prevented by
120 R. Gautam and S. K. Arya

20-30% Gelatinized and liquefied starch (using α-amylase)

Add α-CGTase (under controlled conditions)

A 1-Decanol (to form inclusion complex)
Add

Formation of α-cyclodextrin/1-decanol complex by conversion of starch

Separation of complex by centrifugation

Discard supernatant as it contains by

products

Re- suspension and re- precipitation of recovered complex in water

Centrifugation (to recover precipitate) + steam distillation (to remove 1-decanol)

Crystallization of α- cyclodextrin by concentration and cooling

y
Filtration and drying of crystals

White powder α-cyclodextrin with at least 98% purity

Fig. 4.3 Flow chart is showing the production of α-CD (Lia et al., 2014; Rendleman, 1997)

adding α-cyclodextrin (90 mM). It is one of the most suitable agents for
encapsulating flavours that are encapsulated in powder form by spray drying with
α-cyclodextrin (Shiga et al., 2004). α-Cyclodextrin is also a dietary fibre as it forms
complex with triglyceride at a ratio 1:1 that is typical for dietary fibres and form a
stable complex with dietary fat (Ferrari et al., 2013; Threapleton et al., 2013; Zhang
et al., 2013a, b). It reduces the absorption and bioavailability of dietary fat, which
makes it practical as a weight loss supplement (Suzuki & Sato, 1985).
α-Cyclodextrin is effective in reducing and/or maintaining body weight with
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 121

increasing energy intake for obese patients with type 2 diabetes (Gallaher et al.,
2007; Grunberger et al., 2007). It is effective in improving metabolic syndrome by
reducing serum triglyceride and leptin levels and increasing insulin sensitivity and
faecal fat excretion in rats. α-Cyclodextrin may also be useful as an ingredient for
reducing the glycaemic impact of foods (Penninga et al., 1995).

4.4.5 Alternative Method for the Production of a-Cyclodextrin

Alpha CGTase from Paenibacillus macerans is most commonly used for the
production of alpha cyclodextrin. However, the production of enzyme can be
increased by expressing it in E. coli which can bring a benefit in reducing cost of
alpha cyclodextrin. Alpha CGTases produced in E. coli are accumulated in the
cytosol in the form of inactive inclusion bodies. In this case, alpha CGTase from
P. macerans JFB05-01 is expressed extracellularly in E. coli. The recombinant
enzyme is then purified and characterized with respect to cyclization activity,
including optimum conditions effect of metal ions, etc.

4.4.6 Steps for the Production of Alpha Cyclodextrin

The plasmid pET20b is used for the production of alpha cyclodextrin. At 25
C, very
little recombinant alpha CGTase was secreted into the medium. But after 90 h of
incubation period, the production rate increased rapidly and the cyclization activity
of alpha CGTase also increased which was 42 times higher than the parent strain. A
single colony of E. coli BL21cells harbouring plasmid CGT/pET20B was inoculated
into LB medium containing ampicillin. For inducing the expression of α-CGTase,
different amounts of isopropyl 1-thio-b-D-galactopyranoside (IPTG) were added.
The recombinant alpha CGTase contains a histidine sequence on a C-terminal
domain which can be separated by metal binding affinity. To overcome the hetero-
geneity, the recombinant enzyme is purified by chromatographic separation
techniques which include Q-sepharose and phenyl-superose chromatography. The
purified protein obtained is then separated by SDS page. From the SDS PAGE, it
was found that alpha CGTase migrated as a single band with molecular mass of
approximately 72 kDa. The optimum temperature of alpha CGTase recombinant
enzyme was 45
which was below that of the native enzyme which is 50
C. The
recombinant and native CGTase both showed the best cyclization activity at
pH 5.5 at a pH value; above or below this, the cyclization activity decreases. Enzyme
is stable in Na2HPO4/NaH2PO4 at the same pH. The cyclization activity of alpha
CGTase is inhibited completely by Hg2+ and slightly by Fe2+ or Co2+. The addition
of 2 mM Ca2+, 2 mM Ba2+, or 0.1 mM Zn2+ activated the cyclization activity
considerably. It is also observed that the growth medium has a great impact on the
secretion of proteins in E. coli. However, TB medium was considered the best for
extracellular production of alpha CGTase as TB medium has beneficial effect on cell
growth because of its relative high buffering capacity. Also, the low IPTG
122 R. Gautam and S. K. Arya

concentration (0.01 mM) favoured the extracellular enzyme production. The extra-
cellular productivity of the recombinant enzyme was 40 fold higher than the parent
enzyme P. macerans JFB05-01. The metal chelator like EDTA doesn’t show any
effect on the activity of α-CGTase enzyme even at relatively high concentration
(10 mM), which indicates that a metal cofactor was not required for the function of
the enzyme. Furthermore, α-CGTase could be activated in the presence of Ca2+,
which was probably because of the binding of Ca2+ with its binding sites which are
located at the active site of the protein molecule, resulting in stabilizing the substrate
binding cleft of CGTase (Eastburn & Tao, 1994). Following table represents number
of the sources of alpha cyclodextrin production, optimum temperature, optimum pH,
and main products (Stella & Rajewski, 1997; Buschmann & Schollmeyer, 2002;
Hirose & Yamamoto, 2001).

Optimum
Source of α-CD Optimum temperature
production pH (
C) Main product References
CGTase 6–8 40–50 In this method, alpha- Flaschel
produced from cyclodextrin is the main et al. (1984)
Klebsiella product, produced by
pneumoniae M5 enzymatic degradation of
starch, starch used as a
substrate. The yield of alpha-
cyclodextrin is lower by the
enzymatic method.
CGTase 5.0–8.0 55–65 Main product is α-CDs, Larsen et al.
produced from studied by purification and (1998), Yagi
Micrococcus characterization of et al. (1980)
variant M 849 cyclodextrin
glycosyltransferase from
Micrococcus variant M 849.
CGTase 5.0–7.5 – By using this sp., CGTase Larsen et al.
produced from produces α-CDs as a main (1998),
Klebsiella product. Purification and Bender
oxytoca M5A1 characterization of CGTase (1983)
are done in this article.
CGTase 5.2–5.7 55 In this paper, we study the Larsen et al.
produced from production of α-CDs by using (1998),
Bacillus sp. Bacillus macerans. Fujiwara
macerans IFO et al. (1974)
3490

The concentration of α-CD production is measured by observing the decrease in

absorbance at 507 nm due to formation of methyl orange α-CD complex.
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 123

4.5 Beta-Cyclodextrin

Beta-cyclodextrin (β-CD) has the ability to encapsulate molecules that are hydro-
phobic in nature which helps in improving their aqueous solubility and also reduces
their volatility (Lin et al., 2016).
Production of β-CD can be achieved in both batch and continuous process. But
continuous process is preferred over batch because of its advantages like greater
process control, high productivity, and improvement of quality and yield. Continu-
ous process includes the combination of both CSTR (continuous stirred tank reactor)
and PFR (plug flow reactor) and is considered to be more suitable for long-term use.
Also in case of industrial scale production, CSTR is preferred over batch process
because of its properties like high stability and low by-product formation, but the
disadvantage of using CSTR is the undesired thermal gradients in case of PBR, poor
radial mixing, and low mass transfer rate (Santos et al., 2015; Rakmai & Cheirsilp,
2016; Galan et al., 2011; Kittikun et al., 2008).
Gram-positive microorganisms Bacillus firmus strain and Bacillus sphaericus
strain 41 were immobilized on a loofa sponge and are used as the direct source for
the CGTase and CD production (Moriwak et al., 2014). The CGTase produced by
Bacillus sp. C26 has been reported to have high activity for β-cyclodextrin produc-
tion; immobilized CGTase is then applied for the production of β-cyclodextrin
(Rakmai et al., 2015) (Fig. 4.4).

4.5.1 Alternative Method

CGTase was produced by bacillus firmus isolated from brazillian soil of oat culture.
The medium contained KH2PO4, MgSO4, and Na2CO3 and the pH was maintained
at 10.3. Then the medium is transferred to a 5 L medium and proceeded for
cultivation for 5 days at 37
C. The specific enzyme is isolated using affinity column
chromatography and the protein concentration is estimated using Bradford method
using bovine serum as standard, and using SDS-PAGE, molecular mass of protein is
estimated. The CGTase activity is measured by replacing maltodextrin by corn-
starch and potato starch. The maximum activity of enzyme is seen at a pH greater
than 5 and less than 10 and at a temperature of 50
C (Natalia et al., 2007).
Production of β-CD (10 whole till last): Immobilization of CGTases is more
commonly used as it reduces the production costs and makes it feasible to be used in
various food industries (Garcia-Galan et al., 2011). The enzyme immobilization has
a lot of advantages as the enzyme can be reused, makes downstream processing easy,
and increases thermo-stability of enzyme (Rodrigues et al., 2013; Natividade
Schöffer et al., 2013). Chitosan derivative of chitin has required properties for
enzyme immobilization (Muzzarelli, 1980). Chitosan has reactive amino and
hydroxyl groups in its glucosamine chain (Chiou & Wu, 2004). Chitosan also
exhibits mechanical stability, rigidity, and biocompatibility which permit its use in
food industries (Krajewska, 2004). The following table represents the sources of
β-cyclodextrins from various papers and its optimum pH, temperature, and fermen-
tation methods.
124 R. Gautam and S. K. Arya

Immobilized CGTase at 10 U/g-starch + 20 mL starch solution

(In a batch stirred-tank reactor)

Agitation at 300 rpm using magnetic stirrer.

Collect soluble starch

Add phosphate buffer at varying concentrations of 1–8% (w/v) of pH 7.0

Incubation process ( for measuring the concentration of β-cyclodextrin sample is

collected after every 2 hour)

High yield of β-cyclodextrin production (8.2 g/L)

Fig. 4.4 Flow chart showing production of β-cyclodextrin using entrapped CGTase (Rakmai et al.,
2015; Muria et al., 2011)

Source of β-CD Optimum Optimum Main product and

production pH temperature fermentation medium References
CGTase produced 9.8–10 40
C In this article, Vassileva
from Bacillus cyclodextrin et al. (2007)
circulans (ATCC glucanotransferase is
21783) produced by
immobilized and free
cells of Bacillus
circulans ATCC
21783 in various
types of bioreactors.
Main product is
β-cyclodextrin.
CGTase produced 6–7 40–90
C Bacillus sp. G1 is Mora et al.
from Bacillus isolated from soil. (2012)
sp. (Alkalophilic Inoculum for Bacillus
bacteria) sp. G1 contains 1%
(w/v) tapioca starch,
(continued)
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 125

Source of β-CD Optimum Optimum Main product and

production pH temperature fermentation medium References
0.5% (w/v) yeast
extract, 0.5% (w/v)
peptone, 0.1% (w/v)
KH2PO4, 0.02%
(w/v) MgSO4, and 1%
(w/v) Na2CO3. Its
main product is
β-cyclodextrin.
CGTase produced 8.0 Different B. firmus strain Mazzer
from Bacillus temperatures 37, isolated from the et al. (2008)
firmus strain 37 37, 50, 60, and soil. Composition of
70
C at different culture medium (%,
substrate w/v) is such that it
concentrates. contains 1.0 soluble
starch, 0.5
polypeptone, 0.5
yeast extract, 0.1
K2HPO4, 0.02
MgSO47H2O, 0.01
Congo red dye, 1.0
Na2CO3, and 1.5 agar.
The plates were
incubated at 37
C for
48 h. Main product is
β-CD.
CGTase partially 4.5–5.0 55–60
C Composition of Charoenlap
purified from medium: 10 g/L et al. (2004)
Bacillus circulans soluble starch, 5 g/L
(TISTR 907) by yeast extract, 0.2 g/L
ammonium sulfate MgSO47H2O, 5 g/L
precipitation. peptone, 1 g/L
K2HPO4, and 10 g/L
NaCl at pH 10.0 and
incubation
temperature is 37
C
for complete 24 h
with shaking at
200 rpm. Main
product is β-CD.
CGTases produced 5–8.5 60–65
C Composition of Cheirsilp
by Bacillus sp. C26 medium 1% w/v et al. (2010)
soluble starch, 0.5%
w/v yeast extract,
0.5% w/v peptone,
0.02% w/v K2HPO4,
0.02% w/v
MgSO47H2O, and
1% w/v Na2CO3. Its
main product is
β-cyclodextrin.
126 R. Gautam and S. K. Arya

4.5.2 Applications of b-Cyclodextrin

β-Cyclodextrin is used in inhibiting the browning of potato crude extract and

different fruit juices: β-Cyclodextrin acts as an anti-browning agent in potato extract,
and in case of fruit juices, it helps in stabilizing the flavour, improves mouth feel and
also a mild antimicrobial effect, antioxidant properties in grape juice, improves
retention of volatiles in pear juice, and helps in elimination of off-flavours in
lemon oil (Santos et al., 2015).

4.6 Gamma Cyclodextrin

Gamma cyclodextrin is produced from microbial strain B. thuringiensis GU-2. The

optimum pH and temperature for γ-CD production are pH 8.5 and 37
C, respec-
tively, in the presence of 1.0% starch substrate which results in 95% production of
product with high yield (Goo et al., 2014). γ-CD has the premier solubility and
largest interior cavity. Bioconversion of gamma cyclodextrin (γ-CD) from starch in
the presence of cyclodextrin glycosyltransferase is using a technique aqueous
two-phase system (ATPS). This is a low pollution technique, simple, economic,
and is used for purification and production process in single step (Lin et al., 2016).
γ-CD production was to a great extent enhanced with the use of cyclododecanone,
5-cyclohexadecen-1-one, and glycyrrhizic acid. Optimum pH for reaction conditions
is between 7 and 10 using 15% potato starch. All these parameters resulted in the
corresponding increase in the yield of γ-CD from 21.2% to 41.6%. Optimum
temperature range for the reaction is between 40
and 65
. Higher enzyme dosages
had a detrimental effect on the CD yield, probably because excess CGTase might
have hydrolysed CDs to produce glucose and high quantities of malto-
oligosaccharides, which might have facilitated coupling reaction with the CDs,
thus severely degrading them. A CD was not detected. When soluble starch or
potato starch as a source was used, it becomes very difficult to detect the amount
of CD. Soluble starch and potato starch are responsible for the production of highest
percentage of γ-CD among the total CDs (nearly 88%), when compared with the
other two starches (Loftsson & Brewester, 1996).

Optimum
Source of Optimum temperature Main
γ-CGTase pH (
C) product References
Bacillus subtilis 8.0 65 γ-CD Li et al. (2007), Kato and
strain 313 Horikoshi (1986)
Bacillus 7.5–10.5 55 γ-CD Li et al. (2007), Fugita et al.
sp. strain AL-6 (1990)
Bacillus firmus 6–8 60 γ-CD Li et al. (2007), Veen et al.
strain 290-3 (2000)
Bacillus cereus 8.0 37 γ-CD Li et al. (2007), Lin et al.
(2016), Ng et al. (2011)
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 127

4.6.1 Applications

Cyclodextrins are used in food due to the following reasons:

1. Cyclodextrins are used to preserve the natural flavours, vitamins, and natural
colours of foods
Manufacturing, packaging, and other factors modify the flavours by reducing
aroma. There are wide techniques and processes used for retaining the properties
and flavour of the food like spray drying, freeze-drying, fluidized bed coating,
and crystallization. However, those involving the formation of flavour/CD
molecular-inclusion complexes offer great potential for the protection of volatile
substances throughout many rigorous food-processing methods such as freezing,
thawing, and microwaving. The addition of β-CD has proved to be most effective
in retaining the flavour against heat, evaporation, and also against thermal
degradation (Jouquand et al., 2004). The flavour enhancing complexes consists
of many components, thus it is important that there is proper incorporation of
these components in the complex without causing any change in its composition
(Astray et al., 2009). Natural and synthetic coffee flavours have also been retained
by the use of beta cyclodextrins. When these complex bound flavours come in
contact with water molecules, they get released immediately and thus responsible
for the taste or flavour (Astray et al., 2009; Szente & Szejtli, 1986). In addition to
coffee, β-CD is also used for tea aromatization (jasmine, peppermint, and cinna-
mon) and also results in stability as well as efficient storage.
2. For the removal of undesired taste, flavour, microbial contamination, and other
undesired products
For the rejection of various food items, bitter taste is responsible. It has been
found that there are two classes of chemical compounds like flavonoids and
limonoids that are responsible for bitterness. Fresh citrus juice turns bitter in
case of storage which is dependent on pH and temperature. It is highly desirable
to remove the bitter components without the addition of any other substance to
the fruit juice. Therefore, cyclodextrins are used for masking the undesired
components from food. When the cyclodextrins are added to the fruit juice
which has a peculiar odour and an undesirable taste, it forms complexes and
retains the flavour of the components. For example, this process is used for
deodourizing soybean milk and soy protein which removes the peculiar fish
odours. A mixture of cyclodextrins alpha, beta, and gamma is ordinarily used.
Rice which gives an unpleasant odour when stored for a long time can be made
odour-free by cooking the rice in the presence of 0.01–0.4% CD (Sakakibara
et al., 1985). The taste of cooked ice can be improved by addition of maltosyl beta
CD. When CDs are complexed with sweetening agents such as aspartame; it
helps in stabilizing and improving the taste of product. This complexation process
also helps in eliminating the bitter aftertaste of other sweeteners like stevioside,
glycyrrhizin, and rubusoside. CD itself is a promising new sweetener (Singh
128 R. Gautam and S. K. Arya

et al., 2002). When these cyclodextrins or cyclodextrin-complexed antimicrobial

agents are incorporated into food packaging plastic films, they help in effectively
reducing the loss of the aroma substances and also improve the microbiological
preservation during storage. Various CD complexes can be utilized in food as an
antiseptic or conserving agents; 0.1% iodine-β-CD inhibits putrefying for
2 months at 20
C in fish paste or in frozen seafood products (Hara & Hashimoto,
2002). Incorporating fungicide CD complexes into films-for example, packaging
of hard cheeses-significantly elongates the shelf life of the product by inhibiting
the rapid development of mould colonies on surface of the packaged cheese. The
characteristic colour of muttons and fish and bad odour of bone powder can be
removed by the use of cyclodextrins. The bitter taste of grape juice also reduced
considerably when 0.3% of β cyclodextrin was added prior to heat treatment of
canned juice. Empty beta cyclodextrin has also been used in food industries to
remove undesirable compounds such as bitter taste from tea and coffee (Shiga
et al., 2004) and fruit juices (Ferrari et al., 2013; Threapleton et al., 2013).
3. To protect against oxidative degradation, heat-induced changes, and light-
induced decomposition
The lipophilic components present in food which are sensitive to light- or heat-
induced degradation can be protected because of the formation of inclusion
complexes with CDs. The formation of inclusion complexes with cyclodextrins
removes the polyphenol oxidase which converts the colourless polyphenols to
coloured compounds which then leads to browning reaction in case of preparation
of fruit juices. Cyclodextrins remove the polyphenol oxidase by forming
complexes (Del Valle, 2004).
In case of apple and pear juice, the maltosyl beta cyclodextrin can enhance the
ability of ascorbic acid to prevent enzymatic browning due to protective effect of
maltosyl beta cyclodextrin against ascorbic acid oxidation. Hence, maltosyl beta
cyclodextrin is considered as secondary antioxidant reducing apple and pear juice
browning reaction. During industrial food processing when these inclusion
complexes are heated, they exhibit high thermal stability, are stable, and last for
a longer period of time. This can be supported by taking an example such as in
case of aromatizing substances which are sensitive to some kind of radiation, like
citral (responsible for fresh citrus odour) cyclizes under exposure to UV irradia-
tion. This can be prevented by complexation with beta cyclodextrins. It has been
found that complexation is more effective (protective) in solid state than in
aqueous solution. Another application of cyclodextrins is that since they can
form inclusion complexes with the substances, they can be used to retain or
scavenge substances such as odour, lactose, and cholesterol, thus retaining the
aroma, colours which could enhance shelf life, and also the quality of the
packaged product (Zhang et al., 2013b; Artiss et al., 2006; Suzuki & Sato, 1985).
4. Food preservation
This is the most important application of cyclodextrins in food. Cyclodextrins
help in reducing the volatile organic contaminants present in packaging materials
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 129

and also improve the barrier properties (diffusion rate and transmission rate), thus
maintaining the food quality (Suzuki & Sato, 1985). Cyclodextrins can release
antimicrobials and antioxidant compounds using CDs as carriers. As the humidity
levels in freshly cut vegetables or fruits increase, they can lead to spoilage; this
can be reduced by using cyclodextrins. At high relative humidity, high water–CD
interaction weakens the host guest interaction and consequently there is a release
of antimicrobial molecule which should protect the molecule against microbial
growth (Astray et al., 2009; Ayala-Zavala et al., 2008).
5. Cyclodextrins as stabilizers
Cyclodextrins are used for the preparation of stable water in oil emulsion such as
mayonnaise and salad dressing due to their hydrophobic cavity and hydrophilic
outer surface. In tomato ketchup, natural food colouring components can be
stabilized by the addition of beta cyclodextrins. This ketchup when prepared by
heating at 100
for 2 h did no discolouration, while the control did. Also, the
addition of cyclodextrins to emulsified cheese increased the water retention and
shelf life. However, the control discoloured when heated to this temperature
(Szente & Szejtli, 2004). Cyclodextrins also help in processed meat products in
improving water retention and texture (Ota & Takeda, 1981).
6. The browning or caking formation in the solid compositions containing sugars
and amino acids can be prevented by addition of oligosaccharides such as
cyclodextrin
By the addition of 20% of β-CD to powdered juice containing anhydrous glucose,
aspartic acid, d-alanine, citric acid, etc., the shelf life and also stability of the
product are enhanced. Even after 30–35 days at 40
C, no change in colour was
observed (Fujimoto, 1981). The control started to discolour even on the second
day and turned brown on fourth day (Szente & Szejtli, 2004). Starch gelatiniza-
tion is the process in which heat treatment is given to starch and water, resulting in
swelling of starch granules. However, addition of β-CD considerably improves
the gelatinization of wheat flour. Addition of 1.5% beta cyclodextrin improves the
solubility and swelling properties of starch granules. It has been observed that the
adding up of 1.5% of beta cyclodextrin augmented the viscosity of starch paste to
4%. Milk casein hydrolysate is a readily digestible protein and its bitter protein
can be removed by addition of 10% of beta cyclodextrin (Specht et al., 1981).
Similarly, the bitter taste of ginseng extract and propylene glycol can also be
eliminated by the use of beta cyclodextrin (Akiyama & Miyao, 1979).
7. Extraction efficiency is increased with the use of beta cyclodextrin
When the roasted coffee beans are extracted using beta cyclodextrin, the extrac-
tion becomes more effective and the complex containing beta cyclodextrin and
coffee can be used for effectual preservation of volatile components and also
provide better aroma of the product (Wagner et al., 1988). Low cholesterol butter
is also made by the addition of beta cyclodextrin. Molten butter is blended with
crystalline beta cyclodextrin, which forms stable complexes with triglycerides/
cholesterol and the cholesterol/β-CD is easily removed from molten butter.
130 R. Gautam and S. K. Arya

Ninety percent of cholesterol in fat milk can also be separated by the addition of
beta cyclodextrin; the butter obtained after this process does not retain any of the
cyclodextrin (Szente & Szejtli, 2004). Cyclodextrins are widely used in order to
modify the natural properties of materials, for example simply eggs have been
produced in USA which means a low cholesterol egg which is produced by the
addition of cyclodextrins to reduce cholesterol.
8. β-Cyclodextrin is more extensively used in food applications due to its availabil-
ity at low cost, chemically stable structure, and non-hygroscopic, non-toxic, and
edible nature. Ethyl vinyl alcohol copolymers are widely used in food industry
due to their properties like gas barrier to oxygen and organic compounds and high
transparency (Sanchez-Chaves et al., 2007; Lopez-de-Dicastilloa et al., 2010).
On comparing all the three forms of cyclodextrins, γ-cyclodextrins show the
highest flavour retention capacity among the other three.

4.6.2 Properties

S. no. Properties α-Cyclodextrin β-Cyclodextrin γ-Cyclodextrin References

1. Form Benzene Aromatic or Macrocycles Threapleton
complexes derivatives heterocycles or steroids et al. (2013)
with
2. Water Modest Lowest Highest Threapleton
solubility solubility solubility solubility et al. (2013),
(14.5) (1.85) (23.2) Arya et al.
(2006)
3. Hydrolysis by Resistant to Resistant to Completely Threapleton
human salivary hydrolysis hydrolysis digested by et al. (2013)
and pancreatic amylase
amylases
4. Production 1 (produced in 0.6 0.3 Threapleton
from much higher et al. (2013)
halophilic amount)
α-CGTase
5. No. of 6 7 8 Qi and
glucopyranose Romberger
units (1988), Radi
and Eissa
(2010),
Arya et al.
(2006)
6. Molar mass 972 1135 1297 Arya et al.
(g/mol) (2006)
7. Inner cavity 4.7–5.2 6.0–6.4 7.5–8.3 Arya et al.
diameter (Å) (2006)
8. Outer cavity 14.6 15.4 17.5 Arya et al.
diameter (Å) (2006)
1. 9. Cost per Highest Threapleton
production et al. (2013)
(continued)
4 Production of a, b, and g-Cyclodextrin Gluconotransferase (CGTase). . . 131

S. no. Properties α-Cyclodextrin β-Cyclodextrin γ-Cyclodextrin References

10. ΔH
8.36 9.98 11.22 Arya et al.
(ionization), 7.66 8.31 7.73 (2006)
kcal/mol
ΔH

(solution),
kcal/mol
11. ΔS
(solution), 7.67 8.31 7.73 Arya et al.
kcal/mol 28.3 22.4 17.6 (2006)
ΔS

(ionization),
cal/mol/K

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Enzyme in Milk and Milk Products: Role
and Application 5
Aparna Agarwal, Naman Kaur, Nidhi Jaiswal,
Memthoi Devi Heirangkhongjam, and Kanika Agarwal

Abstract

Enzymes are biocatalysts that catalyse a desired chemical reaction. Enzymes are
specific in their action and yield into products. The enzymes that are utilized in
the dairy industry for processing milk and milk products, like yoghurt, cheese,
and fermented milks, are commonly known as dairy enzymes. These enzymes
mostly aid in coagulation, cheese production, and enhancing shelf life of various
dairy products. The most used dairy enzymes include lactase, amylase, lipases,
transglutaminase, protease, catalase, and rennet. The functions of enzymes vary
with the kind of the product to be processed. Both endogeneous and exogeneous
enzymes are important for dairy application to provide functionality and safety to
the product along with shelf life extension.

Keywords
Enzymes · Dairy products · Cheese · Fermented products

5.1 Introduction

Enzymes, also known as biocatalysts, are proteins that immobilize chemical

reactions (Godfrey & Reichelt, 1983). Almost all the processes that exist in nature
require enzymes in order to occur at significant rates. During an enzymatic reaction,
the substrate molecules present on the onset are converted by the enzymes into
different molecules, called products (Whitehurst & Van Oort, 2009).
The nomenclature of enzymes depends upon the reaction carried out by them or
the substrate that they act upon as enzymes are highly specific for their substrates,

A. Agarwal (*) · N. Kaur · N. Jaiswal · M. D. Heirangkhongjam · K. Agarwal

Department of Food and Nutrition, Lady Irwin College, University of Delhi, New Delhi, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 139
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_5
140 A. Agarwal et al.

and hence, of various reactions; they catalyse only some of the many possibilities.
Generally, the suffix ‘ase’ is added to the substrate name (e.g. glucose-oxidase, an
enzyme which oxidizes glucose) or the type of reaction (e.g. a polymerase for a
polymerization). However, pepsin, rennin, and trypsin are enzymes that were studied
originally, and hence, are the exceptions to this rule. Enzymes can be categorized
based upon the reactions they catalyse. These include, EC 1 oxidoreductases,
enzymes that immobilize oxidation or reduction reactions (e.g. oxidases or
dehydrogenases); EC 2 transferases enzymes (e.g. transphosphorylases and
phosphomutases, example of a phosphate group), transfer a functional group such
as a methyl or phosphate group; EC 3 hydrolases (e.g. hydrolases, including
carbohydrases, deaminases, and proteases; hydrases such as fumarase, enolase,
and carbonic anhydrase) are enzymes which catalyse the process of hydrolysis of
different bonds (involving mostly, addition or removal of water); EC 4 lyase
enzymes split bonds by methods other than hydrolysis and oxidation or form a
C¼C bond; EC 5 isomerases stimulate isomerization modifications within a mole-
cule (e.g. glucose-isomerase); and EC 6 ligases combine two molecules using
covalent bonds (Whitehurst & Van Oort, 2009).
All enzymes reduce the energy required by a reaction to take place. Different
enzymes have different mechanisms to catalyse reactions, which involve creating
conditions in which the transition state is stabilized in order to reduce the activation
energy. This can be accomplished by providing a substitute way which involves
bonding, and consequently, stabilizing the transition-state conformation of the
substrate. For example, temporary reaction with the substrate in order to form an
intermediate enzyme–substrate complex, where an enzyme plays an indispensable
role. Enzymes possess an interesting property which is their specificity. For exam-
ple, certain enzymes display absolute specificity which implies that they immobilize
only one specific reaction or are selective for a certain kind of chemical bond or
functional group. Generally, there exist four types of specificity which are: Absolute
specificity; here, highly specific enzymes stimulate only one reaction; Group speci-
ficity; here, enzymes that are group-specific act only on molecules with certain
functional groups, such as phosphate, amino, or methyl groups; Linkage specificity,
where enzymes act on chemical bonds of specific nature, regardless of the molecular
structure; and, Stereochemical specificity, where enzymes act only on a certain steric
or optical isomer and not on their isomeric counterparts (Whitehurst & Van Oort,
2009). These biocatalysts are highly potent, as even in a very small quantity, they are
capable of augmenting the rate of reactions to several folds. For example, orotidine-
50 -phosphate-decarboxylase is an enzyme that facilitates a reaction to occur in
milliseconds which would generally take millions of years (Callahan & Miller,
2007; Radzicka & Wolfenden, 1995). As a result, enzymes have advanced as a
vital component for the sustenance of life. Evidently, over 5000 types of biochemical
reactions have been found to be catalysed by enzymes (Schomburg et al., 2013).
Enzymes take part in several metabolic functions of living organisms. For
example, they (kinases and phosphatases) are crucial for processes like signal
transduction and cell regulation. They are also responsible for inducing movement,
with adenosine triphosphate (ATP) which hydrolyses myosin in order to produce
5 Enzyme in Milk and Milk Products: Role and Application 141

contraction in the muscles and to also mobilize cargo around the cell as part of the
cytoskeleton (Macrae, 1985). They also serve a vital role in the ‘digestive systems’
of various mammals and animals. For example, amylases cleave starch molecules
and proteases cleave protein molecules (Whitehurst & Van Oort, 2009). Besides,
enzymes have also been utilized at an industrial scale for antibiotic production.
Moreover, there are certain enzymes which are used in household products like
detergents that help in cleaving several stains on clothes. Additionally, as a result of
the ability of enzymes to alter and enhance the nutritional, functional, and organo-
leptic properties of various ingredients and products, they have also found a wide-
spread application in the food industry. They are employed in the food industries to
manufacture bread, alcoholic drinks, and milk-based products like cheese and
yoghurt, and to improve the flavour and texture of dairy-based products (Qureshi
et al., 2015).
The enzymes that are utilized in the dairy industry for processing milk and milk
products like yoghurt and cheese are commonly known as dairy enzymes. These
enzymes mostly aid in coagulation, cheese production, and enhancing shelf life of
various dairy products. The most used dairy enzymes include lactase, amylase,
lipases, transglutaminase, protease, catalase, and rennet. Lactase enzyme is mostly
utilized for hydrolysing lactose molecules to glucose and galactose sugars and
augment the solubility and sweet flavour in various dairy products. Amylases are
biocatalysts which break down starch by immobilizing the process of hydrolysis of
internal α-1,4-glycosidic bonds of polysaccharide sugars like maltose, glucose,
dextrin, and maltotriose, while preserving the α-anomeric configuration in the
products (Takata et al., 1992). Lipase is another commonly used enzyme which is
employed mostly to mature cheese and improve flavours. Another dairy enzyme is
transglutaminase, commonly known as TGases, which plays a pivotal role in the
enhancement of protein functionality in dairy products. Similar to lipases are
different types of protease enzymes which are also utilized in manufacturing of
cheese to increase the rate of cheese aging, as a functional property, and modifying
milk protein to lower the allergic effects in cow milk-based infant food products
(Fox, 2002). Another frequently used dairy enzyme in cheese production is catalase.
It is employed in producing some cheeses in lieu of pasteurization (e.g. Swiss
cheese) to save regular milk proteins that are responsible for providing certain
benefits to finished product and flavour enhancement of the cheese (Fox, 2002).
Rennet, which is also known as rennin, is a combination of pepsin and chymosin
extracted from animals as well as microbiological sources. It is also employed in the
cheese manufacturing industry to curdle milk (Merheb-Dini et al., 2010).
This chapter aims to emphasize on various endogenous and exogenous dairy
enzymes like lactase, amylase, lipases, transglutaminase, protease, catalase, and
rennet which are used widely, not only in the production of milk and milk-based
products, but also lactose-free products. It briefly delineates the structure, sources,
and applications of these enzymes in the dairy industry. Additionally, the chapter
also meticulously explains the use of these dairy enzymes in the production of
cheese, lactose-free milk, fermented milk as well as in packaging.
142 A. Agarwal et al.

5.2 Endogenous Enzymes

All biochemical processes and metabolic pathways are propelled and modulated by a
group of enzymes mobilizing a series of reactions. These enzymes catalyse reactions
and improve the product yield up to several folds. However, in their absence, the rate
of reaction decelerates, and eventually reaches impasse. Enzymes, on the basis of
their source, can be broadly classified into two categories—exogenous enzymes and
endogenous enzymes (EHEs). Enzymes secreted by animals are known as exoge-
nous enzymes. These are used to enhance the process of digestion in animals.
Optimizing the conditions required for exogenous enzymes is a tiresome job,
which include optimum temperature, pH, and enzyme-substrate concentration.
Additionally, these enzymes are expensive, and hence, increase the ultimate cost
of the by-product. Contrariwise, EHEs are enzymes that are added to the animal
feed. The efficacy, ease of use, unique properties, and profitability of these enzymes
make them an appealing candidate for industries for the production of numerous
by-products (Ramawat & Mérillon, 2015).

5.2.1 Lactase

β-Galactosidase, also known as lactase, is an endogenous enzyme which is account-

able for hydrolysing lactose, a disaccharide composed of subunits of galactose and
glucose (Carrara & Rubiolo, 1994; Felicilda-Reynaldo & Kenneally, 2016;
Vandenplas, 2015; Lukito et al., 2015). This enzyme is located in the brush border
of the small intestine of humans and other mammals. It plays a crucial role in the
absorption of lactose. Lactase cleaves lactose by breaking the bond that links the two
monosaccharide sugars—galactose and glucose and eventually resulting in
glycolysis.
This enzyme also results in transgalactosylation of lactose to allolactose, which is
then split into monosaccharides. The binding of allolactose to lacZ repressor
modulates the amount of lactase in the cell by generating positive feedback (Pivarnik
& Senecal, 1995).

5.2.1.1 Structure
Lactase or β-Galactosidase is a tetramer, that is four identical polypeptide chains.
Each chain comprises 1023 amino acids that amalgamate to create five distinct
structural domains. Of the five domains, one is a jelly roll barrel, whereas the rest
comprise β-sandwich, fibronectin, and a central domain. The central domain with
TIM-type barrel is made of tetramer subunits and acts as the active site (Huber et al.,
1976). However, cleavage of tetramer to dimers causes the active site to inactivate.
The α-peptide present at the n-terminal of lactase is involved in α-complementation
and contributes in subunit interface (Corral et al., 2006).
5 Enzyme in Milk and Milk Products: Role and Application 143

5.2.1.2 Sources
Lactase can be found in fungi, bacteria as well as in yeasts. It can be mostly found in
plants, like apples, apricots, peaches, and almonds. However, for industrial and
commercial scale, lactase is usually acquired from fungi like Aspergillus and
Kluyveromyces (Zhou & Chen, 2001).

Bacterial Sources
Lactase derived from bacterial sources possesses high activity, stability, and ease of
fermentation, and hence, it is utilized to hydrolyse lactose. Studies have reported
Lactobacillus and Bifidobacterium species to be an efficacious probiotic. As a result,
they are commonly used to obtain lactase. It has been reported that lactase derived
from bacterial strains like Bifidobacterium longum strain CCRC 15708,
Bifidobacterium infantis strain CCRC 14633, and Bifidobacterium longum CCRC
15708 possess high enzyme activity (Hsu et al., 2007).

Yeast Sources
Lactase is extracted from yeast to produce lactose-free products. Kluyveromyces
lactis, a variety of yeast found in dairy environment, is one of the most commercially
used sources of lactase (Pivarnik & Senecal, 1995). The optimal pH of lactase
obtained from a yeast lies between 6.0 and 7.0. Studies have reported that
Kluyveromyces marxianus, another variety of yeast, can be utilized to produce
homologous enzymes of lactase and other heterologous proteins that have the ability
to thrive on a variety of substrates with lactose as their only source of energy. It has
been found that a cold-active acidic lactase extracted from Guehomyce spullulans, a
strain of psychrophilic yeast species, can be utilized for whey and milk hydrolysis in
food industries (Saqib et al., 2017).

Fungal Sources
Fungi have been known to yield enzymes that are highly stable. Lactases obtained
from fungal sources have an optimal acidic pH range of 2.5–5.4. This makes them
highly potent for hydrolysing lactose of an acidic material such as whey. The most
commonly used fungal sources to produce lactases that have been accepted as
“generally recognized as safe” (GRAS) by FDA are Kluyveromyces lactis,
Kluyveromyces fragilis (Saccharomyces fragilis), and certain Aspergillus species.
Lactase extracted from Aspergillus oryzae is used on commercial scale and has been
reported to be an effective source for whey utilization. Lactase generated from
another fungus, that is, Aspergillus niger, is used to remove galactose residues
from oligosaccharides and polysaccharides that are obtained from plant (Kazemi
et al., 2016).

Plants
Lactase can also be obtained from a variety of plant sources like apple, tomato,
mango muskmelon, avocado, coffee, kiwifruit, and Japanese pear plants (Seddigh &
Darabi, 2014). These enzymes contribute to plant growth, ripening of fruits, and
hydrolysis of lactose. It has been reported that this enzyme reduces galactosyl
144 A. Agarwal et al.

content of cell wall of persimmons, causes cell wall hydrolysis in papaya, and
facilitates the process of fruit ripening (Saqib et al., 2017).

5.2.1.3 Applications in Dairy Industry

Lactase has multiple industrial applications. They are used to produce lactose-free
food products for patients suffering from lactase deficiency (Karasova et al., 2002).
As a result of the hygroscopic nature of lactase, which leads to crystallization in
foods, it is utilized for hydrolysing lactose to avert crystallization of lactose in
desserts that are frozen and concentrated. This helps in reducing the lactose content
of milk to be consumed by individuals with lactase deficiency (Champluvier et al.,
1988). Additionally, on a commercial scale, this enzyme is also used to dispose
whey. Whey disposal is a grave environmental concern as it is disposed into water
streams, which leads to water pollution (Brandão et al., 1987). Hence, lactase is
utilized to convert whey into useful products that have a range of applications in food
industry such as ethanol and sweet syrup (Zhou & Chen, 2001).

Production of Hydrolysed Milk Products

A significant percentage of the world’s population has been known to develop
lactase deficiency which causes lactose intolerance (Sitanggang et al., 2016). The
burden of lactose intolerance among the Asian population is nearly 90%. Individuals
suffering from lactose intolerance can consume food products with either no or least
amount of lactose in them. A solution to this problem is to treat milk or milk-based
products with lactase. Lactase helps in pre-hydrolysing lactose in these products,
thus reducing the concentration of lactose in them (Ramana Rao & Dutta, 1978).
Besides, lactase is also preferred to decrease the crystallization in frozen and
condensed milk and milk products which occurs as a result of high concentration of
lactose. Lactase also helps in improving the texture of milk and milk-based foods
and in making them even more digestible. The by-products obtained from lactose
hydrolysis (i.e. glucose and galactose) have been reported to be fermented easily.
This helps in minimizing the total time required to attain the optimal pH in various
milk-based foods like cottage cheese and yogurts. Additionally, lactase has been
reported in reducing the calorie content of the end product by reducing the require-
ment of adding extra sweeteners (Domingues et al., 2005).

Whey Utilization
Cheese industry has been known to yield a massive amount of whey as a derivative.
The chief constituents of whey include lactose, proteins, and minerals. The lactose in
whey is associated with chemical oxygen demand and biochemical oxygen demand.
Therefore, despite the fact that in developing countries a substantial amount of whey
is used to produce permeates and protein concentrates, whey, when disposed in
water streams, leads to severe water pollution. Nevertheless, whey, when converted
into beneficial products such as ethanol as well as lactase, can be utilized for the
production of lactose-free food products (Kokkiligadda et al., 2016). Additionally,
treating whey with lactase can help in converting it into a substrate that is readily
available for cell cultivation (Parashar et al., 2016). Another application of lactase
5 Enzyme in Milk and Milk Products: Role and Application 145

includes recovering of highly functional whey proteins like lactalbumin through

ultrafiltration and subsequently hydrolysing them to manufacture numerous pharma-
ceutical intermediates (Domingues et al., 2005).

Production of Galacto-Oligosaccharides
Another application of lactase enzyme includes the production of galacto-
oligosaccharides (GOS). GOS are nondigestible prebiotics and hence play a crucial
role in improving human health by modifying intestinal microflora of human and
promoting the growth of useful bacteria in the intestine such as Lactobacillus and
Bifidobacterium species. GOS are produced from the transglycosylation activity of
lactase during lactose hydrolysis. It can be stored at room temperature for longer
period of time due to its acidic conditions. This characteristic of GOS makes it
capable of being used for the production of different products without any decom-
position. The oligosaccharide content in GOS ranges from 1% to 45% and depends
further on the source of enzyme and the total amount of saccharides (Pazur, 1953).
GOS is also present in human milk and is known to help in increasing the natural
microflora of small intestine of a breast-fed infant, that is bifidobacteria. This
bifidogenic activity of GOS plays a vital role in reducing the number of pathogenic
bacteria. In view of this characteristic, companies dealing with infant foods are now
producing milk- and cereal-based food products containing GOS (Matto & Husain,
2006). Additionally, GOS also functions as soluble fibres and gets fermented by the
natural microflora present in large intestine. This fermentation produces end
products that are short-chain fatty acids that help in reducing the pH of fecal matter.
The GOS is indigestible by the bacteria present in mouth; due to this reason they do
not contribute to the development of cariogenicity (Matioli et al., 2003).

5.2.2 Amylase

Amylases are enzymes that degrade starch by catalysing the process of hydrolysis of
internal α-1,4-glycosidic bonds of polysaccharides like maltose, glucose, dextrin,
and maltotriose, while retaining the α-anomeric configuration in the products
(Takata et al., 1992). They can be found in a variety of organisms. These enzymes
are known to have multiple applications, and hence, hold a major share in market for
the sales of enzymes (Vaidya et al., 2015). For centuries together, amylases derived
from microbial and plant sources have been utilized in the brewing industry.
Amylases extracted from fungal sources have been employed in the Asian cuisine
(Hernández et al., 2006). The growing research interest in amylases obtained from
bacterial, fungal, and viral sources in comparison to amylases that are derived from
animals and plants sources can be ascribed to the ease in its large-scale production
and industrial application (Mukherjee et al., 2009).
Amylases (EC 3.2.1.0) are classified into three subtypes that are α-amylase,
ß-amylase, and γ-amylase. The two types of amylases—endoamylases and
exoamylases, are categorized on the basis of the method by which the glycosidic
bond is attacked (Vaidya et al., 2015). α-Amylases (α-1,4-glucan-glucanohydrolase,
146 A. Agarwal et al.

EC 3.2.1.1) are extracellular enzyme (Cherry et al., 2004) that is responsible for
degrading α-1,4-glucosidic linkage of starch and associated products and producing
oligosaccharides (Baysal et al., 2008). β-Amylase (1,4-α-D-glucan maltohydrolase;
glycogenase; saccharogen amylase, EC 3.2.1.2), another form of amylase, works
from its non-reducing end and mobilizes the process of hydrolysis of the second
α-1,4-glycosidic bond by cleaving two maltose units at a time. At the time of fruit
ripening, this enzyme helps in cleaving starch present in the fruit and results in the
sweet flavour of ripe fruit. γ-Amylase (EC 3.2.1.3) (Alternative names: Glucan
1,4-α-glucosidase; amyloglucosidase; Exo-1,4-α-glucosidase; glucoamylase; lyso-
somal α-glucosidase; 1,4-α-D-glucan glucohydrolase), the third sub-type of
amylases, is known to cleave α-(1–6)-glycosidic linkage along with the cleavage
of the last α-(1–4)-glycosidic linkages at the non-reducing end of amylose and
amylopectin to yield glucose. This sub-type has an optimum pH of 3, and hence,
is efficacious in acidic conditions (Saini et al., 2017).

5.2.2.1 Structure
Amylase possesses a three-dimensional structure that enables it to bind to substrates.
The high activity of specific catalytic groups helps in the cleavage of the glycoside
links (Iulek et al., 2000). The human α-amylase is made of 512 amino acids in a
single oligosaccharide chain. It comprises calcium and has a molecular weight of
57.6 kDa (Whitcomb & Lowe, 2007). Amylase possesses three domains: A, B, and
C. The A domain which is the largest exhibits a classical barrel-shaped (β/α)8 super
structure. The B domain is cemented to A domain by disulphide bond and is
positioned between the A and C domains. The C domain possesses a sheet-like
organization and is attached to the A domain by a simple polypeptide chain. The
substrate binding site or the active site of the enzyme is positioned in a long cleft
which is situated between the carboxyl end of the A and B domains. The calcium ion
(Ca2+) is positioned amid the A and B domains. This ion may serve in the process of
stabilizing the three-dimensional structure of the enzyme and act as an allosteric
activator. The active site of the enzyme comprises 5 sub-sites with the catalytic site
located at sub-site 3. Substrate can attach to the first glucose residue present in
sub-site 1 or 2, thereby permitting the division to take place between the first and
second or second and third glucose residues (Whitcomb & Lowe, 2007).

5.2.2.2 Sources
Amylases are a group of enzymes produced in nature by plants, animals, and
microorganisms.

Alpha Amylase
Alpha amylase can be isolated from a variety of sources like plants, animals, and
microbes (Pandey et al., 2000). Although alpha amylase has been extracted from a
variety of bacteria, fungi, actinomycetes, and yeasts, the enzymes obtained from
fungi and bacteria have been found to have major industrial applications. This can be
ascribed to their consistency, cost-effectiveness, less time and space requirement for
their production, and ease of process optimization and modification (Ellaiah et al.,
5 Enzyme in Milk and Milk Products: Role and Application 147

2002). The microbial enzyme derived from the microbial sources is capable of
meeting the industrial demand and a huge amount of them are commercially
available. For centuries, amylases derived from microbes and plants have been
used as food additives (Hernández et al., 2006). The bacterial amylases are mostly
derived from Bacillus sp. like B. subtilis, B. stearothermophilus, B. licheniformis,
and B. amyloliquefaciens. Most of the fungal sources of amylases belong to terres-
trial isolates like Aspergillus species (Jamal et al., 2011). The amylases derived from
fungi are extensively utilized to prepare oriental foods (Popovic et al., 2009). The
alpha amylases have also been isolated from filamentous fungi, which are high-
yielding producers of extracellular proteins (Coradin et al., 2011).

5.2.2.3 Applications in Dairy Industry

Effect of Exogenous Amylase on Lactation Performance of Dairy Cows

Many researches have evidently exhibited the resistance of certain exogenous
enzymes to the degradation in the rumen, which has the potential to augment the
digestibility of feeds and eventually enhance animal performance (Hristov et al.,
1998). Various studies have been focussed on the addition of fibrolytic enzymes to
ruminant diets as fiber digestion is generally never amplified in the rumen
(Beauchemin et al., 2003). Certain in vitro and in vivo evidences have reported
that even though starch digestion in the rumen is not usually considered to be
limiting, amylase enzymes contribute in enhancing the animal performance
(Andreazzi et al., 2018). Intensive lactation augments the intake of feed and passage
throughout the rumen. Although this increases the volume of starch passing the
rumen without degradation, the capacity of small intestine to digest starch is limited.
Researches have reported that supplementing the feed with α-amylase may enhance
ruminal starch digestion without an intensive production of volatile fatty acids and
increased risk for acidosis (Nozière et al., 2014).

5.2.3 Transglutaminase

All transglutaminases (EC 2.3.2.13), also known as protein-glutamine

γ-glutamyltransferase, fall under the class of transferases (Marx et al., 2007;
Trespalacios & Pla, 2007). Transglutaminases (TGases) can be found naturally in
a variety of living organisms. They are known to participate in several biological
functions, which include clotting of the blood and healing of wounds (Jaros &
Rohm, 2016). They mobilize the formation of an iso-peptide bond between the
group of γ-carboxamides of glutamine residues (donor) and the first order ε-amine
groups of different compounds, for example, proteins (acceptors of an acyl residue).
Additionally, in the absence of free amine groups, TGase catalyses deamination,
where water serves as an acyl acceptor. TGase mobilizes the reactions that bring
about substantial modifications in the physical and chemical properties of proteins,
which include viscosity, elasticity, thermal stability, and resilience.
148 A. Agarwal et al.

For the first time, TGases were derived from tissues of mammals and body fluids
to investigate their applications in industry. The first commercially available TGase,
extracted from the liver of guinea pig, was not found to be appealing candidate for
industrial use ascribing to the limited supply and requirement of Ca2þ for activation
which resulted in the high production cost of the enzyme. Likewise, another TGase
known as Factor XIII, which was derived from blood plasma, exhibited a major
drawback of red pigmentation and the requirement of thrombin for activation. In
1980s, during the screening of microorganisms for TGase activity, microbial TGase,
which is independent of Ca2þ and possesses low substrate specificity, was identified
from Streptomyces mobaraensis. Production of mTGase results from conventional
fermentation of the wild-type S. mobaraensis strain (GRAS status) with consequent
extraction of the protein that is secreted. Thus far, mTGase is the only commercially
available TGase in the food industry (Jaros & Rohm, 2016).

5.2.3.1 Structure
mTGase, a monomeric protein with 331 amino acids, has an isoelectric point of 8.9.
Its secondary structure comprises 8 β-strands adjoined with 11 α-helices. The
enzyme acquires a disk-like shape that has a deep cleft at the edge. At this cleft is
situated a single cysteine residue (Cys64). Molar mass of mTGase as evaluated by
the technique of mass spectrometry and amino acid sequencing is 37.86 kDa.
Usually, mTGase exhibits activity at pH range of 4 and 9. However, the optimum
pH of the enzyme lies in the range 6–7. The optimum temperature for mTGase is
50  C. Although it is immobilized at 70  C within minutes, it maintains some
residual activity at near-freezing temperatures. Another characteristic of mTGase is
its stability under high hydrostatic pressure. This characteristic provides the
prospects of alteration of proteins, even in situations where they are not available
for mTGase under atmospheric pressure (Jaros & Rohm, 2016).

5.2.3.2 Sources
TGases are widely spread in nature (Kashiwagi et al., 2002). They can be derived
from various invertebrates and microbial cells (Yu et al., 2008; Griffin et al., 2002) as
well as mammalian tissues (Yasueda et al., 1994). TGases can also be found in
tissues of various plants such as orchard apple, soy, fodder beet, and topinambour
(Falcone et al., 1993). These enzymes have been extracted from microbes belonging
to Streptoverticillium sp. and Physarum polycephalum. TGases are biosynthesized
as an extracellular enzyme by Streptoverticillium sp., Streptoverticillium
cinnamoneum subsp. cinnamoneum (Duran et al., 1998), Streptomyces netropsis
(Yu et al., 2008), Streptoverticillium griseocarneum (Gerber et al., 1994),
Streptoverticillium ladakanum (Ho et al., 2000), and Streptomyces lydicus
(Færgemand & Qvist, 1997).

5.2.3.3 Applications in Dairy Industry

Various researches have reported the commercial availability of TGases and their
crucial role in enhancing protein functionality in dairy products. It has been found
that these enzymes avert syneresis in yoghurts and result in firmer yet soft texture
5 Enzyme in Milk and Milk Products: Role and Application 149

(Kieliszek & Misiewicz, 2014). A study reported that TGases at the level of 0.02%
improve melting, firmness, flexibility, and organoleptic properties of high fat Moz-
zarella cheese. However, in the case of Mozzarella with low fat content, 0.05% of
TGase provides adequate meltability, flexibility, free oil formation, and organolepti-
cally to the cheese. The cross-linking property of TGase helps in augmenting soft
cheese production, reducing syneresis, extending shelf life, and decreasing nutrient
bioavailability for deteriorative microorganisms (Mahmood, 2009). It has also been
reported that this enzyme can be utilized to partially replace fat in ice-creams, and it
is also responsible for increasing overrun, partial amalgamation of fat globules,
consistency, melting resistance, firmness, and viscosity in ice cream (Tafes, 2019).
A study reported that up to 0.5% of TGase was more efficacious to enhance the
functional properties of yogurt that was made from goat milk. The cross-linking
property of TGase was found to be accountable for enhancing the gel consistency
and reducing the whey separation substantially in yoghurt (Farnsworth et al., 2006).
Another study reported that when stirred yogurt was developed through covalent
cross-linking by the combined action of inactivated mTGase and glutathione, no
negative effects on the fermentation of yogurt were observed. However, a substantial
rise in apparent viscosity as well as protein polymerization was observed in yogurt
that was only developed by TGase (Bönisch et al., 2007). The use of TGase in high
concentration was found to reduce syneresis and increase the viscosity of yogurt.
However, a minor bottleneck was observed with regard to the growth of Lactic Acid
Bacteria. This led to reduced production of acid and acetaldehyde in comparison to
those reported for the control. Nevertheless, TGase concentration up to 0.3 g/L was
reported to be optimum and was recognized as an acceptable substitute to stabilizers
to be added in the production of fat-free yogurt (Ozer et al., 2007). Studies identified
TGase as an efficacious candidate to enhance the physical properties of yogurt when
its constitution was modified by the addition of whey (Şanli et al., 2011).

5.3 Application in Dairy Industry

5.3.1 Lactose-Free Milk

Low levels of lactase production a human beings has been known to be a leading
cause of “lactose intolerance”, which is an intestinal symptomatic condition. A
lactose-intolerant individual may experience clinical symptoms like nausea, abdom-
inal pain, flatulence, diarrhea, and bloating after consuming food that contains
lactose. It has been estimated that about 70% of the world population, including
all the age groups, is suffering from lactase deficiency (Xavier et al., 2018). Lactose-
intolerant individuals can consume dairy-fermented products that comprise very
little or no lactose in them (Saqib et al., 2017). Currently, there is an increase in
demand for lactose-free dairy among the lactose-intolerant populations. This can be
ascribed to the presence of little or no lactose in these products and their ability to
provide vital macro- and micro-nutrients present in milk to individuals suffering
from lactose intolerance (Dekker et al., 2019).
150 A. Agarwal et al.

The dairy industry employs different processing techniques to reduce the lactose
content in the dairy products to produce lactose-free products. Cheese production
involves certain techniques which help in reducing the lactose content. For example,
Gouda cheese production involves a curd washing step to reduce the lactose content.
Although the lactose content in most of the cheeses is already relatively low without
ripening, in several other types of cheeses, the lactose content is reduced during the
process of ripening with the help of lactic acid bacteria. Generally, aged and hard
cheeses like Parmesan, Swiss, or Cheddar cheeses have a very low concentration of
lactose. Contrariwise, young and fresh cheeses may consist enough lactose that may
be capable of inducing a reaction among individuals with lactose intolerance,
depending on the quantity consumed. Butter is another dairy product with low
lactose content. The majority of water-soluble constituents of milk inclusive of
lactose are eliminated, during the production of butter resulting in the reduction of
lactose concentration in butter (Dekker et al., 2019).
Besides reducing the lactose content in dairy products, another solution to
produce lactose-free dairy products includes hydrolysing lactose into glucose and
galactose using the lactase enzyme. The monosaccharides produced get easily
adsorbed in the small intestine and avert the incidence of symptoms associated
with lactose intolerance. Currently, neutral lactases and acid lactases are the two
kinds of commercially available lactases (Dekker & Daamen, 2011). The former is
chiefly employed on an industrial scale to produce lactose-free dairy products, while
the latter is provided as a dietary supplement to the consumers for its consumption
along with regular dairy products to induce cleavage of lactose in the stomach
(Dekker et al., 2019).

5.3.2 Production of Lactose-Free Milk

Researches have reported that lactose dosage of less than 12 g per meal may result in
mild symptoms among lactose-intolerant individuals; however, the dairy industry
aims to maintain the lactose content as low as possible (Suchy et al., 2010).
Currently, there does not exist global agreement on the regulatory requirements for
lactose-free claims. In the past, lactose reduction to 0.5% or 0.1% was considered
enough by the dairy producers, but, in some countries, lactose content less than
0.01% is considered to be the current requirement to consider milk to be lactose-free.
To achieve such low lactose concentration in milk production entails special atten-
tion to the milk processing, to the dosage and efficiency of the enzyme used in this
process, and to sensitive analytical methods to determine such low amounts of
lactose (van Scheppingen et al., 2017).
Presently, there exist two processes that are used in the production of lactose-free
milk (Troise et al., 2016; Harju, 2004). These include batch process and aseptic
process and both of them use soluble lactase enzyme. Although various studies have
suggested the processes that rely on an immobilized enzyme, these processes have
not been employed in industrial practice to produce lactose-free milk either in the
5 Enzyme in Milk and Milk Products: Role and Application 151

past or in present due to issues related to the microbial stability of the end product
(Finocchiaro et al., 1980).

5.3.2.1 Batch Process (Pre-Hydrolysis)

The batch process involves the addition of a neutral sample of lactase to a tank
containing raw milk and subsequent incubation for approximately 24 h accompanied
with continuous slow stirring in order to prevent creaming. In order to prevent
microbial growth in the milk, which is not yet sterile, cool conditions with tempera-
ture ranging between 4–8  C are maintained to perform this process. Once the
process of incubation is completed, the milk is then pasteurized, homogenized,
and packaged.
It is important for the enzyme dosage to be adequate to reach the requisite limit for
removing lactose at low temperature of the incubation and during the set time period.
It is because of this reason that the enzyme dosage for this process is kept fairly high
and enzymes are selected based on their high activity at neutral temperature. The
batch incubation is discontinuous as it involves a tank in the factory, delaying the
pasteurization of the milk by a day. This may prove to be a setback for certain
factories during high productivity. In conditions like these, lactase enzyme having
higher specific activity may be helpful in reducing the production time, thereby
increasing the total yield of the factory. Additionally, to prevent microbial spoilage
of milk that may result due to delay in pasteurization of the milk, it is important for
the milk quality to be impeccable.
Generally, the lactose-free milk produced with the batch process is comparatively
unresponsive to the side activities of the enzyme. This can be ascribed to the short
storage period of the milk at refrigerated conditions and to the process of pasteuriza-
tion or sterilization after incubation of the enzyme, which is responsible for the
inactivation of most of the enzymatic activities. In the past, certain lactase
preparations displayed proteolytic side activities; however, these issues seem to be
resolved with only rare complaints occurring for the lactose-free milk produced with
the batch process (Mittal et al., 1991).
Techniques like chromatography and ultra- and nano-filtration have been
employed in combination with lactose hydrolysis to avoid the doubling of the milk
sweetness (Harju et al., 2012; Jelen & Tossavainen, 2003). This results in the
production of lactose-free milk of outstanding quality, with its taste nearly alike
the regular milk.

5.3.2.2 Aseptic Process (Post-Hydrolysis)

The aseptic process involves the sterilization of the milk using the UHT technique
and the subsequent injection of the prepared sterile lactase into the milk immediately
before packaging (Dahlqvist et al., 1977). This packaged UHT milk is then kept
quarantined for almost 3 days at optimum temperature, which provides adequate
time for lactose hydrolysis to occur before the milk is distributed to the retailer. The
aseptic process is not employed for pasteurized milk for the production of lactose-
free milk, due to the absence of quarantine period (Dekker et al., 2019).
152 A. Agarwal et al.

Fundamentally, there exist two techniques to obtain sterile lactase enzyme. The
first technique involves pre-sterilization of lactase by the enzyme manufacturer and
usage of distinct sterile dosing equipment that is required for the sterile injection.
The second technique involves the filter-sterilization of the unsterile enzyme imme-
diately before incorporating it to the sterilized milk at the dairy industry (Dekker
et al., 2019).
Considering the high incubation time and temperature required in this process,
the enzyme dosage can be kept much lower. Process control in this process is not
present as the enzyme is vigorous only in the final milk package. The dairy
manufacturer should also consider the aspects such as deviation in the storage
temperature in un-thermostated warehouses, resulting from the seasonal transitions
such as summer to winter, when dosing the enzyme (Dekker et al., 2019).
During the aseptic process, special equipment and consumable costs are essential
requirements specifically for the in-factory filtration. Highly skilled operators are
employed during the process of lactose injection in order to prevent microbial
contamination in the milk. When organized appropriately, the process can function
as a completely continuous operation, thus making the aseptic process a desired
technique in industries requiring a high yield (Dekker et al., 2019). The aseptic
process for the production of lactose-free UHT milk can only be considered to be a
fully developed technique after the crucial refinement of the lactase enzyme quality.
Researches have reported that the arylsulfatase side activity in the preparation of
lactose may result in serious medicinal off-flavours during storage. This has been
ascribed to formation of p-cresol from sulphonated-cresol, which occurs naturally in
the milk (De Swaaf et al., 2006). Currently, arylsulfatase-free lactase enzymes are
available commercially. It is important for a lactose-free UHT milk producer to
consider utilizing only the supreme quality lactase enzymes for this process in order
to avert shelf life-related complication (Dekker et al., 2019).
Lactose hydrolysis in milk has been known to augment the monosaccharides’
content, resulting in an efficient Maillard reaction, which may be amplified due to the
inadequate proteolysis by proteases. These proteases may be present naturally in the
milk or they may originate from the lactase preparation. The amplified reactions lead
to the increase in the formation of off-flavours and greater browning of lactose-free
milk in comparison to regular milk. When stored at higher temperature, it also results
in a reduced nutritional value of the product (Troise et al., 2016; Jansson et al., 2014;
Evangelisti et al., 1999). The enhanced maillard reaction may be considered as the
critical determinant of the decreased shelf life of lactose-free UHT milk in compari-
son to regular UHT milk. In the past, various researches have suggested that the
batch process for the production of lactose-free UHT milk may result in compara-
tively higher browning than the milk produced using the aseptic process (Mendoza
et al., 2005). However, recent studies have reported the relevance of the storage
conditions (temperature) and lactase quality for determining shelf life (Troise et al.,
2016). Excellent shelf life was found for lactose-free UHT milk produced with the
batch process, and milk browning during storage is, therefore, largely independent of
the production process that is used (Dekker et al., 2019).
5 Enzyme in Milk and Milk Products: Role and Application 153

5.3.3 Other Dairy Products

Besides lactose-free milk, various other lactose-free dairy products are manufactured
with the use of enzymatic treatment. Flavoured milk is one such product that is
produced using a similar process (McCain et al., 2018). The comparatively stronger
production of colour and flavour by the flavoured lactose-free milk has been reported
to reduce browning and production of off-flavours due to the maillard reaction.
Unlike regular lactose-free milk, the flavoured lactose-free milk requires less addi-
tion of extra sugar, due to the lactose treatment; however, in certain flavoured milks
like highly sugared chocolate milk, the process of lactose hydrolysis may not be
adequate for complete replacement of sugar addition (Li et al., 2015), and thus there
may be a requirement to add additional sweeteners. Another lactose-free dairy
product is dairy powder, which can be developed from either milk or whey, which
is made lactose-free with the batch process. A major setback reported during the
production of lactose-free dairy powder is the high monosaccharide content in the
treated milk, which causes a reduction in the glass-transition temperature. This
phenomenon further results in the contamination of the spray dryer when the drying
conditions are not adjusted (Torres et al., 2017). Additionally, packaging of the
lactose-free dairy powder should be done cautiously as the high hygroscopic nature
of the powder results in its caking during storage. It is because of these challenges
that the lactose-free dairy powders are not a part of big markets like regular milk
powders. Cheese is yet another dairy product that can be made lactose-free. This is
done using lactase enzyme to incubate the cheese milk before renneting. This
method is used majorly for cheeses that are young and fresh, as they comprise a
considerable quantity of lactose. In the case of ripened cheeses, lactase incubation is
not required as lactose gets completely consumed by the lactic acid bacteria. The
studies conducted in the past have reported that unlike yoghurt, lactase treatment of
cheese milk stimulates the process of acidification during cheese manufacturing.
Furthermore, the addition of lactase during the process of ripening enhances the
flavour of cheese. It is unclear if these effects are a result of the stimulation of the
cheese microbial flora caused either by lactose hydrolysis or residual proteolytic
activity occurring during lactase preparations that were available commercially in
the past (Marschke et al., 1980). Lactose-free ice cream can also be produced using
either lactose-free milk or powders in the ice cream mix (Abbasi & Saeedabadian,
2015). The production of lactose-free ice cream involves the addition of lactase
during the ageing period before freezing, once the process of pasteurization and
incubation is complete. The lactose hydrolysis increases the monosaccharide con-
centration which reduces the freezing point of the ice cream. This results in the
production of ice cream with a soft texture at the same temperature. Although this
may be a desirable attribute in several frozen desserts, it may also cause faster
melting. Additionally, the sweetness resulting due to the lactose hydrolysis may
help in reducing the addition of extra sugar in the ice creams, and consequently,
raising the melting temperature again. In order to avert crystallization of lactose in
the ice creams which causes a sensory defect known as “sandiness”, lactase treat-
ment is employed to convert lactose into two monosaccharides—glucose and
154 A. Agarwal et al.

galactose, which are highly soluble at high temperature. This treatment is essential
when ice cream is produced using whey powder or WPC as the high lactose content
may result in the formation of crystals during freezing (Abbasi & Saeedabadian,
2015).

5.4 Lipase

Lipase, considered as triacylglycerol acylhydrolase, EC 3.1.1.3, is a ubiquitous

enzyme belonging to the family of hydrolases which reacts with carboxylic ester
bonds and is present everywhere, i.e. in plants, animals, and microorganisms. These
enzymes are obtained from plant and animal tissues or through the cultivation of
microbes. Also, the commercial production of lipase is majorly done with the help of
microorganisms such as by genetic engineering through recombinant bacteria and
yeasts (Houde et al., 2004; Schmid & Verger, 1998). Naturally, lipase enzymes are
useful in hydrolysing fats and oils. They lead to the production of free fatty acids,
glycerol, diacylglycerols, and monoacylglycerols from the hydrolysis of
triacylglycerols by acting as a catalyst in the reaction. Also, this hydrolysis reaction
is reversible in order to allow the enzymes to catalyse the hydrolysis of acylglycerols
through free fatty acid and glycerol (Balcao & Malcata, 1998; Macrae & Hammond,
1985) (Fig. 5.1).
Nowadays, due to high catalytic performance, lipase is gaining wide attention in
industries such as for flavor development in food industry, in paper manufacturing
industry, and more important role in dairy industry. It can help in providing
enhanced digestibility, structure, physical properties, and reduced calorie in dairy
products (Balcao & Malcata, 1998; Bjorkling et al., 1991).

5.4.1 Characteristics of Lipase

Lipase enzyme is considered as a monomeric protein with molecular weight of

19–60 kDa. Its physical properties are dependent upon chain length of fatty acid,

Fig. 5.1 Reaction process of

lipase enzyme (Macrae &
Hammond, 1985)
5 Enzyme in Milk and Milk Products: Role and Application 155

position of fatty acid in glycerol backbone, and degree of unsaturation. Also, its
activity depends upon the pH of the solution such as lipase is stable at neutral pH and
pH 4.0–8.0. For the catalytic activity of enzyme lipase, greater mild conditions and
large surface area are required (Aravindan et al., 2007). Among the different lipases
produced from plants, animals, and microorganisms, the most vital are microbial
lipases. Microbial lipases are gaining greater attention due to their non-toxic nature,
convenience, high catalytic activity, greater yield, rapid growth, and usage due to
absence of seasonal interference as well as availability of cheaper media for growth
(Ray, 2012). Microbial lipases are utilized in different areas such as in cosmetics,
detergents, pharmaceuticals, formation of aliphatic acids, leather industry, and in
treatment of waste excreted from different industries (Seitz, 1974).
Lipases obtained from microorganisms are broadly classified into two major
categories on the basis of their specificity, namely, non-specific and 1,3-specific
lipases. Non-specific lipases are those lipases which catalyse the reactions indepen-
dent of the position esterified in glyceride molecules such as lipases obtained from
Staphylococcus aureus, Pseudomonas spp., Candida cylindracea, and
Chromobacterium viscosum. On the other hand, 1,3-specific lipases are those
which react and catalyse only outer, i.e. sn-1 and sn-3, positions of the glycerol
backbone such as lipases obtained from Rhizopus oryzae, Rhizopus delemar, Rhizo-
pus niveus, Aspergillus niger, Mucor javanicus, Candida lipolytica, and Humicola
lanuginose (Balcao & Malcata, 1998).
Lipases are used as biocatalysts, especially microbial lipases, in various industries
such as food industry due to their various functions and properties such as
regioselectivity, stereospecificity, able to function in different pH and temperature,
substrate specificity, and potential to act upon heterogeneous reactions at the inter-
face between water-soluble and water-insoluble systems (Verma & Kanwar, 2008).
On the other hand, bacterial lipases are stable at a temperature range of 30–60  C and
under neutral or alkaline pH except the lipase produced from P. fluorescens SIK W1,
which is stable under acidic pH of 4.8. Bacterial lipases are also stable in organic
solvents (Verma et al., 2012).

5.4.2 Applications of Lipase in Dairy Industry

Lipase has a wide range of applications in dairy industry such as in improving the
flavor and modifying the fatty acid chain of dairy products including cheese. The
major role of lipases in dairy industry involves the hydrolysis of milk fat. Tissues
from animals such as pancreatic glands, i.e. bovine and porcine, and pre-gastric
tissues of young ruminants, i.e. lamb and calf for lipase production, are widely used
in traditional cheese flavor enhancement process (Ray, 2012).
Recent applications of lipase in dairy industry involve the production of cheese,
its flavor enhancement, and lipolysis of cream and butterfat. Lipases are utilized in
the manufacturing of enzyme-modified cheese for providing concentrated flavor to
cheese. In this process, cheese is incubated with enzymes at high temperature and
thus leads to the formation of enzyme-modified cheese which can be used as an
156 A. Agarwal et al.

ingredient in various products such as sauces, dressings, dips, snacks, and soups.
Addition of lipases in the production of dairy products leads to the release of short
chain fatty acids, namely C4 and C6, which provide sharp and tangy flavor to the
products along with the release of medium chain fatty acids, namely C12 and C14,
which provide soapy taste to the finished products (Hasan et al., 2006). Exogenous
lipase enzyme helps in accelerating the cheese ripening process. On the other hand,
addition of free lipases results in greater lipolysis which deteriorates the texture as
well as flavor of cheese, but encapsulating the lipase helps in maintaining and
stabilizing the enzyme-substrate ratio, thus eliminating the problem of excessive
lipolysis (Houde et al., 2004). It is also used in the manufacturing of chocolates by
production of cocoa butter-equivalent from palm oil with the technique using
interesterification. There are many commercial lipase enzymes which are used for
the production of different milkfat flavor profiles for milk-based products, such as
Snow plum blossom and Palatase 20,000 L (Sarmah et al., 2017).

5.4.3 Other Applications of Lipase

In addition to the dairy industry, lipase also plays a vital role and possesses various
applications in other industries such as cosmetics, pharmaceutical, leather, paper,
food industry, etc. In food industry, lipase is widely used in the preparation of infant
formulas which can provide an alternative of breast milk. The enzyme is also used
for the modification of lipid characteristics of oils rich in omega-6 fatty acids such as
coconut, sunflower, olive, rice bran, corn oil, and oils rich in omega-3 fatty acids
such as linseed and fish oil. In pharmaceutical industries, lipase is used as an
ingredient in the formation of products and also utilized in the interesterification of
vegetable oil. It helps in enhancement of colour and texture of the dough and used as
an emulsifier in many bakery industries. Lipase is also essential in the formation of
cosmetic component, i.e. isopropyl myristate, for manufacturing of cosmetic items
(Sarmah et al., 2017; Houde et al., 2004).
Lipases are also utilized in detergent industry as an additive in household and
laundry detergents in order to vanish the oil stains from the fabrics as these enzymes
can withstand harsh pH (10–11) and temperature (30–60  C) required for washing. It
also plays a vital role in paper manufacturing industry by removing the pitch, which
is a hydrophobic portion of wood, i.e. waxes and triglycerides, from the pulp
required for manufacturing paper (Sharma et al., 2001). In addition to this, microbial
lipases such as Candida rugosa, Mucor miehei, Pseudomonas fluorescens, Rhizopus
oryzae, and Aspergillus niger are used for the production of biodiesel. Presently,
lipase produced from the microbe Streptomyces sp. was considered as most effective
for biodiesel production (Chandra et al., 2020).
5 Enzyme in Milk and Milk Products: Role and Application 157

5.4.4 Introduction of Rennet

Rennet is obtained from the combination of two proteolytic enzymes, namely

chymosin and pepsin, which are secreted from the fourth stomach of ruminants
including calves, lambs, and kids. It is used in the cheese production from historical
times, i.e. 6000 BC. Rennet is majorly available in powder, liquid, and paste forms.
The powder and liquid rennet is produced on industrial scale and is obtained from
calf abomasa, whereas rennet in paste form is obtained from lamb or kid abomasa.
Powder and liquid rennet is most widely utilized in cheese production. Along with
rennet, other enzymes produced from different origin are also utilized in
manufacturing of cheese (Moschopoulou, 2011). Among the two proteolytic
enzymes, chymosin is the most essential protease present in rennet. Chymosin is
an aspartic protease which has the properties like pepsin and it cleaves k-casein in
para-k-casein along with a glycomacropeptide (Fazouane-Naimi et al., 2010).
Clotting of milk with the help of rennet takes place in two step reaction; first
includes the enzymic hydrolysis reaction and second step involves enzyme-
independent protein aggregation reaction. As the concentration of enzyme increases,
clotting time of milk decreases because of the greater amount of proteolysis of k-
casein (Najera et al., 2003).

5.4.5 Different Types of Rennet

Different types of rennet are used in cheese manufacturing which are classified on
the basis of their source such as animal rennet, plant rennet, and fungal rennet. Under
the classification of animal rennet, calf rennet is widely utilized for cheese produc-
tion, the reason being its high amount of chymosin. Presently, adult bovine rennet is
used as an alternative towards calf rennet due to high pepsin content in adult bovine
rennet that results in a product with high pH sensitivity and greater proteolytic
activity (Harboe et al., 2010).
Many proteolytic enzymes are derived from plants such as bromelain from
pineapple, ficin from Ficus spp., and papain from papaya. Other plants include fig,
pumpkin, soybean, ash gourd, and milkweed. The enzymes are embedded in the
buds, leaves, flowers, seeds, latex, and roots of the plants. But the proteolytic
capability of enzymes from vegetable sources is low which makes them unsuitable
for cheese production. And if excessive proteolysis occurs, it will lead to bitterness
in the finished products. Although for cottage cheese and soft cheese production,
extract from berries of Withania coagulans was used, it is also not suitable for
cheddar cheese manufacturing (Garg & Johri, 1994).
Rennet clotting enzymes are also produced from fungi such as rennet from Mucor
miehei and Endothia parasitica. Enzyme obtained from Mucro miehei results in an
excellent quality of cheddar cheese, even with high ripening process due to greater
stability between 4.0 and 6.0 pH with no loss of its activity and also due to its high
heat stability. However, rennet obtained from Endothia parasitica is also useful in
158 A. Agarwal et al.

cheddar cheese production, but its high proteolytic activity leads to 1.2% yield loss
of the cheese (Brown & Ernstrom, 1988).

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Enzymes in Brewing and Wine Industries
6
S. Pati and D. P. Samantaray

Abstract

Enzymes are the bio-catalysts used in breweries and wineries for the biochemical
transformation of a substrate to produce finished products. Several enzymes such
as amylase, glucanase, protease, glycosidase, pectinase, phytase and many more
exogenous enzymes are used in different stages of brewing to improve the
production level as well as the quality of beer. In contrast, lipase, lysozyme,
pectinase, glucanase, glycosidase, urease, protease, phenoloxidase and ester
hydrolase and synthetase are employed in oenology practices for the production
of wine with enormous organoleptic properties. This chapter postulates a bird’s
eye view of different key enzymes used in breweries and wineries, their mecha-
nism of action and their pros and cons.

Keywords
Bio-catalysts · Brewery · Winery · Exogenous · Oenology · Organoleptic

6.1 Background

Beer and wine have concealed a fascinating part of history and our social life.
Although the discovery and development of brewing and wine processing signify
a cornerstone achievement of humankind, its origin is still mysterious. In ancient
civilization, people used to preserve fruits and grains in wooden containers for an
extended period to produce beer and wine. The entire process of production is
termed as fermentation, which came from theLatin word ‘fervere’, meaning ‘to

S. Pati · D. P. Samantaray (*)

Department of Microbiology, CBSH, Odisha University of Agriculture & Technology,
Bhubaneswar, Odisha, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 165
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_6
166 S. Pati and D. P. Samantaray

boil’. The crushed fruits and grains in the wooden container produced bubbles due to
microbial action or activities as if they were boiling. During that period, people
didn’t fully understand that a tiny, eukaryotic fungus was making the entire recipe
work (Alba-Lois & Segal-Kischinevzky, 2010). Researchers took ten decades to find
out how a diminutive microbe makes the entire fermentation successful and resulted
in a potation that became a status symbol in the modern era. Nevertheless, the
concept of brewing and wine processing was established prior to thousands of
years, which was found in the graves and settlements of early civilizations. Ancient
Egyptians made wine 8000 years ago from fruits and labelled it as such by pouring it
in fruit-shaped flasks, while beer came to the limelight 7000 years ago from China.
Initially, Germany, the United Kingdom, the United States, Belgium, Spain and Italy
were the leading countries for beer and wine production. However, in the last five
decades, beer and wine production has confronted significant changes as per the
demand. Global beer production reached 1940 mhl in 2018, with China being the
largest producer as well as a consumer (https://www.statista.com/statistics/270275/
worldwide-beer-production). In contrast, as per the data of the International Organi-
zation of Vine and Wine 2018, the global wine production has reached 292.3 mhl,
with 246 mhl consumption, where, Italy, France and Spain are the highest producers.
Enzymes are the bio-macromolecules that act as catalysts in specific reactions and
play a vital role in food science and fermentation technology. Since 6000 BC,
enzymes have been recognized as a biochemical substance to be utilized in the
processing of various foods, such as cheese preparation, brewing, wine preparation
and meat tenderizing to enhance the nutritional, sensorial and functional values of
the finished products (Gomaa, 2018; Gurung et al., 2013). Amongst all, enzymes
offer a heroic performance in brewing and winery by accelerating the release of
digestive sugars and other nutrients used by the yeasts during fermentation (Spier
et al., 2016). Brewing refers to the oldest fermentation process where complex starch
is bio-transformed to ethanol by the action of yeasts. This traditional process
involves a series of complex endogenous and exogenous enzymes that regulate the
malting of grains, the mashing of grist and rate of fermentation to produce
low-calorie beer with admirable flavour, aroma and texture (Oliver, 2011; Bamforth,
2009). While grapes bio-transformed to wine by yeasts under fermentation. This
process is catalysed by a wide array of enzymes that not only convert complex sugar
to ethanol but also release several volatile and non-volatile substances to enhance the
quality and stability of the wine (Ottone et al., 2020). Traditionally, these enzymes of
brewing and winery were produced naturally by yeast or present in the grains or
grapes. However, recent trends in fermentation technology pave the way for com-
mercial production of enzymes to enhance the quality and productivity of wine and
beer (Claus & Mojsov, 2018). Hence, this chapter offers a comprehensive summary
on the sequential processing of brewing and winery using different enzymes as well
as their mechanism of action. Moreover, the effect of enzymes on the quality and
quantity of the final product and pros and cons have been enlightened.
6 Enzymes in Brewing and Wine Industries 167

6.2 Enzymes Used in Breweries

The process of brewing evolved centuries ago as a result of the resourcefulness and
artistry of the brewers. Traditionally, lager type is the most common method of
brewing, comprising low-temperature fermentation of barley and pure water with an
extended maturation period. However, variations have developed in the young beer
style, where brewing is not possible without the application of enzymes that
hydrolyse complex starch to simple fermentable sugar and then convert it to ethanol
and CO2 by yeast or bacteria. In brewing, the keystones, malted grains and barley are
the sources of enzymes, including α- and β-amylase, exo-peptidase, carboxy-pepti-
dase, proteases glucanases and cellulases (Sammartino, 2015). The detailed key role
or mode of action of these enzymes is discussed below.

6.2.1 Enzymes Used in Different Stages of Brewing

The competitive brewing industry is repetitively sounding forward to improving the

process in terms of quality and manufacturing costs. Brewing that refers to conver-
sion of cereal to ethanol involves two steps: enzymatic hydrolysis of starch to
fermentable sugars and conversion of fermentable sugar to ethanol and CO2.
Depending on the brewing process, raw materials and technical preferences, differ-
ent steps such as malting, mashing, pitching and aging are directly affected by the
enzymes, while others are indirectly affected. The detailed process of beer prepara-
tion is presented in Fig. 6.1. The extent and achievement of each step depends on the
development and stability of native enzymes that ultimately affect the quality and
quantity of the final product.

6.2.1.1 Malting
Malting is the initial and prime step of brewing, which stimulates complex carbohy-
drate and protein-reducing enzymes present in the barley grains. The most prominent
endogenous enzymes of malt include α-glucosidase, α-amylase, β-amylase, car-
boxypeptidase, dextrinase, lipoxygenase, xylanase and glucanase. Besides these,
peroxidase, acid or alkali phosphatases, catalase, polyphenol-oxidases and phytase
enzymes are also responsible for malting reactions (Spier et al., 2016; Van Oort,
2010). The malting process comprises steeping, germination and drying. In steeping,
barley grains are immersed in water for 48–60 h under oxic conditions that initiate
germination and biosynthesis of amylase, glucanase, proteases and carboxypepti-
dase. Amylase acts on the modification of complex starch, whereas β-glucanase
hydrolyses β-glucans for malting clarification and proteases act on the complex
protein content of the grain (Van Oort, 2010). Then malt is subjected to drying
with a temperature between 90 and 140  C to enhance the flavour and colour of the
finished product (Curtis, 2013). The challenge during this step is to maintain the
quality of beer as the application of heat can adversely affect the functionality of
enzymes.
168 S. Pati and D. P. Samantaray

Fig. 6.1 Overview of brewing

6.2.1.2 Mashing
Mashing begins with boiling of malt with water, malt adjuncts and hops at different
temperatures such as 50, 62, 72 and 78  C for proteolysis, gelatinization, saccharifi-
cation and inactivation of malting enzymes, respectively. Mashing embraces α- and
β-amylases and protease, which degrade complex starch to maltose and dextrins, and
the undesirable barley endosperm complex proteins to simple proteins, respectively
(glutelin and hordein) (Dhillon et al., 2016; Spier et al., 2016). Additionally, exoge-
nous enzymes, glucoamylases and pullulanases are added during mashing that break
down the α-1,4 and α-1,6 linkages of starch to produce maltose, dextrin, maltotriose
and glucose (Briess, 2013).
6 Enzymes in Brewing and Wine Industries 169

6.2.1.3 Pitching
Pitching refers to the addition of different strains of Saccharomyces such as
S. cerevisiae, S. uvarum and S. carlsbergensis to the fermenter as inoculum. These
strains own the MEL gene that synthesize α-galactosidases to cleave oligosaccharide
melibiose to glucose and galactose. However, research is still on its way for geneti-
cally modified yeasts that can synthesize glucosidase, amylase and glucanase for
metabolization of wort and soluble proteins (Stewart et al., 2013; Van Oort, 2010).

6.2.1.4 Aging
The beer obtained after completion of fermentation is called green beer due to its
harsh taste and which can be removed by a process called aging. In aging, green beer
is collected from a fermentation vat and stored in refrigerated conditions from a few
weeks to several months. This process enhances the flavour of beer and precipitates
yeasts, proteins and resins. During this period, diacetyl reductase is secreted by
yeast, which reduces diacetyl to acetoin to develop an undesirable butterscotch
flavour in beer. Thus, exogenous enzyme acetolactate decarboxylases is supplied
that directly converts acetolactate to acetoin thereby reducing the undesirable flavour
of beer during aging (Spier et al., 2016). In addition, several other enzymes actively
involved in brewing are presented in Table 6.1.

6.2.2 Types of Enzymes in Brewing

The brewing process requires a prodigious knowledge of enzymology as it not only

improves the quality of beer but also adds innovated attributes to the beer. Thus,
enzymes with varied action and properties are used in the brewing industry.
Enzymes involved in brewing can be either endogenous or exogenous. Endogenous
enzymes are secreted naturally during the process of malting and mashing. Amongst
all, the principal endogenous enzymes like amylase, protease, glucanase and pepti-
dase are involved in brewing (Gomaa, 2018; Bamforth, 2009). On the other hand, at
the pinnacle of enzyme technology, exogenous enzymes are applied to rectify the
issues that evolved during brewing as well as to enhance the shelf life of beer by
maintaining quality. Ficin, alpha-acetolactate decarboxylase (ALDC) and papain are
commercially available enzymes typically used in brewing (Boulton, 2013). Details
of all the enzymes are briefly discussed below.

6.2.2.1 Amylase
During brewing, both α- and β-amylases are involved in the biotransformation of
complex starch to simple sugars such as dextrin, maltose, glucose and oligosaccha-
ride (Bamforth, 2017). Endogenous α- and β-amylases are released from the barley,
when the outer membrane of the granules is hydrolysed by xylanases and
glucanases. During malting and mashing, amylase degrades complex starch, thereby
increasing the availability of fermentable sugar. Amylase (α) strikes the internal
α-(1–4) glycosidic linkage of α-glucose amylose and amylopectin to produce dex-
trin, whereas β-amylase cleaves the external α-(1–4) glycosidic bonds of amylose
170 S. Pati and D. P. Samantaray

Table 6.1 Sources and functions of enzymes involved in brewing

Enzymes Process Sources Function
α-Amylase Malting/ Endogenous/ Improve starch hydrolysis and
mashing exogenous (Bacillus clarification
licheniformis, Bacillus
subtilis)
β-Amylase Malting/ Endogenous/ Improve starch hydrolysis,
mashing exogenous (Bacillus saccharification, malting and
licheniformis) fermentation yield
Protease Malting/ Endogenous/ Boost fermentation and
mashing/ exogenous increase malting, clarification,
storage (Aperguillus sp. and chilling and storage quality
pineapple latex)
β-Glucanases Malting/ Endogenous/ Improve malting and
mashing/ exogenous clarification, lower viscosity
fermentation (Trichoderma sp. and and support clear wort
Orpinomyces sp.) production
Fungal α-amylase Fermentation Exogenous Increase fermentation yield
(Aspergillus sp.)
Amyloglucosidase Mashing Exogenous Increase glucose content in
(Aspergillus niger) wort
α-Acetolactate- Fermentation Exogenous Reduce fermentation time
decarboxylase (recombinant Bacillus
(ALDC) subtilis)
Cysteine Post- Exogenous Improve stability of beer and
endopeptidases fermentation shelf-life of packaged beer
Glucoamylase Mashing Exogenous Increase glucose content in
wort
Phytase Mashing Endogenous/ Reduce initial pH during
exogenous (bacterial mashing of lager beers
origin)
Pentosanases Malting Endogenous Promotes conversion of
pentosans into arabinose and
xylose, also improves
extraction and filterability of
beer

and amylopectin to release maltose. Both the amylases function optimally at

pH 5.2–5.5 and temperatures about 62–74  C (Sammartino, 2015). Moreover,
α-amylase is only used endogenously during brewing, while β-amylase is used
endo as well as exogenously due to its vital role in saccharification.

6.2.2.2 Protease
Proteases have many reimbursements in brewing like digestion of peptide bonds of
proteins to enhance clarification of malting, augmentation of protein solubility,
lowering of beer viscosity and favour rapid yeast growth. Temperature labile
protease has a key role in the malthouse, functions optimally at 52  C and can be
deactivated at 70–75  C, however, it can function at a wide range of pH. Though
6 Enzymes in Brewing and Wine Industries 171

protease is endogenous, it is advisable to use exogenous protease to maintain the

quality of beer. Brewers are acclaimed to use 0.3–1 kg of protease per ton of barley
as an overdose of protease can cause foam instability leading to degradation of beer
quality (Gomaa, 2018).

6.2.2.3 b-Glucanases
It is the key enzyme of malting and mashing involved in the digestion of the outer
membrane of starch that allows other hydrolysing enzymes to act on starch granules.
This enzyme not only lowers the viscosity of beer but also helps protease activity in
the hydrolysis of a matrix of starch granules to make the kernel soften during
germination. Though, β-glucanase are present naturally in barley, in the case of
light beer it is supplied externally (0.3–1 kg per ton of wort) for improvement of light
quality and texture of the beer (Bamforth, 2017; Lyven, 2016). β-Glucanase
hydrolyses the cell wall at an optimum pH of 6 and temperature of 45–50  C,
however, this extremely heat-sensitive enzyme denatures at 60  C (Bamforth,
2017). Thus, heat-stable microbial β-glucanases of Bacillus subtilis, Aspergillus,
Penicillium and Trichoderma are used exogenously in the mashing stage of brewing.

6.2.2.4 Exogenous Enzymes

When the enzyme technology is at its zenith, other than endogenous many exoge-
nous enzymes are also supplied to maximize the fine ability of beer during
processing, storage and transportation. Some of the prime exogenous enzymes,
bromelain, ficin and papain, belong to protease and alpha-acetolactate decarboxylase
(ALDC) from lyases is involved in brewing (Boulton, 2013). Ficin, bromelain and
papain are the chill-proof enzymes extracted from Pawpaw latex and fig plants and
they are responsible for chilling haze by removing polyphenols and polypeptides
leading to hydrolyzation of proteins as well as improvement of colloidal stability of
beer. This chill-proof enzyme functions in a wide range of pH with temperatures
ranging from 60 to 65  C and supplies directly to the storing vessels at a concentra-
tion of 1–2 g/hl of beer (Gomaa, 2018; Boulton, 2013). Besides, ALDC is added to
the beer during pilot-scale production for increasing the product yield at less
fermentation time, without affecting the quality. ALDC attacks the C–C bond in
the α-acetolactate for catalyzing the bioconversion of α-acetolactic acid to acetoin
(Hans, 2008; Dulieu et al., 2000). It remains active at 25–40  C temperature, 5–7 pH
and is added directly during the initial period of fermentation at a concentration of
1–5 g/hl of wort and 0.4–1 g/hl of wort in post-fermentation period. Apart from
these, many other exogenous enzymes such as pullulanases, phytase and diastase is
also used efficiently during malting, mashing and post-fermentation period to
enhance the quality of beer.

6.2.3 Effect of Enzymes on Quality of Beer

One of the most momentous advancements in brewing is to study the use and effect
of different enzymes for beer production in order to fulfil the demand of consumers
172 S. Pati and D. P. Samantaray

for a beer with an appealing taste and flavour. Therefore, it is the need of the hour to
know the effect of enzymes on the quality of beer. Malting is the sole source of
endogenous enzymes like α- and β-amylases that are actively involved in the
conversion of complex starch to simple fermentable sugar. These enzymes are also
responsible for increasing the fermentability by boosting the saccharification, higher
product yield and enhancing of thermal stability of mash during brewing, which
ultimately removes the bitter taste from beer. The poor activity of β-glucanases can
lead to deprived runoff, recovery, spent-grain drainage, filter performance and
sedimentation in beer. However, surpassing β-glucanases activity makes the mash
thermostable and boosts the fine ability and filterability of beer (Aehle, 2007).
Malthouse enzyme protease provides foam stability in brewing. Moreover, the
shelf life improvement, maturation and flavour of beer are also greatly affected by
enzymatic activity. These enzymes ensure protein solubilization during mashing,
together with sugar availability to yeast during fermentation. Enzymes like amylase
also decrease the fermentation period during pilot-scale brewing. Furthermore,
enzymes in combination with substrate and temperature affect the carbonation,
aroma and flavour of the beer. Many commercial enzymes are also cast-off to
boost the quality attributes (clarification, texture, colour and flavour) of beer. The
enzymes utilized in brewing are assorted in their action and properties and are key
factors for the improvement of each step of brewing. Hence, intensive research is
highly essential regarding the mode of action and properties of these brewing
enzymes.

6.3 Enzymes Used in Wineries

Biotechnological advances facilitate the combined use of fruit juice, yeasts and
enzymes to make a surface for wine preparation. Wine is the partial or complete
alcoholic fermentation of grape juice by yeasts where sugars are bio-transformed to
ethanol and other metabolites. These metabolites, including volatile and non-volatile
composites add significant flavour, colour, odour, taste and aroma to wine. Enzymes
are either naturally present in the grapes or supplied exogenously to catalyse
fermentation process as well as enhance the sensory properties of the wine. In this
high-tech era of biotech, endogenous and exogenous enzymes are involved at
different stages of wine preparation for smooth management of pre and post-
fermentation conditions. Several enzymes such as pectinases, glucanases,
glycosidases, xylanases, glucose oxidases, proteases, ureases, peroxidases and
proteases are actively involved in the catalyzation of different reactions occurring
during wine preparation. The role of these enzymes at various stages are momentar-
ily discussed below.
6 Enzymes in Brewing and Wine Industries 173

6.3.1 Enzymes Used in Different Stages of Wine Preparation

Wine production begins with the collection of matured grapes followed by crushing
for extraction of juice. Then, the juice is subjected to maceration for must formation,
where enzymes start functioning as a part of pre-treatment. The must undergo
fermentation by yeasts to obtain raw wine with a precise aroma due to the involve-
ment of specific enzymes. In clarification, pectic enzymes are implied to reduce the
viscosity and turbidity of the wine. Finally, suitable enzymes are used in the ageing
process to get good quality wine, where pertinent physicochemical properties are
incorporated into the wine. The implementation of various enzymes in different steps
of wine preparation is presented in Fig. 6.2.

Fig. 6.2 Overview of wine preparation

174 S. Pati and D. P. Samantaray

6.3.1.1 Maceration
Desired enzymes of maceration not only expedite extraction of colour, aroma and
antioxidant activity but also regulate ethanol concentration of the wine (Ottone et al.,
2020). The endogenous cellulases and hemicellulases catalyse the lysis of grape
cells, whereas pectinases lead to the degradation of polysaccharide to accelerate the
extraction of juice. These enzymes cause activation, release and solubilization of
derivatives of antioxidant activity and colour and fragrance precursors (Garg et al.,
2016). Besides this, the addition of glucose oxidase to the must regulate the ethanol
concentration of the wine, as it maintains the concentration of glucose in the must by
using it as an electron acceptor. However, the by-product H2O2 released during
maceration oxidized the phenolic components of the wine, resulting in reduced
antioxidant activity. Thus, exogenous catalase enzyme is added to the must for
conversion of toxic H2O2 to non-toxic H2O.

6.3.1.2 Fermentation
The overall flavour and aroma of the wine are determined by the concentration of
volatile components synthesized during bioprocess technology. Wine can be
prepared with four types of aromas such as varietal, pre-fermentative, fermentative
and post-fermentative. Production of varietal aroma is due to the partial metabolism
of grapes depending on the variety, ripening percentage, soil and climate, while pre
and post-fermentative aromas are developed during maceration and ageing
(Samoticha et al., 2017). Moreover, the fermentative aroma is generated by acids,
esters, alcohols and sulfur and carbonylated substrates produced by yeasts during
fermentation. Oenology is incomplete without the involvement of yeasts like Sac-
charomyces cerevisiae and Saccharomyces carlsbergensis. These yeasts release
certain enzymes that enhance the rate of fermentation and product yield and add
flavour and aroma to the wine. Invertase is one of the most vital enzymes that causes
hydrolysis of saccharose to fructose and glucose (Ribéreau-Gayon et al., 2006),
whereas β-1,3-glucanases catalyses the synthesis of mannoprotein in fermentation
medium and cell wall hydrolysis. Pectinases and β-glucosidase also found in yeasts
catalyse the degradation of pectic components and improve the aroma of the wine
respectively (Merín et al., 2014; Villena et al., 2007). Except for these endogenous
enzymes, exogenous β-glucanases are also implemented during fermentation for
better stability and structure of the wine (Spagna et al., 2002).

6.3.1.3 Clarification and Stabilization

In wine preparation, clarification steps make the way for bottling. The chemical
reactions that occur during maceration and fermentation release many insoluble,
floating materials that develop a cloudy appearance in the wine. Thus, in clarification
and stabilization all the suspended materials are removed by processes like flotation,
centrifugation, filtration, pasteurization, refrigeration, maturation and racking to
improve the quality of the wine. Pectinase enzymes are added in the initial period
of clarification to increase the juice yield, colour and flavour of the wine and to
reduce the viscosity and turbidity of must (Martín & Morata de Ambrosini, 2014). It
also significantly reduces the filtration time of wine preparation. Though pectinase is
6 Enzymes in Brewing and Wine Industries 175

highly indispensable for clarification, they are not synthesized naturally during wine
preparation. Thus, commercially synthesized pectinase from microbes and plants is
employed during bioprocess technology. On the other hand, protease is added to the
medium to circumvent haze formation. Sometimes the reaction medium gets
contaminated by Lactobacillus sp. and Pediococcus sp. causing acidity, mousy
taint and buttery flavour in the wine (Bartowsky, 2009; Liburdi et al., 2014). Thus,
lysozyme is supplemented, which not only inhibits the growth of these contaminants
but also enhances malolactic fermentation (Ottone et al., 2020).

6.3.1.4 Ageing
Ageing potentially improves the quality of the wine, where it is aged for a significant
time period till the availability of oxygen for the development of premium flavour,
aroma and taste. However, throughout ageing the naturally occurring urea and
ethanol react to form ethyl carbamate, which has carcinogenic activity. Therefore,
acid urease is added in this period for removal of urea that effectively minimizes
ethyl carbamate formation to reduce the toxicity in the wine.

6.3.2 Types of Enzymes in the Winery

Over the past decades enzymes have been established as a processing aid in
oenology. The enzymes offer several advantageous bids during pre-fermentation,
fermentation, clarification, stabilization and ageing steps that ultimately enhance the
juice yield and add colour, odour and flavour to the wine, leading to the generation of
extremely fine-quality wine. Pectinase, lipase, lysozyme, glucanases, glycosidases,
ester hydrolases and synthetases, phenol oxidases and urease are the potential
enzymes widely used in wine preparation (Table 6.2). The above-said enzymes are

Table 6.2 Enzymes of the winery and their functions

Enzymes Functions
Lysozyme Prevents the growth of undesirable lactic acid bacteria in the wine
Pectinase Involved in degradation of pectin components, hydrolysis of cell wall,
and enhances the quality, stability, colour and flavour of the wine
Glycosidase Improves flavour and develops varietal aroma from odourless
precursors in the wine
Glucanases Liberate mannoproteins for the development of varietal flavour and
cause lysis of the microbial cell wall to boost the clarification and
filtration during wine preparation
Protease Prevents haziness and reduces bentonite demand of the wine
Urease Hydrolyses urea to prevent the synthesis of carcinogenic ethyl
carbamate produced during the ageing stages of wine preparation
Lipase Degrades lipids for better extraction of colour during wine preparation
Phenoloxidases Enhance the stability and sensory attributes of the wine
Esterhydrolases and Promote malolactic fermentation and develop fruity flavour in the wine
synthetases
176 S. Pati and D. P. Samantaray

naturally synthesized during oenological practice; however, in some cases, they are
also supplied externally.

6.3.2.1 Pectinase
The grape cell comprises cellulose, hemicellulose, mannan, pectin and xyloglucan
linked with proteins. During wine processing, the highly viscous pectin impedes
juice extraction, filtration, clarification and diffusion of aromatic components into
the must (Claus & Mojsov, 2018). The enzyme pectinase hydrolyses pectin to
enhance the juice yield and ease clarification and filtration. Pectinase treatment
boosts the absorbance of phenols and anthocyanins by the must, as well as intensifies
the colour and clarity of the wine (Mojsov et al., 2011). Polygalacturonase, pectin
lyase, pectinesterase and acetylesterase are the pectinase enzymes actively involved
in wine preparation. However, pectinase is neither present naturally in grapes nor
secreted by the yeasts. Therefore, commercial pectinase solutions composed of
2–5% of active enzymes and additives (preservatives, sugar and salt) are used in
wine preparation (Mojsov, 2013). The pectinase available in the market is generally
of fungal origin or produced by Rhodotorula mucilaginosa and Cystofilobasidium
capitatum. Nevertheless, high amounts of tannin, alcohol (above 17%) and SO2
(above 500 mg/l) render the pectinase activity (Van Rensburg & Pretorius, 2000).

6.3.2.2 Lysozyme
Traditionally, SO2 was added to the medium to prevent microbial contamination in
the wine, but an allergic response caused by sulphites pushes the oenologist for an
enzymatic solution (König & Fröhlich, 2017; Campos et al., 2016). Thus, lysozyme
is used to prevent microbial contamination, which kills the bacteria by cell lysis.
Lysozyme stabilizes the wine by preventing malolactic fermentation. As per the
International Organization of Vine and Wine, hen’s egg lysozyme at a concentration
of 250–500 mg/l can be used in wine preparation. Moreover, after completion of
bioprocess technology, this enzyme can be removed from the wine by the imple-
mentation of fining agents.

6.3.2.3 Glycosidase
Almost 90% of aroma precursors such as phenolic compounds, nerol, terpenes
linalool and geraniol are present in grapes’ skin in conjugated form as odourless
compounds. Glycosidase hydrolyses these precursors to liberate volatile, aromatic
terpenes that ultimately activate the organoleptic properties of the wine. These
enzymes are naturally found in grapes and promote the liberation of aromatic
compounds under optimized condition. However, these grape glycosidases are
inactivated at high alcohol and glucose concentrations and at pH 5. Therefore,
commercially available glycosidases extracted from the species of Saccharomyces,
Pichia, Candida and Rhodotorula are also employed in wine preparation (Claus &
Mojsov, 2018; Ugliano, 2009).
6 Enzymes in Brewing and Wine Industries 177

6.3.2.4 Glucanase
Lactic acid bacteria and some fungus associated with the skin of grapes release
viscous polysaccharides, which hamper the wine filtration. Neither flocculants nor
filtration can remove these polysaccharides; thus, glucanase is used for reduction of
wine viscosity. Both endo andexo-glucanase release mannoproteins to enhance the
varietal flavour in the wine. Endo-glucanase is naturally synthesized by Saccharo-
myces species during fermentation, while exo-glucanase is supplemented to the
fermentation medium. Moreover, the commercially available exo-glucanase is
extracted from fungus like Taleromyces versatilis and Trichoderma sp., different
species of yeast like Kloeckera, Zygosaccharomyces and Pichia and lactic acid
bacteria (Claus & Mojsov, 2018).

6.3.2.5 Protease
Proteins present in must or synthesized by starter culture cause allergic reactions in
the consumer (Van Sluyter et al., 2015; Rizzi et al., 2016). Though proteins are
precipitated after fermentation, the presence of acid, proteolytic and heat-resistant
pathogenesis-related (PR) proteins causes undesirable turbidity in the wine. Gener-
ally, bentonite is used for the removal of PR proteins, but it can adversely affect the
quality and quantity of the wine (Jaeckels et al., 2015). Thus, researchers are now
focusing on protease as an attractive enzymatic solution for the removal of these
undesirable proteins. Protease can not only enhance the quality and quantity of the
wine but also reduce the haziness in the wine. Saccharomyces species doesn’t show
any protease activity; however, some fungus and non-Saccharomyces species depict
the same. So, nowadays researchers are shifting their attention to selecting or
developing protease positive starter culture for wine production.

6.3.2.6 Urease
In wine, fermentation yeast generates urea that is chemically converted to carcino-
genic ethyl carbamate (Lonvaud-Funel, 2016). In 1997, urease was established as an
enzymatic solution that cleaves urea into CO2 and NH3 and prevents ethyl carbamate
synthesis. Generally, urease of Lactobacillus origin is used at a concentration of
25–50 mg/l in the fermentation medium (Pozo-Bayón et al., 2012).

6.3.2.7 Lipase
Lipids are released during wine fermentation as a result of the autolysis of yeast or
grape cells, which cause significant changes in fermentation as well as in the finished
product. Hence, lipase is employed in wine preparation to decompose the lipids
present in the cell membrane for the improvement of the colour and texture of the
wine. It is found in a few wild strains of Lactobacillus and yeasts.

6.3.2.8 Esterhydrolases and Synthetases

Esters are either present in grapes or synthesized by yeasts during fermentation,
which contributes a significant fruity flavour to the wine. Esterhydrolases and
synthetases regulate the concentration of the ester during malolactic fermentation
by promoting its synthesis as well as hydrolysis (Ugliano, 2009).
178 S. Pati and D. P. Samantaray

6.3.2.9 Phenoloxidases
Phenoloxidases affect not only the sensory attribute of the wine but also the final
phenol concentration of the wine. However, these oxygen-sensitive enzymes on
exposure to O2 cause enzymatic browning of wine. Phenoloxidases include tyrosi-
nase, responsible for implementation of colour to the wine, and laccase, which
causes phenol oxidation to enhance the organoleptic properties of the wine (Claus
& Mojsov, 2018).

6.3.3 Effect of Enzymes on Quality of the Wine

Enzymes have a very controversial effect on wine preparation as they are highly
selective. Several studies have been directed to know the effect of different enzymes
on the organoleptic as well as quantitative properties of the wine. The colour
extraction of the wine is greatly influenced by xylanase and glycosidase activities.
During maceration, the enzymatic treatment enhances the extraction of phenolic
compounds while depicting undesirable anthocyanin content and colour parameters.
Similarly, glycosidase helps in aroma extraction in the wine; however, an abundance
of glucose inhibits its action. Lysozyme causes total inhibition of lactic acid bacteria
from wine, but the presence of the residual amount of lysozyme can cause an allergic
reaction in the consumer (Liburdi et al., 2014). Furthermore, protease is essential to
remove PR proteins from wine, but it is only functional under restricted alcohol
concentration, pH and temperature (Espejo, 2020). It can’t be ignored that enzymes
are highly essential for wine preparation, but the sensitivity of these enzymes
influences industry and academia to analyse the pros and cons before further
application.

6.4 Pros and Cons of Enzymes Used in Brewing and Winery

Before taking any step forward towards the enzymology of brewing and wine
preparation, it is highly recommended to study their pros and cons. The application
of enzymes in brewing enhances the maturation of beer, catalyses low-calorie beer
production, stabilizes the beer by improving mashing and clarification and reduces
the viscosity, leading to fine-quality beer production (Gomaa, 2018). Nevertheless,
the cost is the prime barrier in the enzymatic treatment of beer. Besides, the
sensitivity of enzymes towards temperature, pH, alcohol and glucose concentration
also significantly affects the use of enzymes in brewing. Like in brewing, enzymes
also have a significant effect towards the enhancement of the flavour, aroma and
taste of the wine. Some enzymes are used to prevent contamination of the wine by
toxic microbes and compounds. Enzymes also ease the wine processing for higher
yield and lower production cost, leading to a booming profit. On the other hand, it is
always advisable to consider the drawbacks of these enzymes for a better future.
Sometimes specific activity and side effects of a few enzymes have a detrimental
effect on wine quality. Also, the use of commercial enzymes requires a lot of
6 Enzymes in Brewing and Wine Industries 179

pre-treatment, which is time-consuming as well as expensive. Intermittently,

enzymes undergo unwanted chemical reactions with the components of the fermen-
tation medium and cause an allergic response to the consumer. Hence, a detailed
study of individual enzymes is highly essential before their use.

6.5 Conclusion

In the present scenario, breweries and wineries have established themselves as

extremely lucrative businesses in the agriculture and alcohol industries. Earlier
these two were produced by traditional methods, but in the modern era, the estab-
lishment of brewery and winery is not possible without the involvement of enzymes.
On account of this, brewers and oenologists amalgamate traditional and advanced
enzymological techniques for the development of modern methods for beer and wine
production. Amylases, glucanases, proteases and cellulases are the foremost impor-
tant enzymes being used in beer industries. Though the implementation of enzymes
ensures faster and higher beer production, the brewer should screen the appropriate
enzyme with eyes open because a slight modification in enzymatic treatment can
cause devastating effects on the finished product. On the other hand, enzymes have a
flourishing future in the wine industry and a wide array of enzymes are being used
for the furtherance of different stages of wine preparation. Pectinases, protease,
glucanases, lysozyme and lipase are the enzymes used during the different stages
of wine processing for the production of superior-quality wine. These enzymes
accomplish every goal to provide chemical, mechanical and thermal stability to
wine. Moreover, recent advances in biotechnology pave the way for researchers to
articulate a stringent plan of work or phenomenon to overcome various drawbacks of
brewery and winery. This chapter intensely discussed several aspects of enzymes
used in brewery and winery; however, further study is highly essential in this regard.

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Enzymes in Meat, Fish, and Poultry
Products Processing and Preservation-I 7
Khadijeh Abhari and Hedayat Hosseini

Abstract

Enzymes are used in the food industry widespread to modify and enhance the
nutritional value, and functional and sensorial characteristics such as color, smell,
and flavor of different types of food products.
The technological applications of enzymes in the meat industry are a hot topic
nowadays. The most common application of enzymes in red meat products is to
increase the meat tenderness. The toughness of beef meat produced from dairy
cattle is related to the volume of connective tissue, mainly collagen. Proteolytic
enzymes have the potential to tenderize lower-grade meats and increase their
market value. Consumers’ demand for fresh meat without undesirable excessive
fat and salt content leads the manufacturer to consider new possibilities of
enzyme application to increase the carcass efficiency by using more effective
slaughtering techniques.
The utilization of enzymes to maximize the by-products’ efficiency is an
undeniable possibility.
The common by-products of the poultry industry are feathers, which can be
digested by enzymes and applied for animal feed and also nonfood industries
such as films, coating, and packaging due to their high amount of keratins and
hydrophobic amino acids. Fat, bone, and mechanical flesh are common
by-products of meat processing, which can be digested by enzymatic reactions
and used for their special meat properties. Enzymes also can be applied in seafood
processing for deskinning and descaling, production of caviar, and recovery of

K. Abhari · H. Hosseini (*)

Department of Food Science and Technology, National Nutrition and Food Technology Research
Institute, Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
e-mail: hedayat@sbmu.ac.ir

# The Author(s), under exclusive license to Springer Nature Singapore Pte 183
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_7
184 K. Abhari and H. Hosseini

by-products. In the current chapter, the potential application of enzymes in the

meat, poultry, and seafood industry will be discussed.

Keywords

Meat · Poultry · Seafood · Enzymes · Preservation

7.1 Application of Enzymes in Red Meat Industry

7.1.1 Role of Enzymes in Meat Tenderization

Meat tenderness, which is determined by meat toughness, depends on many factors

such as structure, nature, and the volume of connective tissues mainly collagen and
elastin (Lepetit, 2008). Meat tenderness is recognized as one of the main criteria in
scoring quality characteristics of meat. There are several methods and equipment to
assess the tenderness of meat, such as determination of enzyme activity, scanning
electron microscopic (Koohmaraie et al., 1988), analyzing the myofibrillar fragmen-
tation index (Olson & Parrish, 1977), and measuring hydroxyproline (Ashie et al.,
2002).
Tenderization is one of the substantial events in postmortem changes which can
been affected by decreasing the pH value, temperature, time, and the condition of
animal prior to slaughter. During the tenderization, endogenous proteolytic enzymes
become active and attack all muscle proteins such as myofibrillar and connective
tissues. As the result of proteolytic enzyme activity, the structural and biochemical
changes occur, which lead to a significant increase in meat tenderness (Koohmaraie
& Geesink, 2006; Herrera-Mendez et al., 2006; Hwang et al., 2003).

7.1.1.1 Endogenous Enzymes

There are several endogenous enzymes which play a role in postmortem tenderiza-
tion, including calpains, lysosomal cathepsins, and proteasomes. Among these, the
calpain system is the primary key of tenderization process. Cathepsins stands in the
second level of protein degradation and proteasomes have no significant effect due to
unsuitable role of myofibrils as its substrates (Koohmaraie & Geesink, 2006; Kemp
et al., 2010).

Calpains
The calpain system, which is activated by calcium and thiol, contains three enzymes
three protease enzymes: calpain I (μ), calpain II (m), calpain 3, and calpastatin,
which is recognized as calpain inhibitor. Calpain I is linked to myofibrils of muscles,
whereas calpain II and calpain 3 are situated in cytosol and sarcomere (near the Z and
M line in myofibrillar structure), respectively.
It has been suggested that μ-calpain has an actual role on postmortem proteolysis.
Many factors effect on calpain system activity, particularly the calcium ion
7 Enzymes in Meat, Fish, and Poultry Products Processing and Preservation-I 185

concentration; thus, any operation which increases the calcium ion can improve meat
tenderness (Moudilou et al., 2010).
Studies revealed that injection of calcium or application of electrical stimulation
causes an increase in meat tenderness due to increase in calcium level and decrease
in calpastatin activity. The changes in Z line and degradation of myofibrillar proteins
such as I troponins, T troponins, desmins, titins, and nebulins are the most common
phenomenon during caplain system activity.

Cathepsins and Other Enzymes

Cathepsins contain three classes of enzymes according to the presence of different
amino acids in their structure: cysteine, serine, and aspartic peptidase.
The role of cathepsins in tenderness of meat is still uncertain, due to lack of
information regarding their release from lysosomes through postmortem storage of
meat. It is suggested that small amount of myofibrillar proteins such as actin,
myosin, and α-actinin can be affected by cathepsins.
Studies show that other enzymes such as endogenous collagenases, elastases, and
proteases can cause the degradation of the structure of collagen and elastin.

7.1.1.2 Exogenous Enzymes

Furthermore, there are another group of enzymes which are derived from plants,
animals, and microbial sources and have potential to effect on meat tenderization.
The most common exogenous enzymes used for meat tenderization are proteases.
The proteases are vital for cell growth as they are capable to catalyze and hydrolysis
the peptide bond of proteins and provide amino acids for growth and differentiation.
Proteases have diverse variety due to their structure, protein characteristic, and
mode of action. The exogenous proteases are commonly classified by their original
source. The exogenous proteases that have been approved by Food and Drug
Administration (FDA) and recognized as GRAS substances are as follows: plants
(papain, ficin, bromelain), microbes (Aspergillus niger; Bacillus subtilis; Rhizopus
oryzae), and animals (trypsin, pepsin).

Plant Enzymes

Papain
Papain is a protease enzyme which contains cysteine and extracted protease from the
latex of the papaya plant (Carica papaya). This enzyme is a part of defense system of
plant and protects the plant against insect. The optimum temperature and pH for
enzyme activity is 65  C and 5–8, respectively.
Commercial forms of papain contain various ratio of papain, papaya peptidase,
and chymopapain and lead to different biochemical properties. Papain tenderizes the
toughness of meat by acting on amino acids which have aromatic side chain like
tyrosine and phenylalanine. Papain can effectively hydrolyze collagen and myofi-
brillar proteins. Investigations show that injection of papain approximately 30 min
before slaughter can cause meat tenderness. As injection of papain to animal may
186 K. Abhari and H. Hosseini

cause stress symptoms and severe shock, using inactive papain can be an alternative
approach.

Ficin
Ficin is extracted from latex of fig fruits (Ficus glabrata, Ficus anthelmintica). Figs
contain ten different proteases and ficin is most popular for its tenderizing effect.
Studies revealed that application of ficin in temperatures of 60 and 70  C has the
most influence in tenderization process. It is suggested that ficin has the ability to
effect on collagen, elastin, and myofibrillar proteins in addition to increase in water-
holding capacity.

Cucumin
Cucumin is another proteolytic enzyme which is achieved from kachri (Cucumis
pubescens). The researches show that cucumin can be effective on tenderizing
buffalo meat, sheep meat, and chicken.

Bromelain
Bromelain is cysteine proteases which originated from pineapple. It has two sources:
fruit bromelain and stem bromelain; it has been shown that fruit bromelain is more
specific and more effective in comparison to stem bromelain. The optimum temper-
ature for enzyme activity is 50–60  C; however, the studies showed that the enzyme
activity remains at 0  C and it becomes inactive in temperatures more than 80  C.
The bromelain activity starts by degradation of collagen, and then attacks myosin.

Zingibain
Zingibain is a potent proteolytic enzyme which is obtained from ginger. The studies
reported that Zingibain is more effective in meat tenderization when heated. Ginger
is a great and economical source of proteases that begin degradation from the I band
of each sarcomere and go through M line.

Actinidin
Actinidin is a natural enzyme extracted from kiwi fruit which has been recognized as
an agent which can hydrolyze both connective tissue proteins specially collagen and
myofibrillar proteins. Studies show that applying actinidin in brine solution can
tenderize porcine muscles.

7.1.2 Role of Enzymes in Meat Flavor Development

Flavor has a complex definition which includes several phenomenons such as taste,
odor, trigeminal senses, and texture. As well as the mentioned factors, tenderness
significantly affects the flavor and the relationship of all these together creates
consumer acceptability. Generally, the raw meat is tasteless due to absence of
nonvolatile components; however, the flavor precursors are present in the meat
7 Enzymes in Meat, Fish, and Poultry Products Processing and Preservation-I 187

tissue and it is necessary to undergo enzymatic reaction to tastes appears in the meat
(Fernandez et al., 2000; Toldra, 1998; Calkins & Hodgen, 2007).
As the results of several enzymes activity such as lipase, protease, glutaminase,
and peptidase, biochemical reactions occur and lead to enhance the flavor. These
enzymes may be endogenous natural lipases and protease, microbial originated
enzymes or those which added to the product throughout the manufacturing proce-
dure. Due to proteases activity mainly cathepsins, trypsin-like peptidases, and
Micrococcaceae originated enzymes (Fernandez et al., 2000), nitrogenous
complexes such as volatile compounds, small peptides, and free amino acids appear.
As well as micrococci, other species of lactic acid bacteria, such as Pediococcus and
Lactobacillus, are able to produce intracellular peptidases which contribute to
increase the levels of free amino acids. These substances have principal effect on
the taste of the product.
Another group of enzymes which takes part in flavor enhancement are lipases.
Lipases produced by some microorganisms like lactobacilli or micrococci could
effect on the lipids by their lipolytic activity and oxidation, leading to increase in the
level of free fatty acids and short chain fatty acids. The substances produced by
lipases are responsible to develop the flavor in final product. Although the optimum
temperature for maximum activity of microbial lipases is 30–40  C, some thermo-
philic microorganisms such as Pseudomonas, Bacillus sp., Thermomyces
lanuginosus, and Aspergillus niger release lipases which can stand in the higher
temperatures (50–65  C).
Glutaminase also play a role in creating the flavor of sausages by deamidation of
glutamine, leading to production of L-glutamic acid and ammonia through hydrolysis
of the glutamine amide group. Glutamic acid is an amino acid which is responsible
for the flavor enhancement and producing by starter cultures has an important role in
flavor in sausage, seasonings such as soya sauce and miso, and pickles. Glutamic
acid is the substance which increases “umami” taste in food products. The ammonia,
produced by glutaminase, performs as an acid neutralizer. Glutaminase can be
obtained from Bacillus amyloliquefaciens, Aspergillus oryzae, Debaryomyces, and
Rhizobium etli.

7.1.3 Approaches for Utilization of Enzymes as in Meat Tenderizer

There are several traditional approaches to apply tenderizing substances into

carcasses. These methods include injection; dipping and spraying the enzymes
have both advantages and disadvantages. The main concern regarding utilization
of traditional method is incomplete and insufficient distribution of the tenderizing
agents into meat pieces. Neglected penetration of enzymes into the meat pieces
causes surfaces to become over-tenderized while interior parts of meats remain
unchanged (Rhodes & Dransfield, 1973; Lawrie, 1998).
188 K. Abhari and H. Hosseini

7.2 Application of Enzymes in Seafood Industry

7.2.1 Deskinning

The most important application of enzymes in the seafood industry is to deskinning

and descaling. In this procedure the skin is removed with the least damage to the
flesh. If the deskinning process done suitable, the edible portion can increase. The
main source of enzymes to use in the seafood industry is marine enzymes. Applica-
tion of enzymes in combination with mechanical methods has been performed for
processing of tuna, squid, herring, shrimp shells, Pollock, and skate (Simpson et al.,
2012).

7.2.2 Production of Fish Protein Hydrolyzate

Fish protein hydrolyzates are recognized as one of the important by-products of the
seafood industry which can be obtained by enzymatic or chemical reactions. The
wasted of fish industry such as gills, head, skin, bones, viscera and liver are the main
sources for producing fish hydrolyzates which are in the forms of peptides with
2–20 amino acids depends on the type of enzyme, kind of fish and the time of
operation.
Fish hydrolyzates also can be obtained by alkali or acid reactions in high
temperature and pressure in the traditional method. The major acids utilized in
acid hydrolysis are sulfuric acid and hydrochloric acid. One of the disadvantages
of acid and alkali hydrolysis is production of great amount of sodium chloride and
sodium hydroxide, respectively, which decreases its functionality. Moreover,
destruction of tryptophan, a critical amino acid, and production of several toxic
compounds are other concerns regarding using acid hydrolysis approach.
On the other hand, enzymatic hydrolysis has several advantages, such as being
economical, and required slight conditions of pressure, temperature, and pH. Fish
hydrolyzates demonstrate functional properties in food formulation and can be
applied as emulsifier or a substance which increase water-holding capacity, protein
solubility, gelling activity, and oil-binding capability.
Fish hydrolyzates which are mostly produced by action of alcalase are more
constructive compared to poultry by-products.

7.3 Application of Enzymes in Poultry Processing

Poultry industry with various products including chicken, egg, and turkey has a large
portion in food sector and human diet. Due to side effects of red meat consumption,
nutritional benefits of white meat and ease of preparation of such products for
customers and of the poultry industry has been growing fast, as the USA, China,
and Brazil have the maximum products, worldwide.
7 Enzymes in Meat, Fish, and Poultry Products Processing and Preservation-I 189

Consequence of such a large development in poultry industry is production of

high amount of waste mainly feathers, deboning residue soft meat, blood and also
dead birds on arrival. Due to several concern regarding converting the poultry waste
to soil fertilizer such as accumulation of fat in soil, excessive levels of oxygen
requirement and probable occurrence of pathogens in hatchery, dead and litter
compost, utilization of enzymes to use wastes has been recommended. Nowadays,
the waste materials of poultry industry are transformed to feather meal, meat and
bone meal, blood meal, and fats/oils through rendering process. While the mentioned
meals are rich source of protein, their application can be restricted due to their
nutritional loss particularly essential amino acids, as well as high amount of lysine,
calcium, and phosphorous in meat/bone meat and bone meal.
The significant portion of waste products is feathers which is 8% of the weight of
an adult chicken.
Keratin is a protein which constituted approximately 90% of the feathers. Degra-
dation of keratin is a complicated process due to disulfide bonds between polypep-
tide chains and also hydrophobic interactions and several hydrogen bonds.
Additional to feathers, other parts of the bird such as legs, heads, bones, skin, and
viscera contain large amount of various proteins such as collagen, keratin, and
elastin. The dead birds which on the arrival are also rich source of proteins. Utiliza-
tion of poultry by-products helps to avoid environmental pollution; besides, the
extracted and hydrolyzed protein can be used as a nutritious part of animal diet.

7.3.1 Utilization of Keratinase for Bioconversion of Feathers

Feathers are a great source of inexpensive protein; however, due to its low nutritional
value and low digestibility, its application as a feedstuff has various concerns.
Conventional hydrothermal method of degradation for feathers causes losing some
essential amino acids, as well as production of non-nutritious amino acids such as
lysinoalanine and lanthionine. To overcome the mentioned problems, biotechnolog-
ical methods via microorganisms have been offered. The keratinase produced by
microorganisms such as Bacillus spp. has the capacity to hydrolyze keratin and
convert it to peptides which can be used as nitrogen fertilizers or animal feed.
Controlled enzymatic hydrolysis is an appropriate method for production of bioac-
tive peptides that have the potential to play role as ant antimicrobial, antioxidant and
antihypertensive as Fontoura et al. (2014) reported DPP-IV and ACE inhibitor
activities of hydrolyzed keratin obtained by feathers of raw chicken.
Another by-product of poultry industry is fat which is significantly located in the
skin. Chicken fat contains both ω-3 and ω-6 polyunsaturated fatty acids which can be
beneficial to reduce cholesterol. Lee and Foglia (2000) have indicated that Candida
rugosa and Geotrichum candidum are able to produce lipases which act on triglyc-
eride fractions that have been evoked from chicken fat by supercritical CO2.
190 K. Abhari and H. Hosseini

7.4 Application of Enzymes for Enhancement of Flavor

In Addition to marine sources for production of protein hydrolyzates, various meat

by-products such as bones, bovine by-products, sheep visceral mass and chicken by-
products can be treated with different enzymes for this purpose. These protein rich
resources, depends on type of applied enzyme and condition have the capacity to
enhance the meat flavor when not proper for food use. The main concern regarding
using meat by-products as source of protein hydrolyzates is formation of bitter taste.

7.5 Future Prospective

Due to consumer demand for fresh tender meat with minimum fat and salt, and also
the manufactures to produce the carcasses with sufficient profits, enzymatic
techniques have been utilized to achieve these aims. One of these methods which
recently become popular in meat industry is using TGase which is a cold binding is
enzymatic technology.
In this method, the cut and timed pieces of meat from one or several animals can
be attached together with TGase to form meat products with new shape and size,
which can be stored in every temperature.

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Enzymes in Meat, Fish, and Poultry Product
Processing and Preservation-II 8
Sandesh Suresh Karkal, Anushma Venmarath,
Suresh Puthenveetil Velappan, and Tanaji G. Kudre

Abstract

The current chapter provides insights into the applications of enzymes in meat,
fish, and poultry products’ processing industries. The mechanical and chemical
techniques mainly employed to process meat, fish and poultry products are not
eco-friendly, consume more energy, and generate by-products. Hence, the substi-
tution of these techniques with enzymes (biocatalysts) could be the best alterna-
tive. Besides, the application of enzymes in these industries is gaining momentum
since they are eco-friendly, consume less energy, and also aid in obtaining good-
quality products with better yield. The enzymes can catalyze the reaction process
at mild temperature and pH and are highly specific toward substrate molecules.
Enzymes such as proteases, lipases, transglutaminases from plant, animal, and
microbial origin are suitable and have been extensively studied to process meat,
fish, and poultry products. Currently, novel enzymes are also being developed as
a result of technological advancements. The recombinant enzymes being devel-
oped through technical advancements in biotechnology for their application in
meat, fish, and poultry products processing could be an excellent prospect. This
chapter describes the potential enzymes used in the processing and preservation
of meat, fish, and poultry products.

Keywords

Meat industry · Seafood industry · Poultry industry · Proteases · Lipases ·

Transglutaminases

S. S. Karkal · A. Venmarath · S. P. Velappan · T. G. Kudre (*)

Meat and Marine Sciences Department, CSIR-Central Food Technological Research Institute,
Mysuru, India
e-mail: tkudre@cftri.res.in

# The Author(s), under exclusive license to Springer Nature Singapore Pte 193
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_8
194 S. S. Karkal et al.

8.1 Introduction

Enzymes are termed as “biocatalysts” owing to their natural catalytic nature and are
considered to be prime tools in biotechnology and related areas (Fernandes, 2016).
Enzymes are mainly protein molecules known to speed up the biochemical reaction
rate occurring in the cell (Singh, 2018). Enzymes have been utilized in the
processing of food since ancient times, long before their catalytic function was
discovered. Homer’s epic poem, Iliad, written around the eighth century BC,
describes the use of enzyme ficin for milk clotting (Copeland, 2000). Apart from
this, the original inhabitants of the Pacific Islands exploited papaya juice for meat
tenderization. The colonization of these islands by the British led to the growth of
enzyme technology for food processing, especially meat tenderization and also
wound healing in European nations (Gomes et al., 2018). At present, enzymes
have found their potential application in food, leather, animal feed, biofuel, deter-
gent, etc., industries. In these industries, enzymes have been employed as efficient
catalysts (Singh, 2018). The increased demand for enzymes in industries, especially
food industries, may be attributed to its high specificity towards the substrate
molecule. Besides, enzymes can catalyze the reactions under mild pH and tempera-
ture with minimal by-product formation (Shahidi & Kamil, 2001).
Food is one of the necessities of life required for human survival. Despite being
an important necessity, the global report on the food crisis (2019 Edition) by the
European Commission illustrates that across 53 countries, a population of more than
113 million is experiencing a food shortage, nutritional deficiency, and also
improper livelihood assistance (European Commission-GRFC, 2019). Due to the
rapid population explosion, extensive utilization of natural resources, disputes across
the globe, and variation in climate, there has been an increased demand for food
worldwide (Singh, 2018). According to the United Nation Department of Economic
and Social Affairs (UNDESA), New York, the population of the world is projected
to be increased from 6.9 to 9.1 billion (i.e., 32% rise) by 2050 and this would trigger
the hike in food demand up to 70% (UNDESA, 2017; Singh, 2018). Moreover, to
mitigate food shortage, in the year 2015, the United Nations (UN) Member States
adopted the 2030 Agenda, which mainly aims for the contribution of fisheries and
aquaculture in the use of natural resources toward food security and nutrition, and
thereby ensure sustainable development in economic, social, and environmental
terms (FAO, 2016). A considerable increment in the production of meat and poultry
products was also observed to meet the global food demands. According to the Food
and Agriculture Organization of the United Nations (FAO), by 2050, the global
production of meat is expected to increase to 455 million tons (Chandrasekaran et al.,
2015).
At present, combating the current global scenario of the food crisis, elimination of
malnutrition and hunger, and ensuring food security have been the most challenging
research and development fields. Food processing could play a pivotal role in
eradicating the persisting global food shortage (Singh, 2018). Food processing refers
to the process where the agricultural raw material is processed into a consumer-ready
product. The main objectives of modern food processing include (1) to prepare safe
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 195

food (microbiologically and chemically), (2) to deliver products enriched in organo-

leptic properties and nutrients, and (3) to make convenient food. Generally, chemical
treatment methods are implemented to aid production while processing the food for
commercial utilization. The substitution of chemicals with enzymes in food
processing has resulted in quality products with better yields. Besides, it has also
reduced energy consumption and doesn’t emit greenhouse gases (Chandrasekaran
et al., 2015). The application of enzymes in food industries such as meat, fish, and
poultry product processing has been vital in obtaining better-quality products as well
as their preservation. The demand for food enzymes in these industries has been
growing alongside their increased production across the globe. Moreover, the
recombinant enzymes, especially from microorganisms, are also being produced
for their application in these industries. In meat and poultry products processing
industries, enzymes have been predominantly used to improve the tenderness of
beef, pork, sheep, and chicken meat, whereas enzymes in fish-processing industries
find a wide array of applications, including the preparation of products, extraction of
biomolecules, and preservation (Fernandes, 2016; Suresh et al., 2015). The current
chapter primarily focuses on the application of enzymes in the processing of meat,
fish, and poultry products, their significance, and also their future scope in the food
industry.

8.2 Importance of Enzyme in Food Industry

Due to the rapid population explosion, extensive utilization of natural resources,

disputes across the globe, and variation in climate, there has been an increased
demand for food worldwide (Singh, 2018). In the food industry, the application of
enzymes is promising for the processing of food and also in solving food-related
problems (Venugopal et al., 2000; Singh, 2018). At present, enzymes have found
their wide application in various food industries, including beverage, dairy, oils and
fats, brewing, cereals, confectioneries, bakery, fruits and vegetables, juice, meat,
seafood, and poultry (Chandrasekaran et al., 2015). The importance of enzymes in
meat, fish, and poultry product processing is described below.

8.2.1 Current Scenario of Meat Production and Role of Enzymes

in Meat Processing

Animals such as sheep, goat, pig, cattle, and other livestock are slaughtered,
processed, and distributed in the meat industry (Chandrasekaran et al., 2015).
According to FAO, in the year 2018, global meat production increased up to 1.2%
compared to 2017, which could be easily interpreted in Fig. 8.1 (FAO, 2019).
Figure 8.1 depicts the production of meat worldwide from the year 2016 to 2019.
Increased production of pork and beef meat was observed in the year 2018, but there
was a slight decrease in their production in 2019. However, sheep meat production
increased from 2016 until 2019.
196 S. S. Karkal et al.

Fig. 8.1 Global meat production from the year 2016 to 2019. (Source: Statista, Production of meat
worldwide, 2020)

In the past decades, global consumption of meat has increased due to population
explosion and the economic boost in developing nations. Currently, the consumers
consider the quality attributes of meat products rather than their price, which
signifies that the greater the quality traits of meat products, the greater their impor-
tance to meet the increasing consumer demands (Gomes et al., 2018). The most
appreciable quality trait of meat judged by consumers is “tenderness”. The tender-
ness is a textural property of food associated with chewiness or cohesiveness. This
quality trait of food during mastication manifests little resistance to fragmentation
(Ashie et al., 2002; Gomes et al., 2018). The tenderness of the meat is primarily
related to the structural integrity of connective tissue proteins and myofibrillar
proteins. The key constituent of connective tissue is collagen, and the tenderness
of the meat is determined by the amount, type, as well as the degree of intermolecular
cross-linking in collagen. Studies have depicted an increased cross-linking in aged
animals rather than young animals (Ashie et al., 2002). To improve the tenderness of
the meat, the application of enzymes has been found to be desirable in terms of both
producing quality products and the cost (Cazarin et al., 2015). There are two
underlying mechanisms to obtain the desired tenderness in meat products using
enzymes. The first mechanism involves the generation of amino acids or peptide
fragments by breaking the covalent bonds of protein molecules. This would result in
meat structure alteration. The second mechanism includes the formation of new
covalent bonds, which would ultimately change the meat tenderness. The enzyme
mainly involved in the new bond formation is transglutaminases (Cazarin et al.,
2015). Both the endogenous enzymes (calpains, cathepsins, capsases, and
proteosomes) and exogenous enzymes from the plant, animal, and microbial sources
(ficin, bromelain, papain, actinidin, cucumin, zinzibain, collagenase, elastase, animal
protease, and fungal protease) have been found to play a pivotal role in the tenderi-
zation of meat (Ashie et al., 2002; Cazarin et al., 2015; Gomes et al., 2018). Apart
from the application of enzymes in meat tenderization, other techniques involve
shock wave pressure, calcium chloride injection, mechanical tenderization, electrical
stimulation, and elevated temperature storage (Ashie et al., 2002).
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 197

8.2.2 Current Scenario of Fish Production and Role of Enzymes

in Fish Processing

Over the decades, a tremendous leap has been observed in fish production across the
globe. The global fish production in the year 2018 was 178.8 million metric tons, and
in the year 2019, it is projected to be 177.9 million metric tons (Statista, 2019;
FAO-GLOBEFISH, 2020). Figure 8.2 displays the total global fish production from
the year 2016 to 2019. The total global fish production includes aquaculture produc-
tion, which includes the production of even crustaceans and mollusks (seafood). In
the majority of the countries, fresh fish and seafood harvested are mainly sold for
local consumption (Suresh et al., 2015). From the total fish and seafood processed,
about 20–50% is available for edible purposes, and the rest of it is generated as
non-edible by-products or discarded as waste. These by-products are rich in various
biomolecules such as oil and lipids, proteins, bioactive peptides, pigments,
polymers, vitamins, and minerals (Suresh et al., 2015).
The utilization of enzymes in the processing of fish and seafood has gained
momentum due to the advancements in enzyme technology. Enzymes have been
employed in fish-processing industries to obtain a diversified range and improved
quality products. In fish-processing industries, enzymes have been used for
descaling, deskinning, salted fish ripening, production of products such as surimi,
fish sauce, and fish protein hydrolyzates, extraction of oil and biomolecules such as
chitin, carotenoproteins, and flavor compounds, and removal of exoskeleton from
shellfish (Suresh et al., 2015). The enzymes used for fish and seafood processing are
mainly derived from microorganisms, plants, and animals (Fernandes, 2016; Suresh
et al., 2015). Moreover, research has also been carried out on the enzymes isolated
from fish discards and its application in the processing of fish and seafood (Suresh
et al., 2015). This approach would help in waste management of fish and seafood
by-products, the majority of which are mainly dumped into the environment. The
enzymes—both natural and recombinants—derived from microorganisms have been
used in fish and seafood processing. Further, the demand for microbial enzymes is
more than that for enzymes isolated from plants and animals since the microbial
enzymes exhibit better catalytic activity and high yield, and can be genetically

Fig. 8.2 Global fish GLOBAL FISH PRODUCTION

production from 2016 to 180
2020. (Source: Statista, 2020;
FAO-GLOBEFISH, 2020) 178
M illion me tric tons

176
174
172
170
168
166
2016 2017 2018 2019 (P)
Year
198 S. S. Karkal et al.

Fig. 8.3 Global poultry GLOBAL POULTRY PRODUCTION

production from 2016 to 128
2019. (Source: Statista, 126
Production of meat

M illion me tric tons

124
worldwide, 2020)
122
120
118
116
114
112
110
2016 2017 2018 2019
Year

manipulated with ease, regular supply, and rapid microbial growth in low-priced
culture media (Suresh et al., 2015). Besides fish and seafood processing, enzymes
have also been used in the analysis of processed aquaculture and fish products
(Fernandes, 2016).

8.2.3 Current Scenario of Poultry Production and Importance

of Enzymes in Poultry Processing

Similar to meat and fish production worldwide, poultry production has also
increased over the decades. Figure 8.3 displays the global poultry production from
the year 2016 to 2019. From the figure, it is evident that the global poultry
production is increasing every year and is higher than global sheep, pork and beef,
and veal production. Global poultry production has been soaring to meet the global
consumption demands due to overpopulation.
Besides the tenderization of pork, sheep, and beef meat, enzymes have also been
used for the tenderization of poultry products. Studies have been carried out on
improving the tenderness of poultry meat using exogenous enzymes such as papain,
bromelain, proteases, etc., and also endogenous enzymes such as calpains (Bawa
et al., 1981; Kang & Warner, 1974; Whipple et al. 1990).

8.3 Enzyme Sources and Types

As described in the previous sections, the enzymes used in meat, fish, and seafood as
well as poultry products processing may be from plants, animals, or microorganisms.
Apart from exogenous enzymes, the roles of endogenous enzymes are also crucial
for the processing of food products. The enzymes from the microbial source are most
widely preferred since they can be easily manipulated and microorganisms can be
easily cultured. Besides, the trend towards the use of enzymes from marine
microorganisms has been increasing from the last few years (Fernandes, 2016).
Proteases such as ficin, bromelain, and papain, which are primarily plant-based,
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 199

have been widely used in meat tenderization. These proteases possess a sulfhydryl
group in their active sites to carry out their catalytic activity. Apart from meat
tenderization, these proteases have also been used in the preparation of the fish
sauce. Moreover, papain has also been used in the preparation of fish protein
hydrolyzates (Fernandes, 2016). Research has also been carried out on the extraction
of enzymes from fish-processing discards and their application in the processing of
fish products. Enzymes including pepsin, trypsin, gastricin, alkaline proteinase,
acidic protease, calpain, lysosomal cathepsins type B, H, L, cathepsin and other
lysosomal cathepsins, chymotrypsin, collagenases, chymosin, elastase, lipase,
carbohydrases, and alkaline peptidase from fish-processing by-products have been
tested for their application in the processing of fish and seafood products
(Venugopal, 2016). Most of the enzymes used in meat, fish, and poultry product
processing are proteases. Besides proteases, other enzymes (lipases and
transglutaminases) play a major role in flavor modification, hydrolysis of fatty
acids, and other processing methods. The different enzymes used in the processing
of meat, fish, and poultry products, and also their sources are exhibited in Tables 8.1,
8.2 and 8.3, Fig. 8.4.

8.4 Enzymes Involved in Meat Processing and Preservation

8.4.1 Calpains

Calpains are calcium-dependent cysteine proteases mostly found in mammals.

Calpain 1 (μ calpain), calpain 2 (m calpain), and calpain 3 are the major calpain
proteases of skeletal muscle; this calpain system also consists of an inhibitor of
calpain 1 and calpain 2, called calpastatin (Goll et al., 2003). They are activated by
micromolar (1–100) and millimolar (0.1–1 mM) concentrations of calcium, respec-
tively (Suzuki et al., 1992). The intracellular calcium concentration is 1 μm or less at
most. Therefore, it always exists as a proenzyme until enough calcium is present for
activation. Tenderness is an essential aspect of meat quality. The degradation of
myofibrillar proteins (troponin T, troponin I, tropomyosin, connectin, C-protein, and
desmin) results in the weakening of myofibers. This is a crucial event in the
tenderization. μ-Calpain-induced degradation of key myofibrillar proteins is the
cause of postmortem proteolysis, and this is the primary source of muscle tenderness
(Koohmaraie & Geesink, 2006). This can be achieved by increasing the calcium
concentration (Rees et al., 2002) or by lowering the calpastatin activity (Hope-Jones
et al., 2010). Electrical stimulation (ES) can also lead to an increased flow of calcium
ions into the cytoplasm and may lead to μ-calpain activation (Veeramuthu & Sams,
1999). Calpastatins inhibit calpains and thereby reduce tenderness, but after cooking
calpastatin is inactivated, which leads to increased tenderness.
200 S. S. Karkal et al.

Table 8.1 Proteases used in the processing of meat, fish, and poultry products
Source Enzyme Source Applications
Plants Ficin Fig (Ficus glabrata, Meat tenderization
Ficus laurifolia, Ficus
anthelmintica)
Papain Papaya (Carica papaya) Meat tenderization, preparation
of fish sauce, and fish protein
hydrolyzates
Bromelain Pineapple (Ananas Meat tenderization and
comosus) preparation of fish sauce
Actinidin Kiwi fruit (Actinida Meat tenderization
deliciosa)
Cucumin Kachri fruit (Cucumis Meat tenderization
pubescens, Cucumis
trigonas)
Zinzibain Ginger (Zingiber Meat tenderization
officinale)
Animal Porcine Pork Meat tenderization
pancreatin
Microorganisms Fungal Aspergillus oryzae, Meat tenderization, preparation
protease Penicilium of fish protein hydrolyzates
chrysogenum Pg222
Alkaline Bacillus subtilis Preparation of fish protein
protease hydrolyzates
Thermophilic Bacillus strain E.A.1 Meat tenderization
enzyme E
A1 protease Thermus strain Meat tenderization
4-1 A Rt4-1.A Meat tenderization
protease
Caldolysin Thermus strain T-35 Meat tenderization
Elastase Bacillus species Meat tenderization
EL31410
Collagenase Vibrio B-30 Meat tenderization
Source: Naveena et al. (2004), Venugopal et al. (2000), Cazarin et al. (2015)

8.4.2 Cathepsins

Cathepsins are lysosomal acid proteases found in almost all organisms. There are
13 types of lysosomal cathepsins, 7 of which exist in skeletal muscle (Goll et al.,
1983). Cathepsins comprise both exoproteases and endoproteases, and are
differentiated from the active site: aspartate (Cathepsin D and E), serine (Cathepsin
G), and cysteine (Cathepsin B, H, L, and X) (Sentandreu et al., 2002). They exist as a
proenzyme in living tissue. Due to pH fall after cell death, they are released into the
cytoplasm or intercellular space due to lysosomal disruption (Duston, 1983).
Cathepsins B and L are the major cathepsins involved in muscle proteolysis (Jamdar
& Harikumar, 2002). Protease inhibitor cystatin regulates cathepsin B, H, and L
in vivo (Turk & Bode, 1991).
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 201

Table 8.2 Lipases used in the processing of meat, fish, and poultry products
Source Source Applications
Animal Porcine pancreas Preparation of ω-3 PUFA concentrates
Atlantic cod Production of ω-3-enriched triglycerides, flavor
improvement
Sardine Production of ω-3-enriched triglycerides, flavor
improvement
Indian mackerel Production of ω-3-enriched triglycerides, flavor
improvement
Red sea bream Production of ω-3-enriched triglycerides, flavor
improvement
Salmon Production of ω-3-enriched triglycerides, flavor
improvement
Microorganisms Pseudomonas Enrichment of EPA and DHA in sardine oil
species
Candida Increasing DHA content, hydrolysis of menhaden oil
cylindracea
Lactobacillus Synthesis of short-chain fatty acid esters
plantarum
Source: Suresh et al. (2015), Venugopal et al. (2000), Cazarin et al. (2015), Uppada et al. (2017)

Table 8.3 Transglutaminase application in the processing of meat, fish, and poultry products
Source Source Applications
Microorganisms Streptoverticillum sp., Fish meat sheet formation, fish meat film and
Bacillus subtilis, mince formulations, surimi and restructured
Streptomyces sp. fishery products, texture modification of
finfish, collagen and gelatin bond formation,
improve the gel strength of chicken and beef
sausages, improve the gel strength of chicken
and beef meat
Source: Suresh et al. (2015), Cazarin et al. (2015)

Many research groups ignore cathepsins’ contribution to meat tenderization due

to various reasons like no large-scale degradation of the actin and myosin in
postmortem conditioning (Koohmaraie et al., 1991). To access myofibril proteins
and to increase the tenderness, cathepsins must be released from lysosomes
(Hopkins & Taylor, 2002). Whipple et al. (1990) stated that there is only a minor
association between meat tenderness and cathepsin activity. However, the activities
of cathepsins B and L at 8 h postmortem have been found to positively correlate with
beef tenderness (O’Halloran et al., 1997). In the myofibrils of rabbit, beef, and
chicken, the main myofibrillar proteins (nebulin, tropomyosin, troponin T, I, C,
titin) along with actin and myosin are hydrolyzed by Cathepsin L during the
postmortem conditioning period (Mikami et al., 1987).
202 S. S. Karkal et al.

Enzymes (Proteases,
Lipases,
Transglutaminases)

Meat food processing Fish food processing Poultry food processing

Meat tenderization Deskinning and descaling, Poultry meat

fish protein hydrolysates tenderization
preparation, caviar
production, recovery of
proteins, ripening, flavor
enrichment, Preparation of
ω-3 concentrates

Fig. 8.4 Applications of enzymes in meat-, fish-, and poultry-processing industries

8.4.3 Papain

Papain is the most common enzyme used in meat tenderization. Papain, which
belongs to the C1 family of cysteine protease, also called the papain family
(Rawlings et al., 2010), is obtained from the latex of the papaya plant (Carica
papaya), and it plays a major physiological role in protecting the plant from insects
(Konno et al., 2004). Papain’s commercial importance is primarily due to its good
proteolytic activity against a broad variety of protein substrates, and because it is
active in a wide range of operating conditions. Smith and Hong-Shum (2003)
reported that it has a high optimal temperature (65
C) and broad pH range (5–8)
for its activity. Papain predominantly acts on the muscle structural component,
which enhances the meat tenderization (Gracey & Thronton, 1985). It is a highly
effective enzyme that is capable of inducing major degradation of collagen and
myofibrillar proteins (Ashie et al., 2002), and the maximum tenderizing activity
occurs during the cooking process (Tappel et al., 1956). Kang and Rice (1970)
reported that papain showed strong activity for a myofibrillar fraction with greater
solubilizing activity on the connective tissue. Use of papain leads to over tenderiza-
tion of meat which resulted in mushy meat, hence the commercial use of papain is
limited for meat tenderization (Han et al., 2009).

8.4.4 Ficin

Ficin, the proteolytic enzyme from fig trees (Ficus sp.), is a sulfhydryl protease.
About ten proteases are present in crude ficin latex (Kramer & Whitaker, 1964). The
mostly studied ficins are those from Ficus glabrata and Ficus carica. It is reported to
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 203

hydrolyze and increase the solubility of muscle proteins (El-Gharbawi & Whitaker,
1963). The solubility of meat protein might be increased by ficin by degrading the
proteins into units of smaller molecular weight, which, when aggregated, form a
three-dimensional network (Ramezani et al., 2006). El-Gharbawi and Whitaker
(1963) reported that the optimum pH was around 7 for enzyme activity for collagen
and myofibrillar proteins, and about 5.0–5.5 for elastin.

8.4.5 Bromelain

The proteolytic enzymes called bromelain are present in fruits, leaves, and stems of
the Bromeliaceae family of which pineapple (Ananas comosus) is the most com-
monly used enzyme in the processing of meat (Doko et al., 1991). The enzyme
extracted from the stem is called stem bromelain, and that extracted from the fruit is
called fruit bromelain (Vanhoof & Cooreman, 1997). Fruit bromelain is said to have
a higher proteolytic activity and specificity compared to stem bromelain (Barrett
et al., 2004; Grzonka et al., 2007). This enzyme, like other proteases, results in over-
tenderization by degrading myofibrillar proteins and collagen (Melendo et al., 1996).
At first, it degrades about 40% of the collagen in the sarcolemma, and then it
degrades myosin in the myofibrillar region (Kang & Rice, 1970; Wang et al.,
1958). The application of this enzyme is easy and cheap and can be exploited on a
household and industrial scale for tenderizing tough meat; it is also a better alterna-
tive to chemical tenderizers (Sunantha & Saroat, 2011). Ionescu et al. (2008)
investigated the use of bromelain in adult beef with best results at 10 mg/100 g
meat, with a tendering time of 24 h at 4
C, followed by thermal treatment with an
increase of 1
C/ min to 70
C (when enzyme inactivation occurs). These conditions
improved the tenderness of the beef.

8.4.6 Cucumin

Cucumin obtained from kachri (Cucumis pubescens) is a protease. Initially, the dried
coarsely ground fruits of C. trigonus Roxb (locally known as kachri) were tradition-
ally used as a meat tenderizer in some parts of India (Naveena et al., 2004). Cucumin
tenderizes meat to a significant degree, and may serve as a basis for commercial
meat-tenderizing mixtures (Hujjatullah & Baloch, 1970).

8.4.7 Zingibain

Zingibain protease was isolated from the plant Zingiber officinale roscoe (ginger
rhizome) by Thompson et al. (1973). This thiol protease shows optimum activity at
60
C (Naveena et al., 2004). The ginger extracts contain two cysteine proteases with
a molecular mass of 29 and 31 kDa, as reported by Su et al. (2009). This enzyme has
a greater proteolytic activity when heated (Naveena & Mendiratta, 2001). Its
204 S. S. Karkal et al.

proteolytic activity produces more tender meat by degrading collagen and actino-
mycin (Naveena & Mendiratta, 2001; Thompson et al., 1973). Lee et al. (1986)
reported that an extensive degradation of myofibrillar proteins is possible with a high
concentration of ginger extract: the degradation appears to begin at the I band of each
sarcomere and progresses to the M line.

8.4.8 Actinidin

Actinidin or actinidain is a cysteine protease obtained from the kiwi fruit (Actinidai
deliciosa). Actinidin is stable at a pH range of 7–10 and has optimal activity at
58–62
C (Yamaguchi et al., 1982). A pH range of 5–7 is also reported by Boyes
et al. (1997), which reflects variations in the cultivar and assay used to estimate the
proteolytic activity. Both myofibrillar proteins and connective tissue proteins are
hydrolyzed by this enzyme (Christensen et al., 2009; Han et al., 2009). But a higher
hydrolysis of collagen was reported by Wada et al. (2002).

8.4.9 Aminopeptidases

Aminopeptidases are the major enzymes involved in the characteristic flavor devel-
opment in meat products. They hydrolyze amino acids from the N terminus of
peptides and proteins during meat processing. This generates a large number of
free amino acids that produce flavor. In porcine skeletal muscle, aminopeptidase B
(BAP), leucyl (LAP), alanyl (AAP), and pyroglutamyl (PGAP) have been localized
in the cytosol, and are named according to the preference of specific N terminal
amino acid (Flores et al., 1993, 1996, 1997). Except for leucyl aminopeptidase, all
are active at acidic pH. In the cytosol of skeletal muscle, around 86% of the total
aminopeptidase consists of alanyl aminopeptidase (Lauffart & Mantle, 1998). As
reported by Toldra (1992), aminopeptidases show good stability during the
processing of dry-cured ham, with activity still recovered toward the end of the
process.

8.4.10 Lipases

Lipolysis is the process of degradation of lipids in meat during processing (Toldra &
Flores, 1998). Enzymes of fat cells and muscle fibers, as well as bacterial enzymes
are involved in lipolysis. However, it is established that in dry fermented sausages,
the contribution of bacterial lipase is weak because of many differences in the
medium and optimum conditions (Molly et al., 1997). Intense lipolysis occurs in
muscle and adipose tissues during the dry-curing of meat; most of the free fatty acid
generation occurs during the initial 5 months (Motilva et al., 1993). Important
lipolytic enzymes of adipose tissue are lipoprotein lipase, hormone-sensitive lipase,
and monoacylglycerol lipase (Belfrage et al., 1984). Lipoprotein lipase, specific for
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 205

primary esters, is active at alkaline pH 8.5. Hormone-sensitive lipase hydrolyzes

ester bond in triacylglycerols and diacylglycerols. Monoacylglycerol lipase
hydrolyzes monoacylglycerols without any positional specificity and is active during
salting and post-salting stages. Hormone-sensitive lipase is also called neutral lipase
due to its high activity at neutral pH (Imanaka et al., 1984). The muscle lipase,
lysosomal acid lipase, active at acidic pH hydrolyzes tri, di, and monoacylglycerol
(Imanaka et al., 1984). Lipases can be used to remove fat in meat products (Uppada
et al. 2017). Lipase produced from Lactobacillus sp. degrades meat in 72 h
(Padmapriya et al., 2011).

8.4.11 Fungal Proteases

An exogenous serine protease (EPg222) obtained from Penicillium chrysogenum

Pg222, an atoxigenic strain, was isolated from dry-cured ham (Núñez et al., 1996).
This protease shows optimum activity at 45–55
C, pH 6, and 0.25 M NaCl (Benito
et al., 2002). Benito et al. (2003) reported that EPg222 hydrolyzed key myofibrillar
proteins, supporting the tenderization of whole pieces of meat with 5% NaCl. The
dry-cured meat product’s texture could be improved by fungal proteases. The
introduction of EPg222 in dry fermented sausages, ripened with starter cultures, is
of great interest to boost sensory characteristics (Benito et al., 2004). An aspartic
protease from Aspergillus oryzae shows optimal activity at a pH range of 2.5–6, and
less than 20% of its activity remains after cooking at 75
C (Ashie et al., 2002). It
does not affect collagen. Its effect on myofibrillar proteins is limited compared to
plant protease, with self-limiting hydrolyses at higher concentrations (Cronlund &
Woychik, 1986; Ashie et al., 2002).

8.4.12 Elastase

Elastase is the enzyme that breaks down elastin in connective tissue. A new elastase,
a protease with high proteolytic activity, was isolated from Bacillus sp. EL31410 by
Chen and He (2002). It had a marked preference for elastin and collagen, which can
contribute to meat hardness, over other myofibrillar proteins at the pH of meat,
usually ranging from 5.5 to 6.0, and it has the same tenderization effect as papain on
beef meat based on the results of texture, sensory, and structural analyses (Qihe et al.,
2006).

8.4.13 Bacterial Protease

Many strains of bacteria play a major role in protein degradation in fermented meat
and fish products (Bekhit, 2010). Because of relatively specific activity and low
inactivation temperatures, proteases from bacterial sources make them useful for
meat tenderization. Alkaline elastase from alkalophilic Bacillus sp. strain Ya-B
206 S. S. Karkal et al.

shows an optimum activity in the pH range of 5.5–6.0 and a temperature range of

10–50
C (Takagi et al., 1992; Yeh et al., 2002). Compared to plant proteases,
bacterial proteases have less hydrolytic activity towards myofibrillar proteins but
their ability to degrade collagen was intermediate to that found in bromelain and
papain (Yeh et al., 2002).

8.4.14 Animal Proteases

Animal proteases are less used for meat tenderization. Injection of pancreatin on beef
semitendinosus tended to improve overall tenderness without affecting the taste
(Pietrasik et al., 2010).

8.5 Enzymes in Fish Products Processing and Preservation

Various enzymes from different sources have been exploited for the processing of
fish products and their preservation. The main enzymes, which play a key role in the
processing of fish and seafood are described below.

8.5.1 Proteases

Proteases find their wide application in the seafood processing industry, especially in
deskinning, descaling, salted fish ripening, fish sauce production, squid tenderiza-
tion, etc. Besides, proteases have also been examined for the extraction of
compounds (mainly pigments, oils and fats, and flavors) and the preparation of
hydrolyzates from seafood processing wastes (Suresh et al., 2015). Proteases from
plant, animal, microbial, and even aquatic sources have been used for the processing
of fish.

1. Deskinning and descaling: Deskinning is the procedure involving the removal of

fish skin without damaging the flesh. The deskinning process is carried out using
rough mechanical procedures,which results in flesh damage and excess waste
generation. The replacement of mechanical procedures with enzymes i.e.,
proteases could improve the edible yield. Commercial proteases such as Protease
N and Proleather FG-F have been tested for the deskinning of catfish nuggets
(Kim et al., 2014). Descaling of fish involves a harsh mechanical process, which
might result in the tearing of fish skin and thereby lower the fillet yield. To avoid
this loss, enzymatic descaling using the mixture of proteases is carried out
(Fernandes, 2016).
2. Fish protein hydrolyzates: Over more than 50 years, the application of proteases
in the fish protein hydrolyzates preparation has been widely studied. The fish
protein hydrolyzates are generally prepared by treating fish meat or protein-rich
fish processing by-products (fish head, skin, liver, trimmings, bones, and fish
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 207

viscera) with proteases at ideal pH and temperature for a few hours, followed by
drying. The enzymes preferred for the production of fish protein hydrolyzates
include pepsin, trypsin, chymotrypsin, alcalase, etc. The prepared fish protein
hydrolyzates are amorphous powder and hygroscopic in nature. The fish protein
hydrolyzates contain proteins (80–90%), fats (<5%), minerals (5–8%) and mois-
ture (1–8%) (Venugopal et al., 2000; Venugopal, 2016). Traditionally, the prepa-
ration of fish protein hydrolyzates was carried out using either acids or bases.
Hydrochloric acid or sulfuric acid (occasionally) was preferred for acid hydroly-
sis, whereas alkali hydrolysis was carried out using sodium hydroxide. Both acid
and alkali hydrolysis are carried out at elevated temperature. Moreover, alkali
hydrolysis produces toxic compounds during the reaction process, and acid
hydrolysis destroys tryptophan. Besides, fish protein hydrolyzates preparation
using low-cost proteolytic enzymes can be carried out at a mild temperature,
pressure, and pH, and doesn’t produce toxic compounds. Thus, the preparation of
fish protein hydrolyzates using proteolytic enzymes is economical (Fernandes,
2016).
Gajanan et al. (2016) prepared protein hydrolyzates from threadfin breams
(Nemipterus japonicas) a fish frame waste using two different plant proteases,
papain and bromelain. The prepared protein hydrolyzates exhibited high
Angiotensin-Converting Enzyme (ACE) inhibitory and anti-oxidative activity.
The hydrolyzates also showed better functional properties. Similar results were
also exhibited by protein hydrolyzates prepared from the muscle of small-spotted
catshark (Scyliorhinus anicula) using esperase and alcalase (Vázquez et al.,
2017).
3. Fish sauce: Fish sauce is a product produced by the fermentation process using
endogenous or exogenous enzymes. The underlying process mainly involves the
solubilization and digestion of fish proteins by enzymes. The pre-dominant
enzymes involved in protein hydrolysis include chymotrypsin and trypsin, along-
side cathepsins. When the pH of fish sauce drops from 7 to 5, the action of trypsin
and chymotrypsin becomes complicated since they are active at near-neutral pH,
whereas cathepsins are active at acidic pH. Fish sauce production using endoge-
nous enzymes is a traditional process, and it’s a very time-consuming process.
Therefore, to hasten the process, exogenous enzymes such as papain, ficin, and
bromelain, and commercial enzymes such as protamex (Protex 51FP) and
neutrase have been used (Fernandes, 2016). About 80–90% of the Southeast
Asian people consume fish sauce. It is being used as a prime condiment in
Southeast Asian cuisines to improve the taste of food. Currently, Thailand is
the leading producer of fish sauce in the world (Gowda et al., 2020).
4. Recovery of proteins from fish processing waste: Fish waste is a rich source of
proteins such as collagen, myofibrillar proteins, and sarcoplasmic proteins.
Proteases derived from the fish wastes (collagenases and pepsin) have been
employed for the extraction of collagen from various fish offals, including
skins, fins, bones, scales, swim bladders, and heads (Venugopal, 2016).
5. Ripening: Proteases have been known to be involved in the ripening of fermented
fishery products as well as salted fish (Venugopal, 2016; Fernandes, 2016). The
208 S. S. Karkal et al.

exogenous proteases accelerate the development of soft texture and characteristic

flavor in fermented fishery products. In Mediterranean countries, salted anchovy
(Engraulis encrasicholus) is a heavily salted fish comprising around 14–15% of
NaCl and has high nutritional value. In many European countries, salting of
herring (Clupea harengus) is being carried out as a conventional preservation
technique (Suresh et al., 2015). The ripening of salted fishes involves a complex
biochemical process, wherein the muscle proteins undergo degradation by endog-
enous enzymes. The main endogenous enzymes involved in the ripening are
trypsin, chymotrypsin, and cathepsins (Venugopal, 2016; Fernandes, 2016).
6. Caviar production: Caviar refers to the cured fish roes of salmon, trout, white
sturgeon fish, etc. Traditionally caviar was used to refer to riddles and cured roe of
sturgeon fish. The most difficult task in the preparation of caviar is the separation
of roe from the roe sack. This process is generally termed riddling. Riddling
process is generally carried out either manually or mechanically. Recently, the
applications of enzymes in the preparation of caviar from various fish species
have been examined (Suresh et al., 2015; Venugopal, 2016).
7. Other applications: The proteases have also been used in various other seafood
processing operations. Some of these operations include (1) control of curd
formation in canned fish, (2) preparation of bioactive peptides, (3) extraction of
biopolymers, pigments, and compounds such as chondroitin sulfate and
hyaluronic acid, (4) the production of aquafeed and seafood flavoring, (5) reduc-
tion of viscosity of stick water, and (6) control of enzymatic browning
(Venugopal, 2016).

8.5.2 Lipases

Lipases (triacylglycerol acyl hydrolases EC 3.1.1.3) are the enzymes primarily

involved in the hydrolysis of mono, di, and triglycerides and fatty acids. The
hydrolysis reaction takes place in the presence of an excess of water, but when
there is limited water, lipases promote the synthesis of esters. Lipases also exhibit
other esterase type or phospholipase type activities. These activities of lipase have
recognized industrial relevance. Lipases differ in their properties and also their origin
(plant source, animal source, and microbial source). Lipases have been exploited for
isolating oils and fats from seafood by-products. The lipases have also been utilized
for ω-3 fatty acids’ (eicosapentaenoic acid and docosahexaenoic acid) enrichment of
fish oils. Lipases along with other muscle enzymes such as caplains, cathepsins,
aminopeptidases, and peptidases have been known to influence the release of flavor
compounds (Suresh et al., 2015; Fernandes, 2016; Venugopal, 2016).

8.5.3 Transglutaminase

Transglutaminase (TGAs, protein-glutamine γ-glutamyl transferase EC 2.3.2.13)

enzymes primarily catalyze acyl transfer reactions. The reaction includes the
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 209

γ-carboxyamide group of peptide-bound glutamine acting as acyl donor and many

primary amines acting as acyl acceptors. Simultaneously, many intermolecular and
intramolecular covalent bond formation occurs, resulting in the cross-linking of
proteins and peptides, and polymerization. In the absence of primary amines,
water acts as an acyl acceptor, and the γ-carboxyamide group undergoes deamina-
tion to glutamic acid residues. TGase from plant and animal sources are Ca2+
dependent enzymes, whereas TGase from the microbial source is Ca2+ independent
enzyme. Moreover, microbial TGase is active over a wide pH and temperature
(0–70
C). These features of microbial TGase and its cost-effective production
make it the most widely preferred enzyme for processing of fish products. TGase
has been produced by microorganisms such as Streptomyces sp., Bacillus subtilis,
and Streptoverticillium sp. The application of TGases in the fish products processing
industry includes preparation of surimi and restructured fishery products, fish meat
film formulations, processing of shark fin, modification of texture of finfish, the
formation of gelatin and collagen bonds, fish meat mince formulations, drip reduc-
tion in thawed fish, freeze-texturization of fishery products, and binding of
ingredients with fish meat (Suresh et al., 2015; Venugopal, 2016).

8.5.4 Other Enzymes

Besides proteases, lipases, and transglutaminases, other enzymes have also been
exploited for the fish products processing industries. Enzymes such as diamine
oxidase, xanthine oxidase, nucleotide phosphorylase, putrescine oxidase, and gluta-
mine dehydrogenase have been used for the evaluation of the quality of seafood and
freshness. Restriction endonucleases and Taq polymerase have been used for the
identification of fish species and adulteration detection. Furthermore, research works
have also been carried out in increasing the shelf-life of seafood products using
glucose oxidase enzymes.

8.6 Enzymes in Poultry Products Processing and Preservation

Various exogenous and endogenous enzymes have been exploited for the processing
of poultry products. The enzymes used in the processing of pork, sheep, and beef
meat are also being used in the processing of poultry meat. Mainly the enzymes are
used to improve the tenderness of poultry meat. Papain, ficin, and bromelain
enzymes derived from plants have been extensively utilized for the tenderization
of poultry meat. The proteolytic enzymes derived from plants are considered to be
superior to enzymes derived from bacteria (Ketnawa & Rawdkuen, 2011). The
applications of commercial enzymes such as alcalase, neutrase, flavorzyme,
protamex, collupulin, bromelain, and alphalase in softening the chicken meat
(breast) texture have been studied. Of all the enzymes bromelain and collupulin
have shown the marked softening of chicken breast. The exploitation of proteolytic
enzymes from Calotropis procera latex has also shown to be effective in the
210 S. S. Karkal et al.

tenderization of poultry meat. Apart from the application of enzymes in poultry meat
processing, the addition of enzymes in the improvement of barley and wheat-based
feed has also been studied by Perić et al. (2011). They stated that incorporating the
enzyme Allzyme SSF into poultry feed had a positive effect on the growth of
chicken, and observed increased egg production in layers. Thus, enzymes find
extensive application in the poultry industry to prepare poultry feed and poultry
meat processing.

8.7 Future Prospects

Nature is a reservoir of several enzymes with promising potential for a wide

application in human endeavors, but only a few of them have been exploited for
over a century. This scenario’s rationale includes that the enzyme might be from an
undesirable or unreliable source (from unreachable environments such as deep-sea,
or from microorganisms), challenges in production at a commercial scale and
downstream processing, and the requirement of harsh conditions for activity. As a
result, scientists and technologists continue their attempts to uncover novel enzymes
from new origins and experiment with their potential applications, overcoming all
obstacles through laboratory research efforts (Chandrasekaran, 2015).
Enzymes are known to play a crucial role in the food industry in producing both
traditional and novel products. Enzymes have also found their application in meat,
fish, and poultry processing to obtain products with better quality attributes, evaluate
the products’ quality and freshness, and extend the products’ shelf-life. With the
increase in the production of meat, fish and poultry products, the demand for
enzymes has also increased. The enzymes from plants, animals, and microbial
sources have been extensively studied for the processing of meat, fish or seafood,
and poultry products. The complete substitution of the chemical processing methods
with enzymatic methods would help overcome environmental problems arising due
to chemical processing techniques. Further, the application of enzymes in the meat,
fish, and poultry products processing industry has excellent prospects since the
production of enzymes to be exploited in these industries is cheaper. Their utilization
helps to obtain quality products with better yields.
Furthermore, advances in biotechnology, nanotechnology, molecular biology,
instrumentation, and bioinformatics have opened up new horizons for designing
hybrid catalysts and novel enzymes for diverse food industry applications, including
meat, poultry, and fish processing industries. The novel enzymes or recombinant
enzymes developed through the modern biotechnological approach would effec-
tively carry out industrial processes at low temperatures, reduce the cost of produc-
tion and energy consumption, and enhance the products’ properties
(Chandrasekaran, 2015). Besides, the enzymes derived from seafood processing
waste have a promising future for their fish product processing application alongside
the recombinant enzymes being developed for enzymatic processing and preserva-
tion of meat, fish, and poultry products.
8 Enzymes in Meat, Fish, and Poultry Product Processing and Preservation-II 211

8.8 Conclusion

Currently, the meat, fish, and poultry industries play a significant role in meeting the
global food demand. The enzymatic processing of meat, fish, and poultry products
processing is still in the early stage, and the introduction of new techniques is
resulting in progress. The enzymatic processing technique has the potential to
overthrow the chemical and mechanical techniques employed in the processing of
meat, fish, and poultry products processing. This would help to overcome the
environmental problems caused by chemical and mechanical processing techniques.
The most suitable enzymes that could be employed in the processing of meat, fish,
and poultry products are discussed in this chapter. The meat and poultry industries
mainly utilize enzymes to improve the tenderness of the meat, whereas in fish
products processing enzymes have a wide array of applications. Technological
advancements have enabled the development of recombinant enzymes from
microorganisms for their application in these industries. However, recombinant
enzymes are yet to be used in the industries. Thus, the enzymes have a promising
future for their application in the processing of meat, fish, and poultry products.

Acknowledgments The authors are grateful to the CSIR-Central Food Technological Research
Institute (CSIR-CFTRI), Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, and
the Department of Biotechnology (Govt. of India, New Delhi) for the support.

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Enzymes in Functional Food Development
9
Iran Alemzadeh, Asma Sadat Vaziri, Kianoush Khosravi-Darani,
and Pierre Monsan

Abstract

Enzymes are ubiquitous biocatalysts, represent a consolidated strategy in various

food industries for the transformation of one biological compound to another. In
contrast to conventional synthesis, enzymatic synthesis is an important techno-
logical advancement as a consequence of sustainability, substrate selectivity,
nontoxicity, and low energy input. Therefore, there has been a growing trend in
the use of free and immobilized enzymes in various food industrial applications,
including the manufacturing of several functional foods and ingredients. Func-
tional foods with health benefits beyond nutrition have gained much attention due
to some factors such as interest in health-consciousness and the growing market.
A variety of functional foods can be considered to be those fortified, wholes,
enriched, or enhanced foods that represent physiological health benefits beyond
the essential nutrients provision (e.g., vitamins and minerals) when consumed in
adequate amounts on a regular basis. The initial components in functional food
supplements naturally consist of fruit, food, beverage, grains, and supplement
sectors at a low level. Nutraceuticals or functional food could be produced from
the enzymatic reaction utilizable in one of the several areas of food science and
technology, that is experiencing fast growth in recent years. Functional food

I. Alemzadeh (*) · A. S. Vaziri

Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
e-mail: alemzadeh@sharif.edu
K. Khosravi-Darani
National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences and Food
Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran
P. Monsan
Département de Génie Biochimique et Alimentaire, INSA, Toulouse Cedex 4, France
White Biotechnology Center, Parc Technologique du Canal, Ramonville Saint Agnes, France

# The Author(s), under exclusive license to Springer Nature Singapore Pte 217
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_9
218 I. Alemzadeh et al.

could be enzymatically derived from various reactions: L-asparaginase, an inter-

cellular enzyme, is used in the baking industry which suppresses acrylamide
production. Addition of asparaginase is a recommended strategy to decrease the
formation of acrylamide, as it catalysis the hydrolysis of asparagine to aspartic
acid an amino acid with high nutritive value. Carbohydrate active enzymes such
as β-glucanase which is able to proceed on glucose polysaccharides and to further
develop the production of β-glucans, the saccharide made up of multiple sugar
molecules. β-Glucan may offer a number of health benefits, including lowering
cholesterol, improving blood sugar management, and boosting the immune
system which is produced by the enzymatic process of beta D-glucose polysac-
charide naturally occurring in the cell wall of cereals like oat. Chlorophyllase
(Chlase) is widely distributed in the chloroplast, thylakoid membrane, and
etioplast of higher plants, such as ferns, mosses, brown and red algae, and
diatoms. Chlase catalysis is the degradation of chlorophyll to chlorophyllide
(also called Chlide), which is introduced as a food colorant. L-Glutaminase is a
catalyst for the production of L-theanine, a free amino acid with many health
benefits. L-Theanine synthesis occurs from L-glutamine and ethylamine substrates
through the transpeptidation reaction of L-glutaminase, and a demand for this
amino acid is expected to grow rapidly in the industry. Lipids such as CLA
(conjugated linoleic acid) are in the group of functional food, a result of CLA
production from natural oil in the presence of a lipase. CLA is a natural derivative
of essential fatty acid, linoleic acid which shows many beneficial effects on the
immune system, role in obesity control and anticarcinogenic behavior phytases
may find application in food processing to produce functional foods and phytic
acid degradation. Phytase is able to decrease the antinutritional effect of phytate
(inorganic phosphate complex) adverse impact of inorganic P excretion to the
environment while increasing the digestibility of phosphorous (P), calcium,
amino acids, and energy. γ-Amino butyric acid (GABA) is an active protein, in
rice germ using protease or additives: produced by the decarboxylation of
glutamic acid, GABA is catalyzed by glutamate decarboxylase, GABA functions
as the main inhibitory neurotransmitter in the central nervous system. Tannase is
able to catalyze the production of tea beverages while maintaining the tea’s
original deep flavor. Engineering of enzymes is a potential strategy in the design
of food development according to the specificity and enzyme structural changes.
The next future step toward the commercialization of the enzymatic approach in
food processing is to enhance their efficiency and mitigate adverse effects on the
sensory properties of food products. In this chapter, we mainly highlighted the
importance of selected enzymes on food development.

Keywords
Enzymes · Functional food · L-Asparaginase · β-Glucanase · Chlorophyllase ·
Lipase · L-Glutaminase · Phytase · Protease · Tannase
9 Enzymes in Functional Food Development 219

9.1 Introduction

Enzymes are ubiquitous biocatalysts, represents a consolidated technique in green

chemistry for the transformation of one biological compound to another for biotech-
nological purposes. In contrast to conventional synthesis, enzymatic synthesis is an
important technological advancement as a consequence of sustainability, substrate
selectivity, nontoxicity, and low energy input. Therefore, there has been a growing
trend in the use of free and immobilized enzymes in many industrial applications,
including the manufacturing of several functional foods and ingredients. Functional
foods with health benefits beyond nutrition have increased rapidly due to factors
such as interest in health-consciousness and the growing market. A variety of
functional foods can be considered to be those fortified, wholes, enriched, or
enhanced foods that represent physiological health benefits beyond the essential
nutrients provision (e.g., vitamins and minerals) when consumed in adequate
amounts on a regular basis (Alemzadeh et al., 2020). The initial components in
functional food supplements naturally consist of fruit, food, beverage, grains, and
supplement sectors at low levels (Granato et al., 2020). Nutraceuticals or functional
foods involve extraction, purification, concentration, and activation processes in
order to be utilizable in one of the several areas of food science and technology,
which could be processed by an enzymatic reaction. The discovery, characterization,
and stabilization of various enzymes employed in the production of functional foods
have been attended increasingly in many researches.
Food and stress are two major factors that have a direct impact on the gut
microbiome and are responsible for inflammatory reactions in the human gastroin-
testinal. Any dysbiosis in the gut microbiota results in several gastrointestinal-
associated diseases like diarrhea, irritable bowel syndrome (IBS), and colorectal
cancer. The use of functional foods has been increasingly applied as a complemen-
tary therapy to rejuvenate and enrich the beneficial gut microorganisms, thereby,
promoting the health, alleviate, and relief the symptoms associated with gut-related
diseases and side effects of conventional diseases, reduce the risk of cardiovascular
diseases, and improve overall well-being. Microbial enzyme engineers or enhance
the production of certain naturally occurring dietary substances through fermentation
technology (Melini et al., 2019). Functional foods, enriched with prebiotics,
synbiotics, and probiotics, as well as other plants- and animal-derived food
compounds, are also considered beneficial additives to improve the consumers
health and well-being (de Paulo Farias et al., 2019; Vaziri et al., 2019).
Functional food could be enzymatically derived from various reactions; the L-
asparaginase enzyme produced by the enzymatic reaction is used in the baking
industry, which suppresses acrylamide production. The addition of asparaginase is
a recommended approach to decrease the formation of acrylamide since it results in
the hydrolysis of asparagine to aspartic acid, an amino acid with high nutritive value
(Momeni et al., 2015; Torang & Alemzadeh, 2016).
Lactic acid bacteria strains have been shown to produce glucose, galactose, and
oligosaccharides (prebiotics), as a result of lactose hydrolysis and transgalactosidal
activities of beta-galactosidase. Another illustration is the production of
220 I. Alemzadeh et al.

prebiotic-based compounds from cereals, a kind of non-starch polysaccharides

(NSP), such as β-glucan and arabinoxylan as dietary fiber constituents (You et al.,
2016). β-Glucan consists of multiple sugar molecules, which has a significant role in
lowering cholesterol level, boosting blood sugar management, and improving the
immune system. This essential fiber is synthesized by an enzymatic process through
the action of carbohydrase on beta D-glucose polysaccharide, which basically occurs
in the cell wall of cereals like barely and oat (Kumar et al., 2020).
Chlorophyllase enzyme (Chlase, chlorophyll-chlorophyllidohydrolase, EC
3.1.1.14) is a hydrophobic enzyme in the chlorophyll hydrolysis to become degraded
into chlorophyllide (Childe) and phytol. Chlorophyllide can be further considered as
natural water-soluble food colorant alternative to synthetic colorants. Moreover, they
possess biological and therapeutic activities, such as anti-inflammatory, antioxidant,
antibacterial, wound healing, anticarcinogenic, and deodorizing properties (Hosikian
et al., 2010).
Incorporation of lipase enzyme in the esterification of glycerol with CLA and
production of enriched fatty acid derivatives with more health benefits and reduction
in free fatty acids accumulation is a solution for functional food development. Lipids
such as CLA (conjugated linoleic acid) are in the group of functional food, resulting
from CLA production from natural oil in the presence of a lipase. CLA is a natural
derivative of essential fatty acid, linoleic acid, which shows many beneficial effects
on the immune system, obesity control, and anticarcinogenic behavior. Catalytic
action of lipase enzyme in the esterification of glycerol with CLA causes the
production of enriched fatty acid derivatives with more health benefits and reduction
in free fatty acids accumulation. Incorporation of lipase enzyme in the esterification
of glycerol with CLA and production of enriched fatty acid derivatives with more
health benefits and reduction in free fatty acids accumulation is a solution for
functional food development (Kouchak Yazdi & Alemzadeh, 2016a, b; Kouchak
Yazdi et al., 2017).
Synthesis of theanine was examined in the presence of the L-glutaminase enzyme
produced by Trichoderma koningii fungus. Production of L-glutaminase enzyme was
first concerned by T. koningii using sesamum oil cake under solid-state fermentation
(SSF). Then L-theanine production by L-glutamine and ethylamine as substrates and
extracted enzyme solution were evaluated (Sakhaei & Alemzadeh, 2017).
Enzyme-catalyzed degradation to enhance the bioavailability of metal ions like
iron and zinc in cereal-based foods is an example of enzyme-hydrolysis by phytase.
The effects of phytase functionality on amino acid digestibility and energy utiliza-
tion, and the in vivo functional food specificity have been monitored (Dersjant-Li
et al., 2015). Phytases application in functional foods development is recommended
for the increment of phytic acid dephosphorylation and degradation. Phytase can
increase mineral bioavailability and decrease the anti-nutritional effect of phytate
(inorganic phosphate complex) by improving phosphorous (P) digestibility, as well
as reducing the negative impact of inorganic P excretion on the environment (Song
et al., 2019; Handa et al., 2020).
Proteolytic enzymes are ubiquitous in innumerable biological systems and led to
improve functional and biological characteristics of protein byproducts, which can
9 Enzymes in Functional Food Development 221

Fig. 9.1 Enzymes accessible in functional food development and the selected bioactive
components

be further utilized as protein substrates for food applications (Pokora et al., 2013).
Bioactive peptides (BP) are one of the most important bioactive compounds
generated from protease hydrolysis. The protein fragments of BPs act as peptide
inhibitors of the angiotensin-converting enzyme (ACE), and antimicrobial,
anti-aggregating, antithrombotic, anticancer and antihypertensive, and immune-
modulatory peptides. The proteolysis of BPs is required for their release and activa-
tion since they are hidden and encrypted within the parent proteins, such as casein,
lactalbumin, beta-lactoglobulin, and lactoferrin (De Leo et al., 2009). Gamma-
aminobutyric acid (GABA) is another potent bioactive protein catalyzed by gluta-
mate decarboxylase. GABA functions as a major inhibitory neurotransmitter in the
mammalian brain and central nervous system that strongly effects the behavior and
stress management. It is widely distributed in nature and demand for microbial
GABA supply has raised to satisfy the needs of GABA-enriched foods (Cui et al.,
2020).
Tannase is another group of enzymes that are suitable candidates as biocatalysts
in the treatment of phenolic-rich foods. It catalysis the biotransformation of phenolic
compounds in beverages to generate a product with enhanced clarity and color
appearance, as well as promoting its functional activity. As an example, the process
of browning in tea beverages occurs due to the presence of caffeine and catechins in
the tea, which results in the formation of insoluble compounds in cold water.
Biotransformation by tannase reduces the gallic acid-binding and inhibits the
browning effect (Srivastava & Kar, 2009; Cao et al., 2019; Gligor et al., 2019).
The enzyme accessible in functional food development and bioactive components is
categorized in Fig. 9.1. In this chapter, we describe the functional food components,
functional food in health, the role of selected enzymes in functional foods
manufacturing, or food improving quality circumstances. Enzymes, such as
asparaginase, carbohydrase, chlorophyllase, lipase, L-glutaminase, phytase, prote-
ase, and tannase are introduced, and the beneficial health effects are discussed.
222 I. Alemzadeh et al.

9.2 Functional Food Components

Functional foods are enriched or dietary materials that may offer several health
benefits beyond basic nutrition. Functional foods for health are an essential factor of
an overall healthful lifestyle that consists of a balanced diet from various sources.
People should strive to consume a wide variety of foods and food components
including the examples listed in Table 9.1. These food components could naturally
exist in natural food, such as whole grains, fruits, vegetables, beverages, enriched
compounds, and dietary supplements, extracted by chemical approaches or produced
by enzymatic processes.

9.2.1 Functional Food in Health

Obesity and malnutrition are major drawbacks associated with diet habits, which are
strongly influenced by urbanization, and lifestyle alterations (Tanna & Mishra,
2018). Functional food could inherently include bioactive ingredients or
nutraceuticals and could be added from various sources to produce fortified food.
The bioactive ingredients are in the group of carbohydrates, enzymes, protein,
phytochemicals, vitamins, and lipids (Suleria et al., 2015; Maqsood et al., 2020).
These bioactive compounds could be derived from different sources like cereals,
seeds, marine-based, microorganisms, plants, animals, and fishery. Since bioactive
compounds in natural food products are in low amounts, the bioactive ingredients
could be produced by a microbial or enzymatic process in high quantity. The
bioactive ingredients have antibiotic, antiparasitic, antiviral, anti-inflammatory,
anti-fibrotic, and anticancer activities. Health benefit of the bioactive compounds
affects metabolic syndrome (Mets), such as CVD, elevated triglycerides, cholesterol,
and blood pressure, besides insulin resistance (Sirtori et al., 2017). Nutraceutical
ingredients have specificity such as antimicrobial, antiviral, antioxidant, anti-
obesity, anticancer, antidiabetic, anti-inflammatory, nephrotoxic, and arthritis.

9.3 Enzyme Application in Functional Food

Enzymes are catalytic biomolecules responsible for the catalyzing diverse biochem-
ical reactions in several food industries. Enzymatic processes can be a promising
alternative to conventional organic synthesis, as they provide eco-friendly, more
viable and sustainable conditions, safer products, and use of renewable substrates.
Enzymes are therefore possessing a significant role in many biotechnological
applications including food quality improvement, as well as food manufacturing
operations. Selected enzymes representing these concerns are defined and evaluated
respectively.
9 Enzymes in Functional Food Development 223

Table 9.1 Examples of functional food sources selected nutraceuticals, and their biological
activity
Functional food Selected
sources nutraceutical Biological activity Reference
Coriander fruits Fatty oil Antimicrobial, Sahib et al. (2013)
(seed and antioxidant,
pericarp) antidiabetic
Non-starch Dietary fiber Cholesterol reduction Sirtori et al. (2017)
polysaccharides
(NPS)
Pumpkin seeds Traditional Antioxidative, Patel and Rauf (2017)
medicine hypoglycemic,
anticancer
Yeasts Various preventing oxidative Rai et al. (2019)
bioactive stress
components
Probiotics Traditional Intervention of IBD Al Mijan and Lim (2018),
medicine Grom et al. (2020)
Seaweed Metabolite Chronic diseases such Tanna and Mishra (2018)
as cancer, arthritis,
diabetes
Mediterranean Various Antidiabetes Alkhatib et al. (2017)
diet bioactive
components
Plant sterols and Phytosterols Plasma cholesterol Poli et al. (2018)
stanols control
Phytochemicals Various Cancer, CVD, anti- Gul et al. (2016)
bioactive obesity
components
Higher Dietary fibers Anti-cardiovascular Venkatakrishnan et al.
polysaccharide and cerebrovascular (2020)
disease
Food bioactive Polyphenol, Anticancer Adefegha (2018)
compounds, plant flavonoids
foods
CLA PUFAs Anti-obesity, Kouchak Yazdi et al.
antidiabetic (2017), Yazdi and
Alemzadeh (2017)
Cereals Polyphenols Anti-chronic, diabetes, Arab et al. (2011)
anticancer, anti-obesity
Marin Bioactive Anticoagulant, anti- Dewapriya and Kim (2014),
microorganisms polysaccharides inflammatory, antiviral Suleria et al. (2015)
Date fruit and Phytochemical Antioxidant, antifungal Maqsood et al. (2020)
seed and antiviral,
anticancer
224 I. Alemzadeh et al.

9.3.1 L-Asparaginase

L-Asparaginase, also known as asparagine amino hydrolase (EC 3.5.1.1), is an

intercellular enzyme broadly used in various pharmaceutical and food applications
for catalyzing the degradation of L-asparagine into ammonia and L-aspartate. In the
medicinal aspect, the asparaginase role is to inhibit cell starvation by reducing
asparagines availability (Asselin & Rizzari, 2015). The L-asparaginase has been
regarded to be a safe and favorable additive for the mitigation of acrylamide levels
during food processing (Baskar et al., 2019).
Acrylamide is a main component in many carbohydrate/starchy-based foods,
such as cereals, peanuts, potatoes, vegetables, crips, chips, coffee, biscuits, and
lentil, particularly when exposed to high temperatures up to 120
C. Several food
processes including frying, baking and roasting, and process conditions, such as
temperature, type of product, humidity, time, and content of reducing sugars and free
amino acids result in the formation of acrylamide, and the processing variables
influence directly the level of acrylamide content (Zuo et al., 2015). Acrylamide is
generated during Millard reactions in the presence of reduced sugars and asparagine.
The main consequences of acrylamide formation in food products are the strong
color and flavors.
The potential toxicity and adverse effects of this compound on human health were
declared (WHO/FAO). L-Asparaginase has remarkable attention in research from
past decades, because of its vital role in the metabolism of amino acids, and thus, can
be considered for the treatment of different types of cancers and diseases including
acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),
non-Hodgkin’s lymphoma, and food applications (Alam et al., 2019). According
to the Global Asparaginase Sales Market Report, the asparaginase market has
growing demand. The reported market value for asparaginase was 380 million
USD in 2017 and is estimated to reach 420 million USD by 2025 (Global Medical
Asparaginase Sales Market Report, 2018). L-Asparaginase is isolated from various
plants and animal sources, such as animal cells, algae, chicken liver, serum of
rodents, and microorganisms including fungal, yeast, and bacteria. Bacillus sp. and
Pseudomonas sp. are the two main microbial sources of asparaginase. Bacterial
sources have gained more attention for industrial application due to their versatile
characteristics, easy maintenance, and high efficiency (Shi et al., 2017). There are
two types of L-asparaginase; type I and type II. For food and medical applications,
type II asparaginase is more favorable. Type II asparaginase shows a high affinity
toward L-asparagine and hence it has been implicated for the removal of asparagine
before processing the raw ingredients to reduce acrylamide formation (Hendriksen
et al., 2016). Although several types of research emphasized the allergic reactions
due to the high immunogenicity of the asparaginase and its clinical toxicity, such as
headache, dyslipidemia, diarrhea, nausea, mucositis, pancreatitis, liver dysfunction,
central nervous system dysfunction, and abdominal pain, type II asparaginase
proved to be less immunogenic, and therefore, is more suitable for medical
applications (Mitchell et al., 1994; Rizzari et al., 2000; Zuo et al., 2015). Other
microorganisms with fewer adverse effects such as eukaryotic microorganisms have
9 Enzymes in Functional Food Development 225

been explored (Patro et al., 2014). Fungal sources are also considered safe
asparaginase producers (generally recognized as safe, GRAS). It has been reported
that fungic L-asparaginase utilization during food manufacturing leads to acrylamide
reduction without any negative impact on the final appearance and nutritional value
of the product. This approach mainly occurs from two aspects, interference with
Millard reactions and removal of L-asparagine as the main precursor of acrylamide
formation (Batool et al., 2016). However, the efficacy of asparaginase formation is
not sufficient for the industrial demands (FAO/WHO, 2002; Jha et al., 2012). Two
commercial samples of fungal asparaginase in the food industry, PreventAse (DSM)
and Acrylaway (Novozymes) are obtained from Aspergillus niger and Aspergillus
oryzae, respectively (Pedreschi et al., 2011; Xu et al., 2016; DSM, 2018).
PreventAse presents an optimum pH in the range of 4–5 and temperature at 50
C,
while these values for Acrylaway are 6–7 and 60
C. The activity of L-asparaginase
during food preparation and hydrolysis should be high, thus producing a stable
enzyme is of great importance.
The cultivation conditions including media composition, pH, temperature, oxy-
gen levels, and the fermentation technique, namely solid-state fermentation or
submerged fermentation, have considerable influence on the production of an ideal
enzyme (Brumano et al., 2019). Carbon and nitrogen are the most important
substrates in the culture medium used for L-asparaginase production (Cachumba
et al., 2016). Glucose is considered the most appropriate carbon source to achieve L-
asparaginase (Doriya & Kumar, 2016). L-Asparagine, L-proline, urea, yeast extract,
and peptone are some of the important sources of nitrogen for high-yield
asparaginase (de Moura Sarquis et al., 2004).
Determination of the optimum pH and temperature of the fermentation medium is
also an important step for producing high-yield enzymes. The emergence of
engineered enzymes according to the structural and mechanistic methods and direct
evolution is needed to enhance the efficiency of enzymes for the synthesis of various
products (Bornscheuer et al., 2012). Bakery products, French fries, and roasted
coffee are the most widely consumed products that have high acrylamide content.

9.3.2 Carbohydrate Active Enzymes

Glucans are non-starch glucose polymers and one of the most abundant
polysaccharides found in the cell wall of fungi, plants, and bran cereal grains
(Wu et al., 2018). The glucose moieties link through either or both alpha (α) or
beta (β) linkages, so their structures can be either linear or branched, and microfi-
brillar or amorphous. α-1,3-Glucans sensu strictu (pseudonigerans) is known as the
most abundant α-glucans in the cell wall of fungi. Their structure is in the form of
microfibrils which make resistance to the cell wall. On the other hand, the structure
of β-glucans is a complex of linear or branched. In most cases, the structure is in the
form of microfibrils with mostly β-1,3-linear, and β-1,6-branched linkages. One such
example is a water-soluble low molecular weight polymers (LMW), which contains
β-1,2 besides β-1,6 joined branches and has been isolated from black yeast
226 I. Alemzadeh et al.

Table 9.2 The classification of β-glucans and β-glucanases enzymes from various sources
EC
β-Glucans type Structure Description number References
Bacterial Linear β-1,3-glucan Exo: Wu et al.
(Euglena EC (2018)
gracilis) 3.2.1.58
Endo:
EC
3.2.1.39
Fungal Branched β-1,6-branched, and Endo: Sutivisedsak
(Schizophyllum β-1,3-glucan EC et al. (2013)
commune) 3.2.1.6
Yeast Long branched β-1,6-branched and EC Sato et al.
β-1,3-glucan 3.2.1.39 (2012)
endo-β-1,3-glucanases EC
3.2.1.6
Cereal (barley) Linear β-1,3/1,4-glucan Endo Rodríguez-
3.2.1.6 Mendoza
et al. (2019)
Lichen Linear 1,3–1,4-β-glucan Endo You et al.
(lichenase, 3.2.1.73 (2016)
licheniase)

(Aerobasidiumpillulans). β-1,3–1,6-D-glucan represents antitumor, anti-metastatic,

and anti-stress effects (Sato et al., 2012). β-Glucans together with chitin are known to
be the most important structural constituents of fungal cell walls (Ruiz-Herrera &
Ortiz-Castellanos, 2019).
As an example of a β-glucans-producer fungal, Schizophyllum, produces
schizophyllan, which is a homoglycan natural polysaccharide with a β-1,3 linked
backbone and single-1,6-linked glucose side chains at each residue. Its role as a
biological response modifier and a stimulator of the immune system provides a
variety of applications in the therapeutic and cosmetics industry. This biodegradable
polymer has been also used for food preservation by forming oxygen impermeable
films (Sutivisedsak et al., 2013).
β-Glucanase is an enzyme classified in the glycosyl hydrolase family that
catalyzes the release of glucose from the reducing and nonreducing ends of
β-glucan. The glucose released from the catabolic activity of β-glucanase applied
in bioethanol and biofuels manufacturing (Lopez-Casado et al., 2008). Selected
β-glucanases have an essential role in the pharmaceutical and medical industry by
biologically controlling fungal pathogens, synthesizing drug carriers, and producing
in vivo immunomodulatory anticancer drugs (You et al., 2016). β-1,3-Glucanases
have been used in the food industry, mainly for alcoholic beverages processing. The
classification of β-glucans and β-glucanases enzymes is represented in Table 9.2.
The modification of polysaccharides through β-D-glucans hydrolysis results in
gluco-oligosaccharides production which provides several biological activities such
as immunomodulation (Giese et al., 2012).
9 Enzymes in Functional Food Development 227

Another advantage of exogenous β-glucanase is to incorporate it into wheat and

barley-based diets to inhibit the anti-nutritive effect of water-soluble NSP. Benefits
of using exogenous enzymes for nutrient digestibility have been reported previously;
however, the mechanism of action of these enzymes is not fully recognized. One
other application of these exogenous enzymes is to enhance the microbial activity of
the poultry intestine (Giese et al., 2012).
β-1,3-Glucanases have been recommended to apply in the food industry to
improve the organoleptic properties of the final products. Besides, they have a
positive role in the production of feed and animal nutrition (Rodríguez-Mendoza
et al., 2019). The sources of glycoside hydrolase β-1,3-glucanase are extended
among fungi, bacteria, and plants. The hydrolysis position of structural
β-1,3-glucans is on 1,3-β-glucosidic bonds to transfer or hydrolyze glycosides.
β-1,3-Glucanases are normally classified into endo-enzymes (EC 3.2.1.39) and
exo-enzymes (EC 3.2.1.58). β-1,3-Glucanases are able to act as a protective agent
against fungal pathogens and provide immune-modulating and antitumor properties
in plants. They are also capable to suppress fungal growth in fermentation media by
the convention of β-1,3-glucans into (1!3)-linked β-D-glucan oligosaccharides
(Dewi et al., 2016; Wu et al., 2018).
Endoglucanases are generally classified into three categories based on their
modes of action: Endo-1,3–1,4-β-glucanase (EC 3.2.1.73), endo-1,3-β-glucanase
(laminarinase; EC 3.2.1.39), and endo-1,3(4)-β-glucanase (EC 3.2.1.6). They spe-
cifically cleave the blended (1!3) or (1!4) linkages of β-D-glucan. β-Glucanases
are important commercial biocatalysts in various industries, from exogenous
β-glucanase, which are applicable specifically in reducing the unwanted effects of
barley β-glucan in the mashing operation as a step in the brewing industry, and to
improve the β-glucan digestibility in poultry feedstuffs, to endoglucanase to convert
biomass into bioethanol in and xylanase in bioenergy production (You et al., 2016).

9.3.3 Chlorophyllase

Chlorophyllase enzyme (Chlase, chlorophyll chlorophyllidohydrolase, EC 3.1.1.14)

is a hydrophobic enzyme leading to chlorophyll degradation into chlorophyllide
(Chlide) and phytol. Chlorophyllase activity is the most important step in the
chlorophyll (Chl) breakdown and synthesis (Hörtensteiner & Kräutler, 2011).
Another Chl hydrolysis pathway is through pheophytinase, which leads to
pheophorbide (Schelbert et al., 2009). The chlorophyll breakdown pathway
determines the Chl accumulation and various intermediate catabolites in
chloroplasts. Chlorophyll hydrolysis is most evident in fruit ripening, and seasonal
changes as leaf senescence and the color of plants convert from green to yellow.
Chlorophylls are one of the most important plant pigments that determine the
absorption of solar radiation in leaves (Filella et al., 1995). They are a brilliant
source of natural green color and can be an alternative to synthetic colorants.
Moreover, they possess biological and therapeutic activities, such as anti-
228 I. Alemzadeh et al.

Fig. 9.2 The initial steps of chlorophyll catabolism (CLH and MCS are refer to chlorophyllase and
magnesium-dechelating substance, respectively) (Hu et al., 2015)

inflammatory, antioxidant, antibacterial, wound healing, anticarcinogenic, and

deodorizing properties (Hosikian et al., 2010).
The activity of chlorophyllase in fruits and vegetables prolongs during storage
and processing conditions. Chlorophyllide is obtained through enzymatic
conversion of chlorophyll by the cleavage of the phytol group and is used for the
preservation of green color in various canned vegetable products. By the aid of
magnesium-chelating substrates, the induction of chlorophyll breakdown leads to
pheophytins hydrolysis and more pheophorbide formation (Fig. 9.2). A well-known
application of chlorophyllide formation is the formation of pheophorbide during
olive fermentation. For example, the activity of chlorophyllase in ripe olives of
arbequina and picual varieties was studied and demonstrated that the process of oil
extraction and propitiating the pheophytization of chlorophylls in the fresh fruit
cause a significant reduction in the chlorophyll’s pigments (Gandul-Rojas &
Mínguez-Mosquera, 1996).
As shown in Fig. 9.2, the catabolism of chlorophyll to synthesize chlorophyllide
triggers chlorophyllase. In both decoloration processes and the homeostasis of
chlorophylls, chlorophyllase takes part as an effective catalyst in the early steps of
chlorophyll breakdown. The role of chlorophyllase in plant development and
9 Enzymes in Functional Food Development 229

survival is vital because it leads to faster degradation of chlorophyll and its

intermediates, and further detoxification process (Carle & Schweiggert, 2016).
Experimental evidence in epidemiology has shown the association of green fruits
and vegetables with chronic disease prevention, thus, phytochemical foods have
established as bioactive dietary compounds. Chlorophyll and its lipophilic
derivatives are composed of chlorophyll a and b (fresh fruits and vegetables),
metal-free pheophytins and pyropheophytins (thermally processed fruits and
vegetables), and Zn-pheophytins and Zn-pyropheophytins (thermally processed
green vegetables) are believed to be responsible for such associations, as they are
phytochemical compounds. Chlorophyllides, pheophorbides, and sodium copper
chlorophyllin (SCC), a commercial-grade derivative, are hydrophilic compounds
with the predominant contribution to the dietary chlorophyll role. Antioxidant and
antimutagenic activity, mutagen trapping, modulation of xenobiotic metabolism, and
induction of apoptosis are attributed to useful biological activities of chlorophyll
derivatives which assimilate cancer prevention.
Although the medical evidence of chlorophyll derivatives are mainly focused on
commercial-grade SCC, there is significant attention to the cancer-preventive poten-
tial of chlorophyll. Further research is required to define the extent to which
chlorophyll derivatives are responsible for modulating cancer-risk biomarkers
(Ferruzzi & Blakeslee, 2007).
Chlorophyllase catalyzes the hydrolysis of chlorophylls to chlorophyllide and
phytol. This catalytic activity of chlorophyllase has potential industrial and agricul-
tural applications. In this context, the application of chlorophyllase could be useful
in the removal of green pigments (chlorophyll) from edible oils to improve their
oxidative stability. In addition, the products of chlorophyllase, chlorophyllides, and
their derivatives (such as pheophytin) have been demonstrated to have antiviral,
antioxidant, antimutagenic, and anticarcinogenic activities in vitro. Accordingly, in
recent years, trends for purification of natural chlorophyllases or recombinant
production of the enzyme has been increased (Sharafi et al., 2017).

9.3.4 L-Glutaminase

L-Glutamine catalysis is the enzymatic reaction of thiamine. Thiamine (γ-glutamyl

ethyl amide) is a nonprotein amino acid and is known to be responsible for the sense
of relaxation in green tea. L-Thiamine is generally recognized as safe (GRAS),
and apart from its umami taste and unique flavor, has several pharmaceutical and
physiological effects, such as anticancer, anti-obesity, anti-hypertensive and
neuroprotective effects. It may further improve learning ability and mind perfor-
mance. Thus, thiamine is an essential compound for the human body that should be
administrated orally, as it is not synthesized in the body (Haskell et al., 2008; Panesar
et al., 2016).
The natural sources of thiamine include plants, animals, and microorganisms.
Microbial-derived is preferable sources compared to plant and animal-derived thia-
mine, mainly due to the low cost of operation, as well as other technical advantages
230 I. Alemzadeh et al.

Fig. 9.3 Theanine synthesis

reaction catalyzed by L-
glutaminase in the presence of
ethanolamine (EtNH2)
(Sakhaei & Alemzadeh, 2017)

such as higher yields within a limited period of fermentation time. The enzymatic
biosynthesis of L-thiamine takes place in the presence of ethylamine and L-glutamine
as substrate and catalyst (glutaminase). L-Thiamine production was carried out under
solid-state fermentation by the fungal strain Trichoderma koningii. The enzymatic
synthetic reaction for theanine production is presented in Fig. 9.3.

9.3.5 Lipase

Lipases, also known as triacylglycerol acyl ester hydrolases (EC 3.1.1.3), are
capable to catalyze cleavage of carboxyl ester bonds in tri-, di-, and
monoacylglycerols. Most lipases are generally recognized as safe (GRAS) and can
be employed in the food industry, mainly for diary applications with the aim of
ripening acceleration in Italian-type cheeses, and hydrolysis of milk fat to further be
used as a flavor enhancer in cheese products (Afarin et al., 2018).
Further applications of lipases in pharmaceutical and fine chemical preparation
processes include (but are not limited to) lipase-mediated synthesis of optically pure
and optically active polymers, lipase-catalyzed resolution of racemic aqueous
mixtures, monoglycerides production (as an emulsifier in the food and cosmetic
products), fatty alcohols (as lubricants in hydrophobic waste treatment), and sugar
esters as biosurfactants (Tarahomjoo & Alemzadeh, 2003).
Another advantage of lipase incorporation in dairy products is fat and oil-tailoring
for enhancing functional and nutritional properties. One such example is employing
lipase in conjugated linoleic acid (CLA) large-scale production for the modification
of triglycerides and upgrading of low-value oils and fats like human milk fat
replacers, cocoa butter substitutes, and enrichment of nutraceutical fats with poly-
unsaturated fatty acids (Paiva et al., 2000; Yazdi & Alemzadeh, 2017). CLA is one
of the most physiological active fatty acids a natural derivative of linoleic acid, an
essential fatty acid, that possesses a conjugated double-bond system for describing
octadecadienoic acids mixtures (18:2) (Jiang et al., 1998; Lin, 2006; Gofferjé et al.,
2014).
Due to the global rising trend concerning diseases, such as obesity, hyperlipid-
emia, atherosclerosis, diabetes, cancer, and high blood pressure and the established
association of lifestyle on health management, the quality of dietary fat is an
important factor that should be considered for prevention and treatment of early
stage diseases. In this regard, conjugated fatty acids have gained much attention for
their potential health effects on lowering the risk of these diseases, such as
9 Enzymes in Functional Food Development 231

anticarcinogenic activity, immune system enhancement, immune stimulation, low-

ering atherosclerosis symptoms, and cholesterol reduction (Niezgoda &
Gliszczyńska, 2019; Kouchak Yazdi & Alemzadeh, 2016a, b; Afarin et al., 2018).
Natural sources of CLA include dairy products (0.40–0.55% of total dairy lipids)
and beef (0.43% of total beef lipids). Lactobacillus plantarum is a microbial-derived
source of nutritious and valuable components, such as CLA. CLA contains a variety
of positional and geometrical conjugated double bonds of linoleic acid (C18:2)
isomers, including all possible geometric configurations of trans–cis, cis–trans,
cis–cis, and trans–trans isomers (Paiva et al., 2000; Philippaerts et al., 2013).
The 9c, 11t-18:2 and 10t, 12c18:2 isomers of CLA are believed to be responsible
for the beneficial physiological effects of CLA. The 9c, 11t-18:2 isomer (rumenic
acid), is the main isomer, consists of 90% of the total CLA found in dairy and beef
lipids; so, the focus of most research on CLA employment is on cis-9, trans-11 CLA.
Moreover, the multitude health-promoting effects of CLA, such as anti-atherogenic
activity, are associated with c9, t11 CLA (ANDO, 2004). Other isomers, namely,
trans-10, cis-12 (t10, c12CLA), are found in much smaller quantities. c9, t11, and
t10, c12 isomers are determined to represent the highest bioactive property (Ogawa
et al., 2005). Further investigations on optimizing the experimental design for a safe
and selective biological process for CLA preparation to achieve enriched food with
high CLA concentration are required (Afarin et al., 2018).
Although the exact mechanism of anticarcinogenic and atherosclerosis risk-
reduction benefits is still needed to be studied, clinical evidence demonstrates that
the management and prevention of these diseases are achievable through
CLA-functional food on regular basis. It has been recommended that the consump-
tion of 3 g/day of CLA is essential to provide essential health factors. It is notewor-
thy that the attainable CLA from natural resources is approximately 0.3 g/day;
therefore, CLA-based dietary supplements and CLA-rich vegetable oils and dairy
products should be considered as part of a healthy lifestyle. Main commercial
approaches for CLA production consisting of chemical, microbial, and enzymatic
methods are represented in Table 9.3. Each technique implements a different proto-
col and results in a different mixture of CLA isomers. Among them, the alkali
isomerization of linoleic acid has been reported to be the most potential and cost-
effective method (Yang et al., 2002; Afarin et al., 2018).
According to the potential health benefits demonstrated for CLA, it is a preferable
additive for the preparation of functional foods. The synthesis of CLA can be
performed either by isomerization of linoleate-rich oils with the aid of an alkaline
catalyst or by dehydration of ricinoleic acid-rich oils or their esters using an acid
catalyst.
CLA isomers, either in their natural or synthetic forms, have been shown consid-
erable physiological effects on human health. CLA free fatty acid may cause
undesirable reactions in the human gut. Thus, it has been recommended to be
consumed in the form of triglycerides. One way is to apply sunflower oil for
transesterification of lipid, mainly due to its beneficial health effect, via lipase-
catalysis. Enzymatic process is an alternative method to catalyze the
transesterification reaction biologically to produce triacylglycerols with enriched
232 I. Alemzadeh et al.

Table 9.3 CLA production by various techniques

Method Catalyst Substrate References
Chemical Alkaline Linoleic acid Afarin et al. (2018)
Chemical Alkaline Ricinoleic acid Yang et al. (2002)
Chemical Metal catalyst (SnCl2) Methyl linoleate Philippaerts et al.
(2013)
Microbial Lactobacillus plantarum PTCC 1058 Castor oil Kouchak Yazdi and
Alemzadeh
(2016a, b)
Microbial Dairy starter cultures Linoleic acid Jiang et al. (1998)
Microbial Lactobacillus acidophilus Linoleic acid Ogawa et al. (2005)
Microbial Lactobacillus plantarum A6-1F Linoleic acid Zhao et al. (2011)
Microbial Lactobacillus reuteri Linoleic acid Lee et al. (2003)
Microbial Bifidobacteria and Propionibacteria Linoleic acid Hennessy et al.
(2012)
Microbial Lactic acid bacteria Linoleic acid, Ogawa et al. (2005)
castor oil
Microbial Lactobacillus plantarum JCM 1551 Castor oil ANDO (2004)
Enzymatic Crude enzyme from Lactobacillus Linoleic acid, Lin (2006)
delbrueckii ssp. bulgaricus oleic acid,
(CCRC14009) linolenic acid

residues. Generally, the alteration of fatty acids composition results in promoted

nutritional properties of triacylglycerol molecules. Identifying an environmentally
friendly and less energy-intensive strategy for oil esterification by using an enzyme
as a catalyst should be investigated (Ogawa et al., 2005; Hennessy et al., 2012).

9.3.6 Phytase

Phytic acid, designated as phytate when in salt form, is regarded as a primary

reservoir of organic phosphorus in pollen and plant seeds like nuts, cereals, oil
seeds, and legumes. It has a high negative charge and is, therefore, able to form ionic
complexes with essential cationic minerals (Ca2+, K2+, Fe2+, Zn2+, Mg2+, and Mn2+),
proteins, and trace elements (Greiner & Konietzny, 2006). A main anti-nutritional
consequence of this interaction is the considerable reduction in dietary minerals
bioavailability. Phytase, also known as myo-inositol (1,2,3,4,5,6)-hexakisphosphate
phosphohydrolase, act as a dephosphorylating enzyme to transfer phytic acid to
more soluble and lower chelating products to inhibit anti-nutritional factors of phytic
acid in food products and increase mineral absorption (Kuddus, 2018). Depending
on their catalytic mechanism, phytases can be divided into histidine acid phytases,
cysteine phytases, β-propeller phytases, or purple acid phytases (Chu et al., 2004)
and based on their optimum pH, into acid and alkaline phytases. Dephosphorylation
is initiated at the carbon in the myo-inositol ring of phytic acid. There are three
different types based on the carbon; 3-phytases (EC 3.1.3.8), 5-phytases
9 Enzymes in Functional Food Development 233

(EC 3.1.3.72), and 6-phytases (EC 3.1.3.26) (Greiner & Konietzny, 2006). Enzy-
matic dephosphorylation of phytates was originally a strategy to improve the
nutritional value of feed by liberating phosphate, especially for mono-stomached
animals like fish, broilers, shrimp, pigs, and poultry diets (Konietzny & Greiner,
2004).
Recently, there have been numerous studies showing the potential use of these
enzymes for functional food development. One of the major concerns in phytate
hydrolysis content during food manufacturing is the selection of an ideal phytase.
The activity of added phytase has to be high enough during food preparation.
Therefore, a high capability of phytate degradation over a wide range of
temperatures and pH is essential (Greiner & Konietzny, 2006). According to previ-
ous studies the optimum pH range for acid phytase is 3.5–6.0, and for alkaline
phytase is 7.0–8.0. The optimum temperature for high phytate-degrading activity is
in the range of 35–80
C. The optimum temperature and pH of the added enzyme are
strongly affected by the source of phytase.
Phytase has been found in plants, microorganisms, and animal tissues. There are
already available commercial phytases from different microbial sources, such as
Escherichia coli, Peniophora lycii, Aspergillus niger, Schizosaccharomyces pombe.
Commercial production of phytase from Aspergillus, a soil fungus, has several
prominent drawbacks, such as fungal enzymes are catalytic efficiency, substrate
specificity, and resistance to proteolysis. Bacterial phytases have been distributed in
various aerobic and anaerobic species, including Bacillus strains, Pseudomonas
strains, Raoultella strains, Escherichia coli, Citrobacter braakii, Prevotella strains,
Selenomonas ruminantium, Mitsuokella jalaludinii, Megasphaera elsdenii, Lacto-
bacillus sanfranciscensis. The expression of phytase from these bacteria sources is
different and it can be regulated by the nature of the culture composition used for
growth and the nutrient or energy limitations. The level of inorganic phosphate on
the phytase synthesis induction was reported to be effective for all microbial sources,
except Raoultella terrigena and the rumen bacteria. In some phytase producers, the
nature of the carbon source is a significant inducible factor by involving in the
catabolism pathway (Konietzny & Greiner, 2004).
Generally, microbial-derived phytases are more favorable economically com-
pared to plant-derived ones, normally due to their thermostability and pH tolerance
as well as a broad specific activity. The specific activity of characterized phytasea, is
approximately in the range of 10–1000 U/mg at 37
C and various pH optimums
(Greiner & Konietzny, 2006). The highest specific activities are reported for
microbial-derived enzymes. In food applications, it is important that the enzymatic
properties of phytase to hydrolyze phytate in the gastric conditions be stable under
acidic pH and pepsin enzyme. Likewise, for food supplements, an enzyme with
acidic pH optimum and highly resistant to digestive enzymes is indisputably
attractive.
However, when exposed to pH values above pH ¼ 7.5 or below pH ¼ 4.0, most
of the plant enzymes dramatically lose their stability (Greiner & Konietzny, 2006).
Besides, the activation of these enzymes is irreversibly affected when exposed to
temperatures above 70
C within a few minutes. Despite the fact that most of the
234 I. Alemzadeh et al.

plant enzymes are inactivated at pH values lower or higher than the pH optima and
high temperatures, they are more acceptable among consumers as they are rarely
reported as an allergen.
The use of agro-industrial byproducts in the culture media has been suggested as
low-cost substrates for the production of phytase (Pires et al., 2019). Corn meal and
rice bran were used for the production of phytase from the fungus A. zeae B, and the
yeast K. marxianus. The activity of phytases from agro-industrial products strongly
depends on the species of the microorganism. For example, the determination of
total phytase activity in the mentioned study revealed tenfold higher values in the
yeast extraction compared to those of the fungus extraction (Pires et al., 2019).
Engineered phytases via genetic engineering are a promising type of enzyme that
show optimized catalytic features, thermal stability, and broad specific activity for
food application. Enhancement of thermal stability by a shift from 80 to 90 and
optimum temperature from 55 to 65 for Escherichia coli was achieved by using
mutagenesis technology. The basis of the phytase synthesis is to mutation of gene
site saturation by comparing the amino acid sequences derived from both the
available standard program and the homologous proteins (Rodriguez et al., 2000;
Garrett et al., 2004).
The incorporation of phytase in the food industry is a growing trend, mainly for
two purposes; increasing the mineral bioavailability of a given food by reducing its
phytate content and exerting nutritional health benefits in a supplemental product
(Konietzny & Greiner, 2004).
Natural phytate-degrading activity, observed in most plants, results in phytate
dephosphorylation during food processing such as soaking, germination, and fer-
mentation. This capability of intrinsic phytate-degrading activity in plant seeds
exhibits over a wide range of pH values from 3 to 10, and the maximum activity
peak occurs at pH ¼ 5–5.5 (Greiner & Konietzny, 2006). Therefore, phytate
hydrolysis by a certain phytase at optimum pH and temperature might differ greatly
among various plants and microorganisms. Some examples of optimum conditions
for maximum phytase activity are represented in Table 9.4.

9.3.7 Protease

Enzymatic hydrolysis for modification of food proteins in functional food

applications has been explored for several decades. Proteolytic action of an enzyme
results in alteration of molecular conformation of the native protein, and thus, leads
to the production of functional and bioactive components as texture enhancers in
beverages, infant formula, and pharmaceutical ingredients (Adebiyi et al., 2009).
The resultant properties are highly affected by the degree of hydrolysis (DH).
Variations in functionality factors such as solubility, stability, gelling behavior,
water absorption, emulsion, and foaming capacity are best observed following a
low degree of protein hydrolysis (<10%). Low-DH protein hydrolysates are appro-
priate for enhancing food texture, while high-DH protein hydrolysates are suitable
 9

Table 9.4 The effect of phytase sources on the specific activity and pH and temperature optimum for various applications
Specific pH Temperature optimum
Phytase source activity optimum (
C) Application References
Yersinia intermedia 3960 U/mg 4.5 55 Feed industry Huang et al. (2006)
Streptomyces sp. (NCIM – 2.5 70 Plant growth Puppala et al. (2019)
5533)
Sporotrichum thermophile 1190 U/mg 5.0 60 Dietary supplement Maurya et al. (2017)
Aspergillus aculeatus APF1 13.82 U/mg 3.0 50 Biofortified cereals Saxena et al. (2020)
Enterobacter sp. ACSS 805.98 U/mg 2.5 60 Dephytinizing animal feeds, baking Chanderman et al. (2016)
Enzymes in Functional Food Development

industry
Pichia anomala – 4.0 60 Fractionation of allergenic soya Joshi and Satyanarayana
proteins (2017)
Rhizopus oligosporus 31.3 U/gds 5.5 50 Food and feed industry Suresh and Radha (2016)
MTCC 556
Aspergillus niger NCIM 160 IU/mL 2.5 50 Organophosphorus pesticides Shah et al. (2017)
563 degradation
Penicillium oxalicum 12.8 U/g 7.0 40 Food industry Kaur (2017)
EUFR-3
Aspergillus ficuum 5.17 U/gds 6.1 37 Food industry Tian and Yuan (2016)
235
236 I. Alemzadeh et al.

for utilization in medical diets, such as hypoallergenic foods or protein supplements

(Pokora et al., 2013).
The scientific community has declared the important role of dietary compounds
as health-promoting ingredients for food and pharmaceutical applications. Dietary
components are mainly considered bioactive substances if their consumption in
adequate amounts shows beneficial biological effects on human health. Therefore,
the desirable consequences of bioactive compounds incorporation in food produc-
tion are represented provided that their utilization is in the real sense and without the
exertion of side effects (De Leo et al., 2009). Short peptides including 3–20 amino
acids are examples of bioactive peptides (BP) that usually encrypted within the
primary structure of a larger protein. BPs are encrypted within the parent proteins; so
in order to exert their physiologically positive effects on human health, their
proteolysis is required.
Biologically active peptides in the protein sequence are defined as “fragments that
remain inactive in precursor protein sequences, but when released by the action of
proteolytic enzymes, they may interact with selected receptors and regulate the
body’s physiological functions” (Mohanty et al., 2016). Bovine milk contains
approximately 3–3.5% protein of which 80% and 20% are casein and whey proteins,
respectively. It exerts a wide range of nutritional, functional and biological activities
(Fox, 1989). Many milk proteins possess specific biological properties that make
them potential ingredients for health-promoting foods. Many researches have been
carried out on bioactive peptides from casein hydrolysates. The list health benefits of
biologically activity of milk bioactive peptides are radical scavenging, lipid oxida-
tion inhibition, antiproliferation, stimulate proliferation, antimutagenicity,
immunostimulant, immunosuppressant, and antimicrobial (Phelan et al., 2009).
These peptides can be liberated by:

1. Gastrointestinal digestion of milk

2. Fermentation of milk with proteolytic starter cultures or
3. Hydrolysis by proteolytic enzymes like pepsin, trypsin, chymotrypsin, etc.

A great variety of peptides are formed during cheese ripening which has been
shown to exert biological activities. Furthermore, secondary proteolysis during
cheese ripening may lead to the formation of bioactive peptides (Korhonen &
Pihlanto, 2006). The occurrence of the bioactive peptides which are naturally formed
in cheese depends on the equilibrium between their formation and the degradation of
the peptide during cheese ripening. Ripening is a time-consuming process during
cheese making. To overcome these problems, some economic and technological
methods for acceleration of cheese ripening have been reported (Fox, 1989). Accel-
eration of ripening may help to increase bioactive peptides. Encapsulation of
enzymes in liposome can accelerate cheese ripening (Jahadi et al., 2016;
Mohammadi et al., 2015; Zoghi et al., 2018). Vmax and Km and stability of the
enzyme were increased by encapsulation of the Flavourzyme in liposome
(Vafabakhsh et al., 2013; Jahadi et al., 2012, 2015, 2020; Jahadi & Khosravi-
Darani, 2017).
9 Enzymes in Functional Food Development 237

Gamma-amino butyric acid (GABA) is another amino acid that consists of four
carbons widely found in microorganisms, animals, and plants. GABA has been used
in pharmaceuticals and functional foods due to its blood pressure-lowering activity
and its role as a neurotransmitter signal in the central nervous system including the
mammalian brain (Takahashi et al., 2012). It can also be used for the synthesis of
nylon 4 as a monomer (Kim et al., 2007; Park et al., 2013). Its production has been
reported to occur by decarboxylation of glutamate, a reaction catalyzed by glutamate
decarboxylase (Pham et al., 2016).
The ever-increasing demand for GABA has generated the need for efficient
microbial production. Lactic acid bacteria (LAB) are one of the main microbial
sources for GABA production, mainly due to their food-grade property and high
ability in the direct production of GABA-rich components. The progress toward
culture conditions optimization and genetic engineering to achieve physiology-
oriented engineering approaches and co-culture methods for the GABA-producing
LAB species, the biosynthesis pathway of GABA by LAB, and the glutamate
decarboxylase (GAD), as the key enzyme in GABA biosynthesis, is growing rapidly
(Cui et al., 2020). The biosynthesis of GABA is known to exhibit superior perfor-
mance due to high product efficiency, mild reaction conditions, simple process, and
low environmental burden (Gavrilescu & Chisti, 2005; Alcalde et al., 2006;
Carvalho, 2017). Irreversible α-decarboxylation of L-glutamate is an important
pathway in GABA biosynthesis, which is catalyzed by the pyridoxal 50 -phosphate
(PLP)-dependent glutamate decarboxylase (GAD) (Xu et al., 2017).
It has been indicated that the enzymatic hydrolysis of collagen provides structural
cleavage and better functionality. Enzymatic hydrolysis of bovine collagen and beef
bone extract, which were undertaken by Flavourzyme® and Protamex®, respec-
tively, have shown better functional (antioxidant and antimicrobial activity) and
physicochemical properties of the final hydrolysates (Vidal et al., 2018).
Peptides have been proved to offer health-promoting benefits for hypertension, a
primary risk factor in cardiovascular disease. Short-chain peptides are frequently
favored compared to other protein resources like free amino acids combinations, due
to their enhanced absorption kinetics (Jahan-Mihan et al., 2011). Thus, the proteoly-
sis process to achieve short-chain peptides is of paramount importance. Angiotensin-
converting enzyme inhibitory (ACEI) peptides are among the most recognized
peptides. The proteolysis of ACEI peptides is necessary for their bioactivity and
bioaccessibility, as they are encrypted within the parent proteins (De Leo et al.,
2009).
Protein hydrolysis in peptides occurs both in vitro and in vivo. In vitro degrada-
tion takes place during food preparation processes by the addition of isolated or
microbial enzymes. The in vivo process occurs within the gut, in the presence of
digestive and microbial enzymes of the gut flora. Employing a blend of diverse
proteases and a step-by-step procedure should be carried out for consecutive prote-
olysis (Boutrou et al., 2013).
Recently, dietary supplements based on the protein hydrolysates have already
gained much attention in case of being unable to assimilate native proteins due to
specific illnesses, such as Crohn’s disease or pancreatitis (Posovszky, 2016). The
238 I. Alemzadeh et al.

incorporation of protein hydrolysates in sports nutrition has heightened its signifi-

cance (Holm et al., 2017). Another consequence of protein hydrolysates is muscle
anabolic due to their insulin atrophic effect (Yuan et al., 2017; Rittig et al., 2017).
The combination of several effects would couple their nutritional effects with other
“bioactivities,” an area of study which has received increasing attention nowadays
(Tavano et al., 2018).
The enzymatic hydrolysis of soybean protein products has resulted in reducing
allergenicity and improving functional properties. The allergenicity of hydrolyzed
soybean protein isolates (SPI) by means of alcalase, trypsin, chymotrypsin, brome-
lain, or papain was investigated. Assessment of chymotrypsin hydrolyzed samples
indicated a higher allergenicity potential of digested SPI due to the β-conglycinin
protein fragments (Panda et al., 2015).
Allergenicity of food products is regarded as adverse health effects arising from a
specific immune response when exposed to a given food reproducibly (Sicherer &
Sampson, 2014). Both IgE-mediated and non-IgE-mediated allergenic reactions are
included in adverse immune responses (Berin, 2015). In this regard, the selection of
a fit protease with desirable specificity and proper hydrolysis procedure can signifi-
cantly reduce protein allergenicity (Burton et al., 2002). It has been reported that the
allergenicity of milk proteins and gluten decreased significantly by proteolysis
utilization in food process engineering (Rizzello et al., 2007). Another study con-
firmed the amplification of food allergenicity by IgE immunoblots measurement
when chymotrypsin or bromelain was used for the hydrolysis of soybean protein
isolate (SPI) (Panda et al., 2015). A 65% reduction in IgE reactivity was shown for
roasted peanut protein hydrolysis using Flavourzyme for 300 min (Berin, 2015). SPI
hydrolysis was carried out using pepsin, alcalase, and papain. The results showed the
suitability of these enzymes in the destruction of the main soybean allergens.
However, the application of papain and Flavourzyme were more beneficial in view
of less marked bitter taste production (Meinlschmidt et al., 2016). The effect of
enzymatic hydrolysis of SPI by Corollas PP on antihypertensive and antioxidant
activities was evaluated (Guan et al., 2018).
It has been proved that the gluten uptake, from various sources like Triticum
species, rye and barley proteins, and their crossbred varieties, can notably distress
genetically susceptible individuals to inflammation of the small intestine, namely
celiac and nonceliac diseases (Rizzello et al., 2007).
The effect of proteolytic enzyme modification of egg-yolk protein and white
protein during the preparation processes was evaluated and resulted in the produc-
tion of the bioactive peptide with enhanced properties like antioxidant activity,
ACE-inhibitory activity, ferric reducing power, scavenging capacity, water-holding
capacity, and chelating capacity (Pokora et al., 2013).

9.3.8 Tannase

Polyphenolic compounds are naturally present in a variety of plant sources, namely

tea, nuts, and fruits. These compounds are considered to be health-promoting and
9 Enzymes in Functional Food Development 239

disease-preventing bioactive molecules. They have an important role in decreasing

degenerative diseases, such as diabetes, cardiovascular, and cancers by mutagenesis
and carcinogenesis-preventive activities (Kuddus, 2018). Therefore, it seems these
bioactive compounds are a two-edged sword. However, the bioavailability and
bioefficacy of polyphenolic compounds are affected by their capacity to form
complexes with macromolecules like proteins, cellulose, pectin, starch, and
minerals, generally found in polymeric, glycosidic, and esteric compounds (Arshad
et al., 2019). As a result of the formation of complexes with macromolecules, the
absorption rate in the small intestine will reduce dramatically (Kuddus, 2018). The
higher the molar mass of polyphenolics, the lower the bioavailability activities and
fewer nutritional effects (Aharwar & Parihar, 2019).
Hydrolyzable tannins are commonly known as polyphenolic secondary
metabolites, in the form of galloyl esters or their derivatives. The attachment of
galloyl moieties or their metadepsidic derivatives of tannin to a variety of polyol,
catechin, and triterpenoid units is called gallotannins. Through hydrolysis, glucose
and gallic acid are produced, which are more beneficial for human health (Chung
et al., 1998; Khanbabaee & van Ree, 2001).
Tannin acyl hydrolase, also called tannase (EC 3.1.1.20), is an inducible extra-
cellular enzyme belonging to the esterase superfamily. The molecular weight of
tannase is in the range of 31–310 kDa, depending on their source. Generally, fungal
tannases have a higher molecular weight range (45–310 kDa) compared to bacterial
tannases (31–90 kDa) (Aharwar & Parihar, 2018). The ranges of pH and temperature
optima of tannases are 4–8 and 30–70
C, whereas the stability ones are 3–8 and
20–90
C, respectively (Selvaraj et al., 2019). Tannase catalysis is the hydrolysis of
depside and ester bonds, including gallyol ester of gallic acid and alcohol
components of hydrolyzable tannins, like chlorogenic acid, epigallocatechin gallate,
epicatechin gallate, and tannic acid. Through hydrolysis, other oligomeric tannins
and other useful and absorbable compounds were yielded. A famous example is a
gallic acid which is an essential intermediary constituted in the synthesis of trimeth-
oprim antibacterial drug and is applied in the pharmaceutical industry. Propyl gallate
is another substrate for the synthesis of antioxidants in the food industry, or even
other components applied for clarifying, as well as treating tannery effluents in the
juice industry (Aharwar & Parihar, 2018, 2019).
Generally, tannase has extensive applications in the chemical, brewing, cosmetic,
beverage, pharmaceutical, and food industries. Tannase is able to degrade plant cell
walls, cleaving cell-wall cross-links, and facilitate the absorption of polyphenolic
compounds in the small intestine by enzymatically hydrolyzing them into simpler
and smaller phenolic compounds (García-Conesa et al., 2001; Shoji et al., 2006;
Georgetti et al., 2009). Increasing the antioxidant, anti-inflammatory, immunomod-
ulatory, antiproliferative, and anticarcinogenic activities are the consequences of
these modifications (Kuddus, 2018). It is also a well-known commercial enzyme in
the elaboration of instantaneous, gallic acid production, and acorn liquor (Aharwar
& Parihar, 2018).
Tannase production has been studied extensively. Several microorganisms (fungi,
yeast, and bacteria) are used for microbial tannase production; among them, several
240 I. Alemzadeh et al.

fungal genera, including Aspergilli, Penicillium, Trichoderma, and Rhizopusare are

more typical. Among different microbial sources, fungi, such as Aspergillus ficuum,
Aspergillus oryzae, Arxula adeninivorans, and Aspergillus fumigates, are the most
common sources of the tannase-producer genus in the absence of tannic acid
(Cavalcanti et al., 2020). Based on the strain type and culture conditions, submerged
fermentation (SmF), or solid-state fermentation (SSF), the enzyme can be inducible
or constitutive. Tannase synthesis is induced in the presence of several phenolic
compounds such as gallic acid, pyrogallol, tannic acid, and methyl gallate (Aharwar
& Parihar, 2018). Agricultural residues have also been used as alternative substrates
for economic tannase production (Selvaraj et al., 2019).
However, the synthesis mechanism involved is a controversial issue that should
be addressed. SmF has several advantages in sterilization, incubation time, process
control, and ease of extracellular recovery (Kumar et al., 2016). In contrast, the
advantages of SSF are related to its cost-effective downstream and upstream pro-
cesses, water-dependent conditions, ease of agro-waste substrates usage, the mini-
mum amount of effluents, and high efficiency, so in most cases, SSF is preferable
(Selvaraj et al., 2019). The potential of Lasiodiplodia plurivora CAN-10 for tannase
production was compared under these two fermentation methods by utilizing
Terminalia catappa (almond leaves) and Magnifera indica (mango leaves) as
agro-waste substrates to provide carbon and nitrogen sources (Ire & Nwanguma,
2020). The results of this study revealed that SSF yielded higher tannase production
compared to SmF. SmF was used for the production of tannase in the presence of
tannic acid (2% w/v) from Aspergillus sp. and culture conditions like temperature,
inoculums size, moisture content, aeration rate, and substrate consecutively affect
the process and the fermentation products (Pandey et al., 1999). More examples of
tannase producing sources and the corresponding mechanism that results in the
functional enhancements are shown in Table 9.5. More studies are required for the
identification of high-yield strains as well as low-cost and efficient processing
development (Aharwar & Parihar, 2018). Recombinant DNA technology and
genetic engineering for tannase production have been investigated in several new
researches. Tannase-encoding genes of Aspergillus oryzae were used for producing a
wide variety of hydrolytic enzymes, namely proteolytic and amylolytic enzymes. In
a study, AotanB (AO09002300047), one of the encoding genes of A. oryzae, was
overexpressed using a glucoamylase gene promoter, PglaA142, and a recombinant
enzyme was characterized. Based on the results, the recombinant enzyme produced
by A. oryzae showed better thermal stability and enzyme activity as compared to
Pichia pastoris-tannase producer strain (Koseki et al., 2018). In another study,
transferring tannase gene into Aspergillus niger resulted in better yield of enzyme
production and improved characteristics (Liu et al., 2018).
Enzyme stability maintenance during shelf life, handling, transport, and storage at
room temperature is an essential property for enzyme commercialization in the food
industry. According to the current literature, several techniques such as hydration by
spray drying and freeze-drying, immobilization through nanoparticles, with the aid
of various additives (adjuvants/carriers), such as carbohydrates, proteins, lipids,
gums, and surfactants, as protectors against oxidative denaturation, have been
 9

Table 9.5 Some examples of tannase production from various microorganisms and their related properties (tannase activity, pH, and temperature)
pH
optimal
Source Activity stability Substrate A half-life (t1/2) temperature Application Reference
Aspergillus 390.4 U/mL 6.0 Catechingallate 5.4 h at 60
C Green tea Shao et al.
niger rAntan1 3.0–8.0 0.5 h at 70
C extraction (2020)
Bacillus 2868.75 U/mg 5.0 Tamarind seed 4.5 h at 60
C Bacterial tannase Jana et al.
subtilis PAB2 3.0–8.0 production (2013)
Penicillium 34.7 U/mL 7.5 Keekar leaves (3% w/v) – Selwal and
atramentosum 32.8 U/mL 5.5 Amla leaves (2% w/v) Selwal (2012)
Pestalotiopsis 98.6 U/mL 6.9 Pomegranate seeds – Reges de
guepinii – Sena et al.
(2014)
Aspergillus 111.5 U/mL 7.0 Propyl gallate – – Liu et al.
niger Bde14 – (2018)
Enzymes in Functional Food Development

Aspergillus 28.5 U/mL 5–6 2% tannic acid 40–60
C (in the presence Sorghum and Cavalcanti
fumigates (B-cyclodextrin) – of B-cyclodextrin as an leather effluent et al. (2020)
CAS21 adjuvant) treatment
Propyl gallate
production
Talaromyces 32.18 U/gds 8.0 Acacia nilotica bark 60
C Fruit juice Aharwar and
verruculosus 4–8 detannification Parihar
(2019)
Bacillus 0.265 U/gds – Triphala (11.532 g) – – Selvaraj et al.
gottheilii – (2019)
M2S2
Aspergillus 21.94 4.0 1% tannic acid 30
C Industrial Cavalcanti
niger 31.89 4.0 30
C applications et al. (2017)
Aspergillus
fumigatus
241

(continued)
Table 9.5 (continued)
242

pH
optimal
Source Activity stability Substrate A half-life (t1/2) temperature Application Reference
Klebsiella 39.72 5.52 Indian gooseberry leaves 39.72
C – Kumar et al.
pneumoniae – (2016)
Serratia 42 U/mL – 2% tannic acid – Antibacterial Nsayef
marcescens – activity Muslim et al.
(2017)
Bacillus 49.32 U/L 4.74 4% tannic acid – Clarification of
gottheilii – acidic beverages
M2S2
Aspergillus 148.7 U/g – Rice bran and spent coffee ground – – Mansor et al.
niger PN1 116.52 – Coconut residue (2019)
88.64
Enterobacter 4 U/mL 6.0 Glucose 50
C – Govindarajan
cloacae – et al. (2019)
Aspergillus 12.26 U/g 5.5 Cashew bagasse 30
C Tannase Tatiana et al.
UCP1284 – production (2016)
Aspergillus 139.22 IU/mL 4.89 Pomegranate rind, Cassia 34.91
C – Varadharajan
oryzae – auriculata flower, black gram et al. (2017)
husk, and tea dust
I. Alemzadeh et al.
9 Enzymes in Functional Food Development 243

employed to enhance tannase activity and stability. The properties of the additive
compounds have a great influence on the properties of the final enzyme powder, like
glass transition temperature, density, particle size, and solubility. Therefore, the
adjuvants should be chosen considerably (Cavalcanti et al., 2020). The immobiliza-
tion method in multi-walled carbon nanotubes for tannase has increased tannase
stability (Ong & Annuar, 2018). Apart from the processing method, statistical tools,
mainly response surface methodology, have been considered for the optimization of
tannase production to reduce the cost and the time (Bhoite & Murthy, 2015).
Five main tannase applications are tea processing, fruit juices clarification and
de-bittering, animal feed treatment for nutritional improvement, gallic acid, and
propyl gallate production used in food, leather, dye, and chemical industries, and
wastewater treatment in agro-industries for decolorization and polyphenolic com-
pound reduction (Aharwar & Parihar, 2018; Cao et al., 2019).

9.4 Conclusion

In this chapter, we have reviewed some of the main enzymes incorporated in food
development, influencing the bioavailability and bioactivity of nutraceuticals.
Worldwide companies have been established to do high-tech research and develop-
ment of functional foods that have potentially positive effects on human health
beyond basic nutrition. However, scientists should bear in mind that in vitro and
in vivo experiments and clinical trials are essential steps that should be considered to
guarantee any health claims based on the major factors limiting their
bioaccessibility. According to traditional knowledge as well as scientific evidence,
enzymes have a dominant role in food biotechnology for the development and
commercialization of functional foods. Enzymes utilization in the production of
bioactive metabolites and development of bioprocesses has shown an increasing
trend. Several bioprocesses which involve enzymes have been established for the
development of commercially important nutraceuticals. Yet, many traditional
fermented foods have remained unexploited from technological aspects of functional
foods and nutraceuticals development. Enzymes, such as L-asparaginase,
chlorophyllase, beta-glucanase, L-glutaminae, lipase, and proteases are key enzymes
in nutraceutical and functional food production.
Exploring for high-efficient and cost-effective technologies should be concerned
in parallel with optimizing the experimental conditions to enhance the yield and
physiological stability of these bioactive compounds. Recently, food and agricultural
wastes have intensely attended as potential substrates to produce high-value
products, namely free and immobilized enzymes. They represent ideal sources of
proteins, lipids, and carbohydrates, and therefore, offer remarkable advantages from
economic and eco-sustainability aspects. Nevertheless, the large-scale applicability,
potential repeatability, and operation costs in real-industrial implementation are
some of the challenges that need to be considered more systematically. Another
emerging aspect of food enzyme application is the alteration of DNA encoding
native enzymes for the development of genetically modified enzymes. Although the
244 I. Alemzadeh et al.

safety of genetic engineering approaches for food processing is a severe concern,

there have been promising results for the beneficial employment of genetically
modified enzymes in the modern food industry.
More investigations should be done on the development of efficient and sustain-
able protocols, as well as enhanced properties such as pH and temperature stability,
multifunctionality, surface property, catalytic efficiency, eco-friendly and economi-
cally efficient to achieve precise and sophisticated food matrixes. In addition, their
impact on the resultant food characteristics such as sensory, rheological, physico-
chemical, and functional properties. All in all, there is a far distance to thoroughly
comprehend the relationships between functional chemical compounds, and their
dosage, stability, safety, cost, and delivery vehicles. In fact, further considerations
should be taken into account for investigating the marketing standpoints of the
functional food market based on the current scientific advancements in food science.

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Enzymes as Active Packaging System
10
Amir Heydari and Negin Mahmoodi-Babolan

Abstract

Standard food packaging is the first step that has a vital role in protecting the
product from moisture, water vapor, odors, gases, dust, microorganisms,
vibrations, shocks, and pressure force, which preserved the freshness, quantity,
and quality of the packaging to reach the customer. Active and smart packaging
approaches enhance the communication and protective functions of food packag-
ing to fulfill the demands of consumers for high-quality, safe, and convenient
food products. Recently with the technology expansion, novel enzymes with
extended applications and specificity have been advanced, and new solicitation
areas are still being investigated. This chapter has focused on oxygen-reducing
enzymes in food-packaging material, and intelligent packaging technologies are
reviewed. The main lines are active packaging materials, enzymes as catalysts in
active scavenging systems, active releasing systems, antimicrobial packaging,
time–temperature indicators, removal of undesirable food components, oxygen-
sensing applications, and the effect of the coating process and composition on the
enzyme activity. Also, enzymatic reactions with a brief discussion investigated
and explained how enzymes are used in advanced smart and food-packaging
systems. Eventually, a comprehensive list of enzymes used in food smart pack-
aging and their application are presented.

Keywords
Enzyme · Intelligent and active packaging · Antimicrobial packaging · Shelf life

A. Heydari (*) · N. Mahmoodi-Babolan

Chemical Engineering Department, University of Mohaghegh Ardabili, Ardabil, Iran
e-mail: Heydari@uma.ac.ir

# The Author(s), under exclusive license to Springer Nature Singapore Pte 253
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_10
254 A. Heydari and N. Mahmoodi-Babolan

10.1 Food Packaging

Food packaging is necessary to keep the safety and quality of the foods. Excellent
and well-designed packaging protects the product from external and natural agencies
that may cause damage to the quantity and quality of the product, tampering
resistance, contamination and spoilage, physical and chemical damage. It can main-
tain the freshness of the food. For making products more applicable and favorable
used to reclosable openings, dispensing caps, sprays, and other features. Packaging
in a suitable method assists transportation of the packaged goods with easiness and
reduces marketing costs (Robertson, 2016). Good packaging has less handling losses
and is easy to be handled by one person during loading; therefore, characteristics of
packaging cusses that marketing costs to be less (Han, 2003).

10.2 Food Package Properties

According to the degree of protection, food can influence maximum moisture gain or
oxygen uptake. Different packaging materials such as metal cans and glass
containers can be regarded as nearly impermeable to the passage of odors, gases,
and water vapor, although in comparison packaging materials based on the paper can
be considered penetrable. Also, packaging materials based on the plastics supply
varying degrees of protection, and be contingent mainly on the nature of the
polymers utilized in their manufacture.

10.3 Shelf Life

For many foods and drinks in which quality declines with time, it pursues that there
will be a limited length of time. Shelf life is when the product becomes undesirable,
suitable, and safe to use in specified storage conditions (Labuza, 1982). Shelf life is a
usual scuffle for food companies. Manufacturers and producers want foodstuffs to
stay fresher to lengthen the time to sell or custom. Therefore, researchers are
believed to improve shelf life to give foods and drinks a better fortuity at high
quality when consumers are prepared to eat them (Labuza & Schmidl, 1985).
Provisions to enhance food product safety and quality, increase, or stabilize food
quickly become market demands (Labuza, 1982).

10.4 Active Packaging Systems

Active packaging, smart packaging, and intelligent packaging denote packaging

systems utilized with products, such as foods (fruit and vegetables, fish, and seafood
products), pharmaceuticals, and several other kinds of products (Ozdemir & Floros,
2004). Active packaging refers to the embedding of special additives into the
package to expand or maintain the quality. Active packaging effectively increases
10 Enzymes as Active Packaging System 255

the shelf life of foods by Realini and Marcos (2014). In active packaging, different
additives may be used based on the packaging film and packaged food (Lorenzo
et al., 2014). Active packaging contains ingredients that can absorb carbon dioxide,
releasing oxygen, ethanol emitters, ethylene scavengers antioxidants, and other
preservatives (Conte et al., 2007).
Active packaging systems supply various solutions to be controlled by quality
attributes that are to be protected. This theme came up, for example, in discussions of
decelerating oxidation food products be considered, so the packaging must utilize an
active system that includes an oxygen scavenger or antioxidants. Also, if the decay
of the product is created by condensation or moisture, the packaging can include a
moisture absorber. Eventually, the motivation for the organization of any novel
technology is cost.
It should point out that the unique decay mechanism of different foods should be
considered previously designing the packaging. The reality, when we have active
packaging that it carries out some role other than supplying an inert obstacle to the
exterior environment. On the other hand, active packaging is identified as a system in
which the product, package, and environment interact in a positive method to expand
the shelf life or to obtain some properties (Conte et al., 2007).
The shelf life of packaged food is reliant on abundant features such as the food
properties or product-intrinsic (e.g., pH, product color, nutrient content, aroma,
texture, water activity, viscosity, biological structure and breathing rate, the exis-
tence of antimicrobial compounds) and product-extrinsic or related to the packaging,
receptacle, or external environment factors (e.g., visual, and tactile properties of food
packaging or dishware, smell, surroundings lighting, temperature and aroma, storage
temperature, relative humidity, and the ambient gaseous composition) (Lorenzo
et al., 2014).
These features will straight affect the physical, chemical, biochemical, and
microbiological deterioration of products and their shelf lives.
Active packaging aims to improve the maintenance of food in the package, and
delaying shelf life involves the application of various plans like oxygen removal,
temperature control, moisture control, besides chemicals, for example, sugar, salt,
carbon dioxide, or natural acids or a blend of these with active packaging.
These developments in active packaging have caused developments in numerous
fields, including controlled respiration rate in agricultural products, postponed
oxidation in muscle foods, microbial growth, and moisture movement to dried
products. Furthermore, active packaging similarly employs choosiness to modify
the atmospheric concentration of gaseous combinations interior of the package by
coating, lamination, micro-penetration, or polymer blending (Realini & Marcos,
2014).

10.4.1 Types of Food Active Packaging Systems

The active packaging can be utilized in various forms involved as shown in

Fig. 10.1.
256 A. Heydari and N. Mahmoodi-Babolan

Fig. 10.1 Different types of

food active packaging systems
(a) using the active pouches
and pads placed inside the
packaging, (b) active
compound coated onto the
polymer, (c) blending of the
active compound on the
packaging surface, and (d)
straight incorporated into the
package structure (Almasi
et al., 2020)

10.4.2 Active Compound Release Mechanisms

The direct incorporation of active combinations into the polymer matrix of the
packaging system is the most regular kind of active packaging system. In this
form of the active packaging system, polymer and the active agent form a composite
matrix. In recent years, this type of active packaging system is the most frequently
considered for food active packaging systems. There are three types of release
mechanisms suggested for directly incorporated food active packaging systems.

1. Diffusion due to release: In this type of release, the active agent diffuses through
the macro-porous or micro-porous polymer matrix structure and is moved from
the surface of the film into the food. This mechanism is usual for releasing active
composites for water-resistant and synthetic polymers. The rate of this kind of
release is depended on polymer chemistry, permeability, and porosity.
2. Swelling due to release: low diffusion coefficient causes the incorporated active
agent unable to penetrate the polymer matrix. When polymer swell, the diffusion
coefficient of the active agent increases; therefore, it penetrates out. Since most
foods have moisture and the water is the most popular penetrant, this type of
release commonly happens in packing materials moisture-sensitive such as
polysaccharides or protein-based films.
3. Disintegration due to release: Deformation of polymer, cleavage, and degradation
are the main reasons for this kind of release. Absorption of liquids commonly
from aqueous fluids through the polymer matrix influences degradation and
deformation rate.
10 Enzymes as Active Packaging System 257

Fig. 10.2 Schematic illustration of different approaches used for release controlling from food
active packaging (Almasi et al., 2020)

This kind of release happens in both nonbiodegradable and biodegradable

polymers. Several approaches have been suggested to obtain a more controlled
diffusion of the active agent from the packaging material. Figure 10.2 summarizes
these approaches by a schematic representation.

10.5 Antimicrobial Packing

Antimicrobial packing is a type of active packaging that use some agents which kill
or inhibit the growth of microorganisms that may be existing in the packaged
product or packaging material itself. For control of unfavorable microorganisms
on foodstuffs, antimicrobial materials can be combined and coated inside food-
packaging materials (Motta et al., 2004).
For example, antimicrobial polymers provide a good antimicrobial plan for
combating pathogens and have already drawn the attention of industrial and aca-
demic research (Prazak et al., 2002). Antimicrobial mechanisms demonstrate either
active or passive action and polymer material categories, including bound or
leaching antimicrobials used. Antimicrobial polymers are used in some industries,
such as medical, food, and textile (Motta et al., 2004). Compounding polymers and
258 A. Heydari and N. Mahmoodi-Babolan

their blends with antimicrobial compounds or preservative agents reveal a new

approach for designing antimicrobial packing films.

10.6 Enzyme

Enzymes are macromolecular biological catalysts (typically proteins), which signif-

icantly are available in all living organisms, which cause reactions to happen
naturally, and can be seen in open self-organizing life (Liu et al., 2014). The first
feature is its catalytic factor. Enzymes permit living systems to perform reactions
that would happen so slowly. Enzymes accelerate (catalyze) chemical reactions; in
several cases, enzymes can make a chemical reaction so much faster than it would
have been without it (Wierschem et al., 2017).
Enzymes catalyze biochemical reactions with different pathways (Narayanan
et al., 2009). A substrate is named chemical species observed in a chemical reaction
and reacts with a reagent to create a product. It can also be called for the surface that
other chemical reactions are played, or achieved a supporting role in microscopic
and spectroscopic techniques. The substrate and enzyme schematic are shown in
Fig. 10.3.
Nonenzymatic or chemical reactions are more slower than enzymatic reactions
(Brody & Budny, 1995).
The second characteristic of enzymes that makes them significant as diagnostic
and tools for research is the specificity they display regarding the reactions they
catalyze (Brody & Budny, 1995). Enzymes exhibit absolute specificity, which is,
they will catalyze only one particular reaction. Other enzymes will be specific for a
specific type of functional group or chemical bond. Overall, there are four different
kinds of specificity (Blixt & Tiru, 1976):

Absolute specificity that the enzyme will catalyze only one reaction.
Group specificity in which the enzyme will affect performance only on molecules
with particular functional groups, such as phosphate, amino, and methyl groups
(Budny, 1989).

Fig. 10.3 Schematic of enzyme and substrate

10 Enzymes as Active Packaging System 259

Linkage specificity that the enzyme, irrespective of the rest of the molecular struc-
ture, will act on a specific type of chemical bond and the best technique to
immobilize enzymes or other bioactive composites on polymers. The chemical
steps included in covalent bonding are complex and timewasting. Therefore, are
not presently practical for joint commercialization for food contact packaging.
Enzymes are effective biological catalysts frequently utilized in the food industry
to get better performance, quality, and shape of food. Using enzymes as active
agents in the food packaging system is not novel technology (Adrian, 1959).
Stereochemical specificity is the enzyme performance with an optical isomer or
specific esters. Enzymatic specificity requires two different forms: the first one is
the type of chemical reaction and the second one is no chemical reaction,
specificity for the reactant, or substrate. Therefore, for each chemical reaction
in a biological system, there is a particular enzyme demand for the optimal
production of reaction products. It is essential to consider many different
biological reactions to pursue numerous different enzymes (Brody & Budny,
1995).

Enzymes, similar to other chemical catalysts, do not change equilibrium

conditions. An enzyme enhances a forward reaction similarly, and the reverse
reaction means that enzymes speed up the rate a chemical equilibrium is obtained.
It considers that the enzyme does not interfere with the equilibrium concentrations
ratio for the products to reactants (Nestorson et al., 2008).
In an active package system, the enzyme may be incorporated into the package
structure or added straight to the packing to affect a reaction. The enzyme in
packaging material should be motionless, and the substrate with a reactant role or
a constituent circulated in the site to initiate a reaction. If the enzyme is placed in a
static position or immobilized, it may be accomplished by making the enzyme the
main part of the packaging system (Klibanov & Dordick, 1989).
Generally, active packaging commonly includes the combination of a chemical
into the packaging material. Active packaging providing enzymes contains the
incorporation into the packaging material of the specific enzymes in much the
same way as the incorporation of a more formal chemical to make the active
package. The significant differences are not changing enzyme with reaction and
can continue indefinitely; the enzyme is sensitive to changes in some parameters,
such as pH, temperature, etc. The range of environmental properties for the perfor-
mance of the enzyme is a relatively narrow band (Kothapalli et al., 2008). Also,
enzymes may be categorized according to the substrates. For example, lipases are
used for lipids and proteases for proteins, etc. These are determination for the vast
group to break proteins of entities that are lipid under a specific set of conditions or
specific to a single protein.
Based on the several researches, the utilized enzymes incorporated in the
polymers can act as oxygen scavengers (Labuza & Breene, 1989). One of the most
widely investigated enzymes in active packaging is Glucose oxidase plus catalase.
When the enzyme is immobilized, the surface carrying the active principle should be
directly contacted with the food for activating the redox reaction, and it caused the
260 A. Heydari and N. Mahmoodi-Babolan

application to be limited (Brody et al., 2001). Scavenging reaction happens in the

interface of product–packaging.
Glucose oxidase/catalase systems convert glucose into oxygen peroxide and
gluconic acid, which is quickly decreased by the catalase enzyme, even though the
food may become oxidized. Consequently, the scavenging reaction is suggested to
be produced distinctly from the product. The liquid must also contain the reactant
(ethanol, glucose). These systems are still being expanded and implemented, but, at
present, there is no commercial application (Effront, 1917).
The immobilization can switch the activity of the adopted enzyme. Realty,
immobilize the enzyme in the package while maintaining enzyme activities, the
chemically modified polymers simplify the covalent bonding with the enzyme. To
insert the enzyme into the matrix of polymer, prior chemical purification of the
polymer is frequently required. The majority of enzymes are used directly in food
processing, and some of them have already been utilized in active packages. For
example, Soares and Hotchkiss (1998) presented naringinase in a plastic package.
Hydrolysis of naringin reduced the bitterness of contained grapefruit juice (Wu et al.,
2019), in Pharmacal biotechnologies industry used from immobilized lactase and
cholesterol reductase in a package for liquids. Also, the reduction of lactose in milk
has been used lactose content of milk or yogurt during storage. Another example is
converting cholesterol reductase into coprostanol (Brody & Budny, 1995). A sche-
matic drawing of the enzyme immobilization process in both immobilization in the
dispersion phase and immobilization on the surface of the polymer film is shown in
Fig.10.4.

Fig. 10.4 Schematic drawing of the enzyme immobilization process. (a) Immobilization in the
dispersion phase and (b) immobilization on the surface of polymer film formed from a latex
dispersion (Nestorson et al., 2008)
10 Enzymes as Active Packaging System 261

10.6.1 Oxygen Scavenger in the Enzyme Packaging System

An oxygen scavenger is a substance that reacts enzymatically or chemically with

oxygen, thus safeguarding the packaged food versus oxidative decay and quality
changes owing to oxygen (Ahvenainen, 2003). Generally, oxygen scavenging
systems follow the oxidation of iron powder or enzymes like glucose oxidase,
with oxygen existing in the headspace of the packaging system. These reactions
do some activities such as absorbing the great majority of free oxygen in the
package, reducing the interaction of oxygen with food, and avoiding deterioration.
Usually, oxygen scavengers are placed inside the package with a sachet too perme-
able to oxygen.
It should be noted that these types of sachets can be utilized for meat products,
fresh pasta, and baked kinds of stuff, such as pizza dough, bread, sponge cake and
cookies, nuts, coffee, and snack foods like chips. Some security issues are related to
utilizing sachet types of oxygen scavenging systems in packaged foods that may
ingest sachets by consumers, and the probability of sachet contents leaking into food
packaging (Lopez-Rubio et al., 2004). Careful investigation and improvement
should be carried out in choosing the suitable sachet material and its seal correctly
before commercial application, and suitable alerting must be proclaimed on the
package. The combination of catalase and glucose oxidase is a general enzymatic
oxygen scavenging system that has been shown in the Fig. 10.5 (Shi et al., 2010).
Glucose oxidase in the presence of oxygen reacts with glucose. This enzyme system
has been embedded into acrylate matrixes of polymer for the deoxygenation of apple
juice (Kothapalli et al., 2008).

10.6.2 Time–Temperature Integrator-Indicators

Time–temperature integrators (TTIs) are indicators that can display food safety and
quality changes during distribution, handling, and storage (Taoukis & Labuza,

Fig. 10.5 The enzymatic reactions in the oxygen scavenging system using the combination of
glucose oxidase and catalase. The mechanism complicated here is adapted from Shi et al. (2010)
and Tian et al. (2013)
262 A. Heydari and N. Mahmoodi-Babolan

1989). TTIs are necessary for temperature-sensitive foods to evaluate and control the
distribution chain (Giannakourou et al., 2005). TTIs must be low-priced, readable,
reliable, and without any poisonous materials. Most of the TTIs are very similar to
labels and can be used for food products. Several TTIs have been expanded based on
different operating principles. Enzymatic TTIs utilize α-amylase (Guiavarc’h et al.,
2004), lipase (Agerhem & Nilsson, 1981), or b-glucosidase (Adams & Langley,
1998). Presently, lipase is an available enzymatic TTI that can be utilized to control
quality assurance. Enzymatic TTIs that have been investigated to date have utilized
synthetic and then rather expensive chromogenic substrates hydrolyzed to generate a
color. Consequently, if an enzyme that uses natural dyes as its substrate were
available, a TTI produced using that enzyme would supply various benefits,
counting developed visibility, less expensive, and extensive use of dye coding.
Laccases are conventionally used for numerous industrial processes counting
paper processing, detoxification of environmental pollutants, oxidation of pigments,
prevention of wine discoloration, and production of chemicals from lignin (Kim
et al., 2012).

10.7 Conclusion

Active packaging technologies suggest novel chances for the industries for
protecting their products. Different active packaging systems are currently available
based on the chemical and physical structure of the foods. Oxygen, moisture, carbon
dioxide, ethylene, and ethanol are considered for the active packaging. The structure
of the active packaging and the potentials of the enzymes are mentioned. The
importance of this subject has produced many investigations to make more useful
systems. In the following, oxygen scavenger in enzyme packaging and time–tem-
perature integrator-indicators were investigated. Finally, investigation shows the
improvement of novel indicators and enzymes that are much more precisely
designed for the active packaging system.

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Enzymes as a Tool in Food Analysis
and Foodborne Pathogen Detection 11
Preethi Sudhakara, Jerrine Joseph, S Priyadharshini, Jemmy Chirsty,
Alex Anand, Davamani Christober,
and Aruni Wilson Santhosh Kumar

Abstract

Enzymes are widely and progressively utilized in food processing industries.

Enzymes find tremendous application in fruits and vegetable processing, dairy
processing, and cereals processing. Nowadays, there is a wide application of
enzymes in food analysis. Enzymes serve as an analytical tool for the detection of
food-borne pathogen and allergens. These may be utilized as biosensors and are
applicable in food industries. This chapter gives detailed information related to
application of enzymes in food analysis and detection of food-borne pathogens.

P. Sudhakara
School of Medicine, University of Florida, Gainesville, FL, USA
J. Joseph
Center for Drug Discovery and Development, Sathyabama Institute of Science and Technology,
Chennai, India
S. Priyadharshini
Department of Food Science and Nutrition, The American College, Madurai, India
J. Chirsty · A. Anand
Center for Molecular Data Sciences and Systems Biology, Sathyabama Institute of Science and
Technology, Chennai, India
D. Christober
Department of Food Science and Nutrition, The American College, Madurai, India
The American College, Madurai, India
A. Wilson Santhosh Kumar (*)
Sathyabama Institute of Science and Technology, Chennai, India
Musculoskeletal Research Center, US Department of Veteran Affairs, Loma Linda VA, Loma
Linda, CA, USA
School of Medicine, Loma Linda University, Loma Linda, CA, USA
e-mail: vcaum@mum.amity.edu

# The Author(s), under exclusive license to Springer Nature Singapore Pte 265
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_11
266 P. Sudhakara et al.

Keywords

Enzymes · Food processing · Food analysis · Food-borne pathogen

11.1 Introduction

Enzymes are defined as the biological materials responsible for fastening the reaction
rate. Enzymes are the so-called multifaceted biomolecules that are indispensable for
biochemical interconversions. They act as a catalyst for all the metabolic processes
in the body. The advancement in rDNA technology and protein engineering has
enabled us to capitalize on the various functions of enzymes from industrial to
biomedical applications. Enzymes are exceptionally effective biocatalysts
investigated for wide-reaching catalysis due to a few particular preferences that
run from their activity in milder response conditions to their excellent component
selectivity and lower ecological and physiological harmfulness (Chapman et al.,
2018). Enzymes are initially involved in the dissolvable or immobilized structure for
isolation, purification, and production to benefit humanity (Chaplin & Bucke, 1990).
The demand for the enzyme increases by 6% yearly, especially in food and feed
production (Nampoothiri et al., 2002). Enzymes are widely and progressively
utilized in exploration and food processing as significant handling biocatalysts
(Ermis, 2017). Enzymes are intricated in a living organism. Enzymes formed in
live cells can enhance reaction rates in vitro. The acclimatization of enzymes in the
food sector is known widely, and intense investigations consistently unravel the
global food crisis. This chapter highlights the fundamentals of enzymes, sources of
diverse enzymes, and their applications in the food sector.
Microbial enzymes have been catapulted to fame due to their inherent properties
of the ease of their production and yield manipulation in laboratory settings. This
highlights their significance and the opportunities that pave the way for
microbiologists by harnessing the biomolecules’ potential. The thoughtful consider-
ation of the enzymes in the food sector has amended the primary methods to afford
improved marketplaces with high quality and safety. Previously, this information
about enzymes has been used in meat tenderization, baking, brewing, etc. Mainly the
enzymes are produced by microbial synthesis. The microbial method for enzyme
production has been favored over other methods for quick and inexpensive
production.
Furthermore, microbially produced enzymes’ harmful content is lesser than other
sources. The first commercial application of enzymes was in the production of
cheese. Enzymes in the food sector alter traditional and toxic methods, substitute
them with sustainable methods, and reduce energy consumption levels. It may be
worthwhile to unclose an unexplored trail for enzyme applications, which could be
advantageous in the food industry.
Foodborne pathogens influence human health negatively and are comprehended
to induce financial losses. Therefore, it is essential to rapidly detect foodborne
pathogens and execute steps to assure their inactivation. Immunological, molecular,
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 267

and cultural approaches are often exploited to detect foodborne pathogens. High
expense, lengthy analysis duration, and the need for technical personnel are some of
the drawbacks of these approaches. Biosensors are known as analytical instruments.
Biosensors can be used as novel techniques for detecting toxins and pathogens.
These biosensors restore various signals (like biochemical signals) to electrical
signals that can be measured with the help of a transducer. Various kinds of
biosensors are being utilized for the detection of pathogenic bacteria. Biosensors
are prompt and cost-effective devices and are employed in multiple areas such as
food safety, medicine, pharmacy, measurement of environmental pollution, and
military defense. Electrochemical and optical biosensors and piezoelectric
immune-sensors are among the most repeatedly used biosensors in detecting
foodborne pathogens.
There are many viruses and bacteria, some that cause animal disease, and others
that contribute to foodborne human illness and disease that can be tested for the
occurrence of foodborne pathogens. Some of the significant subjects of testing
include several swine viruses, including swine influenza virus and porcine repro-
ductive and respiratory syndrome virus. Bovine viruses include bovine viral diar-
rhea virus, bovine syncytial virus, bovine rotavirus, coronavirus, and rhinotracheitis
virus. In addition, the Hepatitis A virus, flaviviruses, West Nile, and the Usutu virus
(USUV) that have recently been on the rise in Europe and some parts of South East
Asia are of much importance. The diagnosis and surveillance of bacterial pathogens
encompass the full spectrum of animal and foodborne pathogens. These include
Escherichia coli and bacteria in the Salmonella, Klebsiella, Clostridium, Listeria,
Staphylococcus, Streptococcus, Pseudomonas, Pasteurella, Actinobacillus, and
Mycoplasma genera.
Rapid, efficient pathogen detection and fingerprinting are crucial and usually
lifesaving to control the foodborne ailment. Presently, detecting and subtyping the
pathogens are distinct operations, but merging these two phases via a technique
known as “metagenomics analysis,” the detection is much prompter and more
sensitive as many microorganisms are now employed to produce food enzymes.
Enzyme technologies have been thoroughly revolutionized with the recent novel
trends in biotechnology applications to fulfill the new and changing requirements of
the food industry. There is an extensive tendency toward novelty in enzyme discov-
ery and creating tailored enzymes to make them inclined for industrial food
applications.
In the recent past, developments in enzyme informatics tools can simplify
designing the enzymatic structure. Also, its computational dynamic simulations
can produce the kinetic and energetic course of its mechanism. Enzyme informatics
can also recreate an exceptional part in mutant library design and other screening
platforms, but its execution of molecular approaches is still time consuming. Recent
improvements in the subject will hover around novel frontier in enzyme technologies
that could attribute various functional aspects to these enzyme biocatalysts. This
study will consolidate the extensive characteristics of food enzymes, their diversified
functions in the food industry, the destructive outcomes of foodborne pathogens, and
their current detection techniques.
268 P. Sudhakara et al.

11.2 Enzymes as an Analytical Tool

Human health and healthcare predominately revolve around the quality of food
consumed. Appealing enzymes in the food industry can manage the quality food
supply problem. Hence, evaluating food quality and ensuring food safety standards
are highly important. This crucial factor plays a vital role in evaluating food quality,
safety, and the turnaround time to ascertain this. Enzymes are biomolecules that
inherently possess specificity and sensitivity traits (Fig. 11.1).
Hence enzymes are a better bet than the conventional diagnostic methods that are
dreary and overwhelming and require correlative modes to detect food quality and
safety. Enzyme-based analytical tools to measure biological functionality are more
reliable and consistent. The analytical tools should be high throughput and compe-
tent to authenticate and authorize the study of particular aspects of biology, either
qualitatively or quantitatively, which is possible by enzymes. Also, a crucial part of
developing such tools is to source or amalgamate the desired enzymes with the
required activity. Thus, the utilization of enzymes as analytical informers is an
inexpensive, simpler, and quicker manner of diagnostics. Microbes have been used
from bygone days; yeast is the first reported organism to produce alcoholic
beverages using barley in 6000 BC. The microbial enzymes have earned acknowl-
edgment globally for their extensive uses in several trades. Nevertheless, recombi-
nant DNA technology and protein engineering are assembled to confound the
demand. It improves consumer goods, expense deduction, natural resources short-
age, and environmental safety (Liu et al., 2013; Choi et al., 2015b).
Microbial enzymes play a crucial role (Raveendran et al., 2018). In terms of
analytical procedures in the Food and Pharmaceutical industries, enzymes play a
vital role. As indicated by food and regulatory agencies, food safety and quality are
the censorious criteria recurrent to all food constituents, and these criteria are of
substantial lucrative significance. Food safety depends on toxins, dangerous
microorganisms, toxins divulged by microbes, insecticides, and pesticides (Terry
et al., 2005). Processing of foods with biological enzymes is chronicled inveterate
approach. Earlier in 6000 BC, bread, brewing, wine, and cheese making were done

Fig. 11.1 Enzyme activity

11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 269

with biological agents, but the initial microbial fermentation was from 2000 BC for
vinegar production (Vasic-Racki, 2006). In 1930 for juice clarification, pectinases
were used. Invertase was used during World War II for the invert sugar syrup
production in the sugar industry that developed the use of an immobilized enzyme
(Vasic-Racki, 2006). In 1960, the extensive use of enzymes established during the
traditional acid hydrolysis was restored by slant based on utilizing amylase and
glucoamylases (Panesar et al., 2010).
The pattern for the plan and execution of procedures and creation of products
moored in the utilization of enzymes has consistently expanded (Norus, 2006).
Enzymes from microbes play a significant part in food ventures since microbial
enzymes’ stability is higher than plant and animal enzymes. They can be fabricated
through fermentation strategies in a lucrative way with less reality prerequisite and
high consistency and process adjustment (Gurung et al., 2013). In food analytical
strategies, food safety and quality control are essential, and they can be analyzed by
numerous analytical procedures like HPLC, GC-MS, and LC-MS (Castillo et al.,
2004). In any case, sample preparation strategies are the significant downside of this
technique. Progressing diagnostic strategies in the food industry are tedious and rely
upon skilled laborers, and these procedures depend on extended division techniques,
costly instruments, and exceptionally unadulterated chemicals. In the field of quick
screening, there is a requirement for complementary strategies to recognize food
safety and quality issues. A more specific and sensitive method for screening food
safety and quality issues is enzymatic analysis (Prodromidis & Karayannis, 2002).
Enzymes play a crucial role in the analytical process in the food, pharmaceutical, and
environmental industry. For governing the compound concentration that performs as
an activator, substrate, or inhibition of specific enzymes, enzymes are used as typical
analytical devices. For the analysis of modification induced physically and nutrition-
ally and quality criterion of food, the concentration and activity of specific enzymes
are used. The list of enzymes and their application, along with the microbial
enzymes, are listed in Table 11.1.

11.3 Important Enzymes as Relevant Markers for Food Analysis

Enzymes are extensively used as an important marker, especially in the food and
pharmaceutical industry. The broad scope of enzymes has been boon to the food
analyst who investigates the different constituents of the food. Food, being more
complex with increased macronutrients and very few micronutrients analyzing these
food constituents, requires extremely simple and affordable, less time-consuming
feasible, and specific techniques. Enzymes are used as markers for food analysis and
their lustiness, adaptability, and cost-effective nature (Ashie, 2012).
270 P. Sudhakara et al.

Table 11.1 Microbial enzymes with their source and application

Microbial
enzyme Microbial source Application References
α-Amylase B. licheniformis, Liquefaction of starch, fruit Lépine et al.
B. subtilis, juice clarification, (2019), Luang-
B. stearothermophilus, improvement of bread In et al. (2019)
B. megaterium and quality, brewing, and baking
B. circulans
Glucoamylase Aspergillus oryzae, Glucose fructose syrups, Bilal and Iqbal
Aspergillus niger and production of beer (2020), Karim
Rhizopus oryzae et al. (2017)
β-galactosidase Lactobacillus delbrueckii Reduction of lactose Gibson and
subsp. bulgaricus and intolerance Wang (1994),
Streptococcus Saqib et al.
thermophilus (2017)
Protease Photobacterium, Bacillus, Tenderization of meat, milk Aruna et al.
Vibrio coagulation (2014), Zhang
et al. (2015)
Lipase Bacillus sp. Pseudomonas Cheese production Aravindan et al.
(2007), Feng
et al. (2011)
Esterase Bacillus subtilis Flavor enhancement, dietary Faulds (2010),
fiber de-esterification Sayali and
Satpute (2013)
Pectinase Aspergillus niger, Fruit juice clarification Pasha et al.
A. flavus, and Penicillium (2013),
chrysogenum Abdollahzadeh
et al. (2020)
Phospholipase Listeria monocytogenes Development of cheese flavor Law (2010),
Titball (1993)
Cellulase Pseudomonas fluorescens Feed of animal, fruit juice Sukumaran
Bacillus subtilis, E. coli, clarification et al. (2005),
and Serratia marcescens Islam and Roy
(2018)
Xylanase Bacillus, Cellulomonas, Enhancement of beer quality Camacho et al.
Micrococcus, (2003),
Staphylococcus, Chakdar et al.
Paenibacillus, (2016)
Arthrobacter,
Microbacterium,
Pseudoxanthomonas, and
Rhodothermus
Glucose A. niger and Penicillium Improvement of food flavor Hanft and
oxidase notatum Koehler (2006),
Nakamatsu
et al. (1975)
Catalase Staphylococcus Hydrogen peroxide removal Sîrbu (2011)
for production of cheese
(continued)
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 271

Table 11.1 (continued)

Microbial
enzyme Microbial source Application References
α-Acetolactate Enterococcus faecalis, Precise beer maturation Choi et al.
dehydrogenase Bacillus licheniformis, and (2015a)
Lactococcus lactis
Peroxidase Bacillus sphaericus, Flavor development Regalado et al.
B. subtilis, Pseudomonas (2004)
sp., Citrobacter sp.
Asparaginase Escherichia coli and Shortening of acrylamide Mohan Kumar
Erwinia chrysanthemi formation while baking et al. (2014)

11.4 Enzyme Marker in Pasteurization of Milk

Enzymes play a vital role in detecting milk pasteurization. For increasing the shelf
life of milk and milk products, heat treatment is essential. In many countries, an
alkaline phosphatase test is used as a standard technique for milk ratification.
Non-appearance of alkaline phosphatase in the heat-treated milk shows efficient
pasteurization. It is based on the inactivation of the alkaline phosphatase enzyme
using heat.
On the other hand, it reduces the pathogenic bacterial population in the milk. Not
only for milk, but this rapid detection method can also be used for other dairy
products like cheese production. Other primordial enzyme markers for milk include
lactoperoxidase and γ-glutamyl transpeptidase (Rankin et al., 2010).

11.5 Enzyme Marker in Blanching

The main aim of blanching is to remove the enzyme polyphenol oxidase. Apart from
this, another equitable goal is to decrease microbial growth and increase shelf life.
Time taken for blanching is enough to destroy the indicator enzyme. Blanching of
frozen foods, are done at different time intervals in order to recognize the relation-
ship between enzyme activity and sensory scores (Williams et al., 1986).

11.6 Novel Analytical Tools

In these decades of higher technological development, the improvement in the food

analysis techniques will have greater insight. Analysts and chemists will have greater
value in using novel analytical tools. Traditional high-performance liquid chroma-
tography system analyzes the hydrophobic and hydrophilic constituents in reversed
phase and the normal phase. However, the conventional method is less susceptible
and time consuming. So, to analyze the higher and lower molecular weight analytes,
272 P. Sudhakara et al.

a novel polymer-based stationary phase is introduced. Capillary electrophoresis that

detects the analyte based on the molecular weight and ionic moiety is introduced as
an alternative. There is a higher technique named capillary electrochromatography
which is better than HPLC and capillary electrophoresis (Huck et al., 2000). The
solid-phase extraction can attain a low concentration analyte. Spectroscopic
strategies at this phase of the systematic technique either incorporate mass spectrom-
etry (MS) or vibrational spectroscopy individually. Electrospray ionization is used
chiefly with mass detectors like time of flight (TOF), ion trap, ion cyclotron, and
quadrupoles. Laser desorption ionization methods determine higher molecular
weight samples (Feuerstein et al., 2006).

11.7 Enzymes in the Food Industry

Enzymes are used in a variety of ways in the food industry. Enzyme activity aids in
the safe digesting of food in various situations. The thermal stability of enzymes has
been used extensively to quantify heat treatment; peroxidase activity, for example, is
used to determine the sufficiency of blanched vegetables. Lactoperoxidase has also
been used to test the effectiveness of the pasteurization process in milk. In some
cases, residual enzyme activity affects the flavors or color of products while they are
being stored. Lipoxygenase, for example, is responsible for off-flavors in frozen
veggies that have not been adequately blanched. The staining of wheat flour and
noodles and fruit liquids and pastes is catalyzed by polyphenol oxidase (Nielsen,
2017).
The enzyme is used to test the components of enzyme-substrate foods that are
commercially accessible. The glucose content of a complex dietary matrix
containing additional monosaccharides can be determined using commonly avail-
able enzymes. Enzyme activity measurement concerning food quality is also note-
worthy. Milk from mastitic udders, for example, has considerably increased catalase
activity. The quantity of bacteria in milk is also connected to catalase activity.
Another technique to use enzyme testing to determine food quality is to estimate
protein nutritional value by measuring the activity of added proteases on dietary
protein samples. Enzymes can track the appearance of breakdown products such as
trimethylamine in fish during storage. Enzymes are also used as preparatory tools in
food analysis. In fiber analysis, amylases and proteases are used, while enzymatic
hydrolysis of thiamine phosphate esters is used in vitamin analysis (Lee et al., 1991).
Table 11.2 lists some of the most often used enzymes in the food business.
Food scientists must understand that the environment significantly impacts
enzyme activity. At high temperatures or under other situations that are not too far
off from ideal, that enzyme can deteriorate and lose its activity. As a result,
therapeutic enzymes must be stored and handled with care, usually in the refrigerator
or freezer. To effectively carry out enzyme analyses in foods, certain basic enzymol-
ogy principles must be understood. Following a brief overview of these concepts,
instances of enzymatic analysis in food systems are discussed. Under physiological
conditions, enzymes are protein facilitators with high specificity and reactivity.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 273

Table 11.2 Application of microbially produced enzyme in industries

Industry Enzymes Uses Microorganisms
Baking Maltogenic Enhance shelf life of bread Bacillus stearothermophilus,
Amylase Flour adjustment, bread Aspergillus sp., Bacillus sp.
softness
Transglutaminase Strengthening of dough Streptomyces sp.,
Streptoverticillium sp.
Xylanase Conditioning of dough A. niger
Glucose oxidase Strengthening of dough A. niger, Penicillium
chrysogenum
Lipase Conditioning of dough and A. niger
its stability
Dairy Acid proteinase Coagulation of milk Aspergillus sp.
Neutral proteinase Ripening of cheese, B. subtilis, A. oryzae
debittering
Lactase (beta- Whey E. coli, Kluyveromyces sp.
galactosidase)
Aminopeptidase Ripening of cheese Lactobacillus sp.
Catalase Cheese making A. niger
Transglutaminase Cross-linking of protein Streptomyces sp.
Beverage Pectinase Depectinization A. oryzae, P. funiculosum
Glucose oxidase For the removal of oxygen A. niger
Pullulanase Saccharification of starch Bacillus sp., Klebsiella sp.
Naringinase Debittering A. niger
Alpha-Amylase Hydrolysis of starch Bacillus, Aspergillus
Beta-Amylase Hydrolysis of starch Bacillus, Streptomyces,
Rhizopus
Beta-Glucanase To inhibit haze formation B. subtilis, Aspergillus spp.
Cellulase fruit Liquefaction A. niger, Trichoderma
atroviride

Enzymatic analysis determines the condition of a biological system, including meals,

by measuring substances with the help of added enzymes or by measuring endoge-
nous enzyme activity. Because enzyme catalysis occurs under relatively favorable
circumstances, it is possible to measure relatively unstable molecules that would be
difficult to measure using other methods. Furthermore, due to the specificity of
enzyme reactions, it is possible to assess the components of complex mixtures
without the time and expense of using expensive chromatographic separation
procedures.

11.8 Beverage and Dairy Industry

Enzymes regulate the brewing process, ensure consistent, high-quality beer; enhance
animal and vegetable proteins’ functional and nutritive properties through enzymatic
hydrolysis; and increase juice yield while improving color and aroma. Enzyme use
274 P. Sudhakara et al.

in the food business is divided into several categories, including baking, dairy, juice
production, and brewing (Mojsov, 2011). Microbial enzymes are widely used in the
baking business, the most prominent application market in the food industry, to
increase dough stability, crumb softness and structure, and product shelf life. The
usage of enzymes in the dairy industry (Qureshi et al., 2015), which is the next
largest application area, is primarily due to the increased use of microbial enzymes in
cheese manufacturing (Qureshi et al., 2015). The laboratory must identify appropri-
ate enzymes with the required activity and understand the target analyte and the
complexity of the matrix in which the analyte must be measured (Kieliszek &
Misiewicz, 2014). Foods are typically very complex matrices, posing a significant
technical barrier to the analyst tasked with determining and establishing the levels of
various components. As a result, numerous analytical methods, including enzymes,
have been developed to meet this issue over time. The usage of enzymes is based on
specificity, which allows for the selective, sensitive, and precise measurement of
macro- and micronutrients in meals. The development of biosensors with greater
robustness and versatility has boosted the use of enzymes as an analytical tool in
recent years. Starch is a critical ingredient in baked goods because it aids in the
browning and flavoring of the crust. It is found primarily in plants in bread
production.
Many enzymes are used for milk processing and cheese production in the dairy
industry. Rennet is a coagulant enzyme mixture of chymosin and pepsin used in milk
clotting and cheese preparation. They have been extracted from animal sources,
mainly from the stomach of young calves (Theron & Divol, 2014). Plant extracts
also contain many enzymes, such as leaves of Sodom apple, berries of Solanum
dubium, Calotropis procera, and cardoon extracts, from microbial source
Rhizomucor miehei and Rhizomucor pusillus, contain protease enzyme that helps
in clotting. Protease enzyme obtained from Cryphonectria parasitica aggregate and
transferred milk into curd and liquid part.

11.9 Necessary Enzymes as Relevant Markers for Food Analysis

Enzymes are ubiquitous, and their application has been used from the olden days in
human edification. Food processing methods modify the food materials and make
them appropriate for consumption (Monteiro et al., 2011). In processing, it has been
used in wine, beer brewing, cheese, and bread making from 6000 BC. Based on the
enzyme properties, the International Union of Biochemistry approved a class of
enzymes used in the food industry, shown in the figure below (Fig. 11.2).
Food enzymes are categorized into two types: food processing and food additives.
Most of them are used for food processing purposes, whereas lysozyme and inver-
tase are used as food additives. The main aim of these food processing agents is to
increase the edible foods’ shelf life and preserve the health and nutritional contents
of the food without affecting their taste and aroma. Uses of enzymes in the food
industry process carbohydrates, proteins, and fats.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 275

Lyases
Oxido
Hydrolases
reductases

Isomerases Pecnase

Types of
Enzyme
Ligases Invertase

Amylase
Protease
alpha, beta
and Gluco

Oxido
Transferase
reductases

Fig. 11.2 Types of major microbial enzymes used in the food industry

11.9.1 Alpha-Amylase

The most important industrial enzymes account for around 25% of the global
enzyme market, which comes from various sources, including plants, animals, and
microbes (Kandra, 2003; Reddy et al., 2004). Enzymes catalyze the hydrolysis of
internal 1,4-glycosidic bonds in starch to produce low molecular weight compounds
like glucose, maltose, and maltotriose (Gupta et al., 2003; Rajagopalan & Krishnan,
2008). The baking sector currently employs a thermostable maltogenic amylase from
Bacillus stearothermophilus. It is also used to clarify beer and fruit liquids and
improve fiber digestion in animal feed (van der Maarel et al., 2002; Gavrilescu &
Chisti, 2005; Ghorai et al., 2009).

11.9.2 Beta Amylase

Beta amylase is an enzyme with the systematic name 4-alpha-D-glucan

maltohydrolase. It acts on starch, glycogen, other polysaccharides, and
oligosaccharides, producing beta-maltose by an inversion, resulting in the sweet
flavor of ripe fruit. This enzyme generally presents in bacteria, fungi, and plants. The
molecular weight of this enzyme is 223 and 8 kDa and has various applications in the
starch, sugar, brewing, and beverages industries. In bacteria, Clostridium, Bacillus,
and Pseudomonas are the major groups of organisms wherein fungi, Aspergillus
276 P. Sudhakara et al.

sp. can produce these enzymes used to manufacture organic acids (Djekrif-
Dakhmouche et al., 2006; Hernández et al., 2006).

11.9.3 Glucoamylase

Exoenzymes, also known as scarifying enzymes, are found in a wide range of

species. It is used in the food industry for a variety of purposes, including lowering
dough staling and increasing bread crust color, converting starch to maltose and
other sugars, assisting in the creation of glucose in the fermentation process, and
making alcoholic beverages and soy sauce (Blanco et al., 2014). Most glucoamylase
enzymes lose their activity at higher temperatures and become stable at lower
temperatures. The principal producers of this enzyme are A. niger and A. awamori.
However, Rhizopus oryzae is widely used in industrial applications (Karim et al.,
2017; Bilal & Iqbal, 2020).

11.9.4 Cellulase

This enzyme is generated mainly by microbes with a molecular weight of 54 kDa

(bacteria and fungus). It is a complex enzyme that hydrolyzes 1,4 links in cellulose
chains and contains endo-1,4-D-glucanase, exo-1,4-glucanase, and D-glucosidase.
Fungi produce more cellulase than bacteria (Gaur & Tiwari, 2015). Trichoderma
reesei, Penicillium, and Aspergillus are the three species. Bacillus cereus, Bacillus
megaterium, Bacillus mycoides, Clostridium straminisolvens, Flavobacterium
balustinum, and Acetobacter xylinum (Wachinger et al., 1989; Tomme et al., 1995;
Gaur & Tiwari, 2015; Sharma et al., 2015; Tabssum et al., 2018). In the food
industry, cellulase increases the nutritional characteristics of consumable food
products. Macerating enzymes, in addition to the combination of pectinase and
hemicellulase (Bhat, 2000), are essential in extracting olive oils to generate superior
quality with less waste (Galante et al., 1998). It is also utilized to break up roughage
in the dough (Chandrasekaran, 2013).

11.9.5 Pectinase

Pectin is a branching heteropolysaccharide with a high molecular weight. Pectinase

has a molecular weight of up to 31 kDa. Fruit juice is made with enzymes that
rapidly operate on hard pectin. It splits pectin molecules by making juice less
viscous, producing pectin-free starch, refining vegetable fibers, and degumming
natural fibers (Christopher & Kumbalwar, 2015). It can be found in higher plants
and microbes in their natural state. Pectinase, employed in tea fermentation,
eliminates pectin’s foam-forming ability and removes the mucilaginous layer from
coffee beans in tea and coffee (Oumer & Abate, 2018). Commercial pectinase can be
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 277

found in microbial strains such as Moniliella SB9, Penicillium spp., and Aspergillus
spp. (Priya & Sashi, 2014).

11.9.6 Invertase

Invertase is a non-pathogenic and non-toxic enzyme also known as beta-

fructofuranosidase, and the molecular weight of this enzyme is 205 kDa (Romero
Gomez et al., 2000). The primary source of this enzyme is plants and
microorganisms. Where Saccharomyces cerevisiae, Candida utilis, A. niger are the
primary source for the production of invertase commercially. It is used in alcoholic
beverages, lactic acid, and glycerol production. It is also used in the chocolate
preparation of infants’ food to improve the taste of the food (Christopher &
Kumbalwar, 2015).

11.9.7 Protease

Proteases represent one of the major groups of industrial enzymes, with a molecular
weight of 126 kDa. It breaks the peptide bonds present in the protein and separates
them as amino acids. Bacillus and Aspergillus spp. is the primary microorganism
that produces protease enzymes. It is used for several purposes such as brewing,
meat tenderization, baking, hydrolyzed animal proteins, functional meat
proteins, etc.

11.9.8 Lipase

Lipase enzymes deconstruct lipids into fatty acids and glycerol. This is something
that can be used in the baking sector. It separates milk fat and gives terrific flavors to
the cheese. The flavor of the cheeses improves the creamy texture and taste of the
products. Excessive lipid lysis leads to unavoidable odors. Most commercial lipases
develop flavor in dairy products, meat, vegetables, fruit, baked goods, milk, beer,
and others (Nagodawithana & Reed, 1993). The major lipase enzyme producers are
Pseudomonas sp., Bacillus sp., and Penicillium sp.

11.10 Enzymes as Biosensors

11.10.1 Enzymatic Biosensors Role in Food Technology

Food quality and safety-related assessment technologies need a necessary improve-

ment since they directly impact the consumers’ health. Reported microbial contami-
nation, pesticides occurrence, food adulteration, mycotoxin presence, freshness
analysis, presence of claimed antioxidants, health hazards caused by foodborne
278 P. Sudhakara et al.

microorganisms demonstrated the need for improved and faster technology to ensure
the safety and quality of the food products. Conventional techniques for food quality
assessments were replaced with fast and easy techniques like biosensors. Biosensors
are more extraordinary than other demonstrative instruments due to different food
quality and security innovations. Enzymatic biosensors are commonly utilized in the
food business (Table 11.3). Different properties of biosensors, especially optical,
mechanical, and electrochemical, can be adjusted by utilizing nanomaterials.
Biosensors are an appropriate detecting tool for quality control and revealing the
contamination in food. It can be a systematic and feasible detecting method in the
food chain.
The most important part of the food industry is maintaining food safety and
quality control. Biosensors have numerous food safety and quality control
applications. Enzyme biosensors are an essential technique as it has a notable
usage compared to the conventional method. It is effortless and less time consuming,
and it does not involve skilled labor and costly equipment (Santos et al., 2006;
Hooda et al., 2018). There are two main biosensors’ main constituents: the
bioreceptor and the transducer (Viswanathan et al., 2009). Biosensors work on
three principles based on the device used to create biosensors, segments of molecular
remembrance, and transducer. There are few enzymes for specific foods to act as a
biosensor. There are numerous applications in the food industry in which biosensors
are involved in quantifying different food constituents to determine their shelf life.
Biosensor technologies have the insightful potential to screen adverse substances
in foods on-site and in-situ phases. Biological agents like enzymes usage in
biosensors improved the specificity due to their binding affinity to a specific sub-
strate and related catalytic effect. In addition, they lack interference with the
compounds present in a complex sample. Due to sensitivity and specificity, enzy-
matic biosensors are considered an inevitable technology for food quality screening.

Table 11.3 Enzymes utilized in biosensors (Modified from Akyilmaz et al., 2010)
Enzymatic biosensors Food constituents
Glucose oxidase, glucose dehydrogenase Glucose
Oxalate oxidase Oxalate
Tyrosinase Tyrosine
Malate dehydrogenase Malate
Galactose oxidase Galactose
Cholesterol oxidase Cholesterol
Alcohol oxidase Alcohol
Peptidase, glutamate oxidase Aspartame
Tyrosinase Phenols
Lipoxygenase Essential fatty acids
Fructose-5-dehydrogenase Fructose
Glucose oxidase, mutarotase, invertase Sucrose
Galactose oxidase, peroxidase Lactose
Glutaminase Glutamine
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 279

This book chapter briefly highlights the recent advancements of enzymatic

biosensors’ role in food quality analysis.
The food industry participates in the mandated food quality and safety assessment
since they are essential for consumer health and nutritional value claims. The need
for reliable and straightforward analytical procedures to ensure the quality of the
food product from the production stage to consumption has recently been
highlighted by an increase in demand for food preservation (Antiochia et al.,
2013). The food manufacturing process is divided into primary and secondary
processing. During main and secondary processing, toxicants from food additives
may infiltrate food items. Toxicants such as drug residues, pesticides, and disease
microbes may interact with the chemical compounds in food products during
storage, causing food poisoning and rendering them unfit for human consumption.
Enzymes are biological molecules that can sense the substrates and catalyze specific
biochemical reactions. Like biosensors, devices can identify the specific material or
analyte in the food samples. In a biosensor, bioreceptor and the transducer are
considered essential components. Bioreceptor is used to screen the biomolecule,
and they may be enzymes, antibodies, DNA, or cell. Enzymes were the most used
bioreceptor molecules in biosensor applications. Enzymes enhance the reliability of
biosensors since their catalytic effects are more specific and sensitive (Monosik
et al., 2012). An enzymatic biosensor contains an enzyme to recognize the target
analyte and mediate the reaction to produce the chemical signal. This transducer
results in a physical signal from the chemical signal and an electronic amplifier. In
addition, enzymes are considered promising bioreceptor molecules due to the
broader role in the various analytical techniques, the flexibility in terms of
transducers types like electrochemical, optical, and thermal modes, required in a
low quantity, and the availability of high-quality commercial enzymes. However,
enzymes were also associated with certain limitations like size, due to which they
were not accessible to the relevant substrates, which in turn diminished the enzy-
matic activity. Enzymes reported with a limited lifespan may be deactivated by the
enzyme-specific components in the sample. Various physicochemical parameters
also influenced the enzymatic activity and cost of the commercially available
enzymes (Marazuela & Moreno-Bondi, 2002; Borgmann et al., 2012). According
to Vargas-Bernal et al.’s studies, enzymes used in the biosensor were classified into
six major classes, and the oxidoreductases class was the most used enzyme in
biosensor design (Vargas-Bernal et al., 2012). The first enzymatic biosensor was
launched in 1962, and glucose oxidase was the first enzyme incorporated in the
biosensor (Clark & Lyons, 1962).
Biosensors comprise biological receptors that can recognize a target, such as
enzymes, aptamers, cells, antibodies, and so on. Electrochemical, optical, mass, and
thermal methods are used in the binding process. Chemical biosensors based on
enzymes are based on biological recognition. The enzymes must catalyze a particular
biological reaction and be stable under the biosensor’s usual working settings to
function. The target analyte and matrix complexity in which the analyte must be
quantified are used to build biosensors. Combining MBs (magnetic particles) with
screen-printed electrodes (SPEs) is the best electrochemical biosensing compound
280 P. Sudhakara et al.

for food safety (Vasilescu et al., 2016). They are cheaper and can be obtained from
various materials with a minimal level of sample volumes (Power et al., 2018). The
enzyme-based sensor detects food contamination in various sectors of the food
industry as shown in Fig. 11.3.

11.10.2 First-Generation Enzyme Sensors

In 1956, Leland Charles Clark Jr published research on the electrode used to measure
oxygen levels in the blood (Clark et al., 1958). Electrochemical sensors and enzyme
transducers were described as membranes at a 1962 Academy of Sciences sympo-
sium. Clark described the functioning enzyme electrode with glucose oxidase
attached to the oxygen sensor later in 1967. Guilbault and Montalvo first described
the potentiometric enzyme electrode in 1970. It works by immobilizing the enzyme
urease on an ammonium-selective liquid membrane electrode. In 1973, they
described a hydrogen peroxide-based glucose and lactate enzyme sensor using a
platinum electrode. Klaus Mosbach invented a thermistor (heat-sensitive sensor) in
1974. In 1975, Clark introduced the glucose analyzer from Yellow Springs Instru-
ment Company, which used an amperometric sensor to detect hydrogen peroxide.
Lubbers and Opitz described a fiber-optic sensor with an immobilized indication to
monitor carbon dioxide or oxygen levels. Later, by immobilizing alcohol oxidase,
the optical biosensor concept was expanded to detect alcohol levels.

11.10.3 Second-Generation Enzyme Sensors

With the help of an artificial pancreas, Clemens and his colleagues built an electro-
chemical glucose biosensor. Miles later marketed it in 1975 as the Bio-stator (Gouda
et al., 2002). Bio-stator was not accessible on the market. VIA Medical introduced a
new semi-continuous catheter-based blood glucose analyzer. The lactate analyzer
LA 640 was introduced by La Roche in 1976 to transport electrons from lactate
dehydrogenase to an electrode.

11.10.4 Third-Generation Enzyme Sensors

Advanced biosensors have both immobilized enzymes and mediators in the same
electrode. Liedberg used the surface plasmon resonance (SPR) method to track
affinity interactions in 1983. In 1984, ferrocene and its derivatives were employed
as immobilized mediators with oxidoreductases in the manufacture of low-cost
enzyme electrodes based on screen-printed enzyme electrodes, according to Turner
and his coworkers.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 281

Fig. 11.3 Schematic representation of enzyme-based biosensor

282 P. Sudhakara et al.

11.11 Types of Biosensor

11.11.1 Electrochemical Biosensors

This biosensor employs an electrochemical transducer, in which metabolic reactions

generate electrochemical signals, which are then captured using potentiometric,
amperometric, or conductometric systems. It is a chemically distinct electrode with
a biological coating covering the conducting substance. These sensors work with
electrochemical sensors, which produce an electrochemical signal detected by an
electrochemical detector when the electroactive analyte is oxidized or reduced.
Voltammetric and potentiometric biosensors are two of the subgroups.

11.11.2 Voltammetric Sensors

Voltammetric sensors are subdivided into amperometric sensors. It measures the

current potential features of a specific reaction’s reduction or oxidation. Ampero-
metric sensors work on the premise of applying a fixed voltage to an electrochemical
cell, which results in a current due to an oxidation or reduction reaction. This current
aided in the identification of the reaction’s species. Unlike amperometry, which
detects current using redox reaction products, this sensor employs glucose, lactate,
and sialic acid analyzers. Electrochemical activity is seen in ion channel sensors. The
cationic marker of electrostatic attraction or repulsion is improved or suppressed
when these analytes are bound to a receptor molecule on an immobilized electrode
surface. Voltammetric biosensors such as fumaric, maleic, and adenine nucleotides
are employed with these sensors (Fig. 11.4).
The enzyme biosensor is characterized based on the substrate used for the food
and drug samples (Amine et al., 2006; Tudorache & Bala, 2007). The hypoxanthine-
sensitive sensors are used to detect fish freshness. Similarly, the xanthine oxidase-
based biosensors are used to disclose the levels of hypoxanthine and xanthine,
mainly involved in meat spoilage. To test the quality of the food products,
oxygen-based biosensor has been used. Other than that, enzyme-based biosensors
are used in food to maintain the freshness of edible products, detection of glucose
level in beverages, analysis of cholesterol in butter, food components of sugars, and
the detection of pathogenic organisms (Luong et al., 1991; Mello & Kubota, 2002).
Some commercial biosensors used in the food industry are listed in Table 11.4
(Chapman et al., 2018).

11.11.3 Novel Analytical Tools

The nanotechnological interventions for food sensors provide significant benefits in

identifying contaminants, especially in the food sector. Chemical nanosensors
(chemical nanosensors) and biosensors (nano-biosensors) based on nanomaterials
(nanosensors) can be utilized online and incorporated into existing production
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 283

Fig. 11.4 Biosensors and their physical and biological elements.

284 P. Sudhakara et al.

Table 11.4 Commercial biosensors used in the food industry

S. Biosensor used to detect the
N. Company materials
1 Affinity sensors Toxin’s detection
2 Ambri Ltd Detection of Salmonella
3 Biacore AB Detection of mycotoxins
4 Biosensor systems design Detection of toxins
5 Biosensores S. L. Detection of toxins
6 Chemel AB Detection of lactose and saccharose
7 Georgia Research Tech Institute Campylobacter and Salmonella
detection in pork
8 IVA Co. Ltd Detection of heavy metals
9 Massachusetts Institute of Technology E. coli O157:H7 detection in lettuce
10 Michigan State University’s electrochemical Salmonella and E. coli O157:H7
biosensor detection in meat
11 Oriental Electric Fish deterioration
12 Texas Instruments, Inc. Detection of allergens
13 Universitat Autonoma de Barcelona in Detection of atrazine traces
collaboration with CSIC

processes and distribution lines or offline as quick, simple, and disposable sensors
for food contamination. Food contaminants may be challenging to detect in this
environment of chemicals. Nanosensors are an emerging technology that could be
used to detect a variety of food pollutants, including mycotoxins and food allergies,
due to their unique applications. Nanosensors are thus more cost-effective, quick,
and sensitive than instrumental and traditional methods. Recent advancements in
nanosensor technology may increase demand for its use in food contamination
detection. As the food laboratory faces increased pressure to minimize cost, time,
and complexity, the role of nanosensors will become increasingly more crucial. This
chapter seeks to provide a broad overview of the potential applications of
nanosensors in the detection and analysis of food contaminants. In recent years,
the traditional PCR technique has been superseded by a simplified application in
POC devices using multienzyme bioreceptors to determine DNA fragments’
properties from allergens or adulterants. Surface platform resonance (SPR)
characteristics and high conductivity are attributes of nanomaterials used in these
sensor systems, such as Au NPs and AgNPs. This will make it easier to incorporate
optical and electrochemical sensors. Magnetic nanoparticles, such as Fe2O3 NPs,
will aid in the separation of analytes and will boost their performance.
However, carbon nanotubes and graphene-based nanomaterials enhance electro-
chemical signals due to their outstanding electrical conductivity. In 2017, electro-
chemical affinity biosensing was utilized to test food allergies, adulterants, and
gluten. Andrei et al. (2016) used Nanoceria, a nanosensor device with oxidase-like
activity, to covert phenolic antioxidants caffeic acid, gallic acid, and quercetin to
quinones to measure wine’s antioxidant content. He et al. (2018) developed manga-
nese dioxide nanosheets to detect ascorbic acid levels in orange fruit and juice. The
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 285

hazardous chemical biphenol A (BPA) is released from the polycarbonate and epoxy
resin during the food packaging process. Tyrosinase converts BPA to quinine to
detect BPA NPs such as Au, Ni, or Fe2O3 (Alkasir et al., 2010).

11.12 Food Packaging

Biosensors play a vital role in detecting foodborne pathogens, and it plays a crucial
role in food packaging as a part of smart packaging, which can be used in packaging
materials to check their freshness and shelf life. As a part of smart packaging, the
freshness indicator, time temperature indicators, integrity indicator, and radio fre-
quency identification are used (Park et al., 2015).

11.13 Enzymes for Detecting Food Adulterants

Food is required for the survival of life. We all eat food to obtain energy for various
metabolic functions. Food is required for all living species to grow, function, heal,
and maintain life processes. There are many different types of food on the market
today, and we all rely on diverse food sources daily, such as vegetables, fruits,
grains, pulses, legumes, etc. Small stones in cereals and grains, darkly stained
vegetables like cabbage and broccoli, fruits, dark red meat, and other items may
have been encountered while shopping for fresh vegetables and other foods. Food
adulteration is described as polluting food or food materials by adding a few
substances, collectively known as adulterants. Adulterants are substances or
products of lower quality that are added to food for economic or technical reasons.
The inclusion of these adulterants lowers the nutritional value of food and
contaminates it, rendering it unfit for ingestion. These adulterants can be found in
various foods that we regularly consume, including dairy, cereals, legumes, grains,
meat, vegetables, fruits, oils, and beverages. In underdeveloped countries, contami-
nation or adding to food components is typical. Milk, for example, can be diluted
with water to increase its volume, and starch powder is frequently added to increase
its solid content.
The urease microbial enzyme from Arthrobacter creatinolyticus was immobilized
on a poly (acrylonitrile-methyl methacrylate-sodium vinyl sulfonate) (PAN) mem-
brane to create a potentiometric urea biosensor. Glutaraldehyde was used as a cross-
linking agent in the biosensor. The following reaction is catalyzed by urease. The
production of ammonium ions produced by the breakdown of urea by urease was
used to detect urea in samples. A commercially accessible electrochemical worksta-
tion was used to perform potentiometric experiments. Milk samples were spiked
with known urea concentrations and evaluated using the biosensor for real-world
analysis. The measured potentiometric measurements were in good agreement with
the spiking concentrations. The biosensor had a detection limit of 0.3 mM and
linearity over the range of 1–100 mM. It was able to maintain appropriate sensitivity
for 13 cycles in a row. It could be kept for 70 days at 4  C.
286 P. Sudhakara et al.

Adulteration of food items is caused mainly by food firms’ marketing strategies,

mimicked food goods, a lack of awareness of proper food intake, and gaining profit
with fewer inputs.

11.14 Methods of Food Adulteration

Adulteration is the illicit practice of increasing the number of high-quality products

by introducing raw and other low-cost materials. This tainted food is highly hazard-
ous and causes various health problems, including nutrition deficiency diseases,
kidney disorders, and organ failure, including heart, kidney, and liver failure.

11.14.1 Melamine

Melamine is a commercially manufactured chemical molecule (1,3,5-triazine-2,4,6-

triamine). Inflating nitrogen content and proteinaceous substances in food is a
significant food adulteration. Melamine’s specificity and ability can be determined
using two approaches. First, commercial enzyme-linked immunosorbent assay
(ELISA) kits were tested using ammeline, ammelide, or cyanuric acid, revealing
that 4,6-diamino-1,3,5-triazine structure, or 1,3,5-triazine-4,6-diones were required.
Melamine detection limits in infant powder ranged from 0.2 to 3 mg/g per sample
(Kim et al., 2010). This melamine produces significant toxicity and kidney stone
formation (Hau et al., 2009). Brown et al. (2007) labeled it a hazardous substance
because it causes renal failure by forming insoluble melamine cyanurate crystals in
the kidneys. Nanosensor to detect melamine in various feed matrices quickly, Au
nanoparticles, cadmium telluride (CdTe) quantum dots, SWCNTs, and other sensors
are used (Li et al., 2010). Zhou et al. (2011) employed an Au nanoparticle biosensor
to detect melamine in various milk and dairy products. Milk powder, infant formula,
lactose, whey protein, wheat bran, and wheat gluten are all examples of foods that
include it (Mecker et al., 2012). Melamine was also detected using water-dispensable
cadmium telluride quantum dots coated with thioglycolic acid.

11.14.2 Meat Adulteration

The demand for ready-to-eat halal foods such as burgers, pizzas, hot dogs,
sandwiches, soups, cookies, sweets, and creams has risen recently (Jeddah, 2011;
Mascini, 2015). Pork is being used more frequently in Halal meals, including beef
meatballs, to maximize profits (Mascini, 2015). It was identified utilizing NPs as a
colorimetric nanobiosensor, which changed the color of the meat from pinkish-red to
purple with a 525 nm absorption peak. Ali et al. (2011) used hybrid biomaterials
with functionalized nanoparticles that are noncovalently or covalently coupled to
biomolecules such as peptides, proteins, and DNA to detect pork adulteration in
processed meat.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 287

Several adulterants such as food coloring compounds and preservatives have

been found with modern technology. Olive oil, milk, honey, and saffron are some of
the most commonly reported contaminated foods, which can be discovered using
HPLC and infrared spectroscopy (Moore et al., 2012). The adulterants included in
butter, pork, and beef meatballs were detected using Raman spectroscopy and FTIR
spectroscopy. Adulterants such as sunflower and corn oils commonly replace olive
oil and can be detected using FTIR spectroscopy.

11.14.3 Enzymes for Detecting Contaminants

Food adulteration can arise from numerous sources, such as natural toxic
compounds, bacterial contamination, pesticides, veterinary drugs, and chemicals
used in food dispensation. Identifying this substance is essential for people affected
by diseases (Viswanathan et al., 2009). It has been mostly separated into two classes:
biological and chemical contaminants. Some critical assays analyze the
phytocompound such as sugars, alcohols, amino acids, flavors, and sweeteners.
Chromatography and spectroscopy have been used to detect contaminants from
the olden days. Currently, many sensors have been developed to detect the presence
of these contaminants in food without affecting their taste and flavor.

11.14.4 Biological Contaminants

Toxic and pathogenic biological substances created during food preparation, pack-
ing, and storage are biological pollutants. Toxins such as aflatoxins, ochratoxins, and
other foodborne microorganisms are the most common pollutants that cause
foodborne illnesses. Pathogenic bacteria include Campylobacter, Salmonella,
Listeria monocytogenes, E. coli, Staphylococcus aureus, and Bacillus cereus. Toxi-
cology can be detected using a variety of approaches. The most prevalent
mycotoxins generated by Aspergillus spp. are aflatoxin and ochratoxins. It is
cancer-causing, teratogenic, mutagenic, and immunosuppressive. A nanosensor
has been created to detect this mycotoxin using the immunosensor approach.
(Sharma et al., 2010). Using antibody-conjugated MNPs and a magnetic field,
Salmonella in milk was detected. Sack et al. (2004) defined Vibrio cholera as an
acute intestine infection induced by eating tainted food or water. Cholera toxin, a
colorimetric nanobiosensor detected with glycol nanoparticles, is secreted by this
bacteria (Schofield et al., 2007).

11.14.5 Chemical Contaminants

Unwanted dangerous chemicals are introduced into foods directly or indirectly from
natural sources, pollution, or formation during food processing, distribution, and
storage. Pesticides, heavy metals, and melamine are just a few chemical pollutants
288 P. Sudhakara et al.

Table 11.5 Nanobiosensors used in the detection of chemical and biological contaminants
Nanomaterial Contaminants Biosensor
Biological
Gold nanoparticle Aflatoxin Immunoelectrode
Silver core and a gold shell AF B1 Immuno-dipstick assay
Antigen-modified and antibody-functionalized AF B1 and OTA Immunosensing probes
DCNPs
Nanostructured zinc oxide Mycotoxin ITO glass plate
Magnetic nanoparticles and TiOz nanocrystals Salmonella Optical nanocrystal
probes
Oligonucleotide-functionalized Au nanoparticles Escherichia coli Piezoelectric biosensor
Glyconanoparticles Cholera toxin Colorimetric bioassay
Chemical contaminants
Gold nanoparticle Carbofuran Electroimmunosensor
Antibody competitive nanoparticle Chloramphenicol Immunosensor IPR
MTT-stabilized gold nanoparticle Melamine Colorimetric sensor
Quantum dots Melamine Colorimetric sensor
CNTs Penicillin Enzyme-based
biosensor

that harm human and animal health. Nanosensors have the potential to be a helpful
instrument for detecting chemical pollutants. Table 11.5 shows a variety of
nanosensors and nanobiosensors for detecting chemical and biological pollutants.

11.14.6 Biosensors and Bioamines to Quantify the Freshness of Food

Products

Chemical compounds produced in surrounding storage due to the decomposition of

food components served as a vital signal for food freshness. The concentrations of
xanthine, hypoxanthine, and biogenic amines, for example, have altered over time.
The breakdown of inosine monophosphate in seafood produces chemicals like
xanthine and hypoxanthine, which act as biomarkers for fresh food. Biogenic amines
(BA), such as histamine, putrescine, and cadaverine, are also generated by the
decarboxylation of amino acids and are used as freshness markers in meat, drinks,
and cheese (Calvo-Pérez et al., 2013). Bioamines are essential for numerous physio-
logical activities at low concentrations, but they can cause health problems in
consumers. BAs were produced in plants, animals, and microbial populations by
pathogenic microbial decarboxylation of specific amino acids, such as histidine, or
by the amination and transamination of ketones and aldehydes.
In contrast, others have aromatic structures, such as Phenylethylamine (PEA) and
Tyramine (Tyr) (Tyr). Then there are a few BAs that have heterocyclic structures,
such as Tryptamine (Tryp) and Histamine (Hist) (His). The presence of BAs in
dietary samples was determined using the time-consuming high-performance liquid
chromatography (HPLC) method (Hernández et al., 2006). BA’s biosensors were
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 289

designed as an effective strategy for facilitating quick and exact estimation in the
search for a steady approach to accurate discoveries. Many amine oxidase enzyme
designs have been used to construct biosensors, to increase precise biorecognition
and signal transduction.

11.14.7 Biosensors and Histamine Oxidase Applications

Substances like histamine were biogenic amines. Their presence varies with degrees
in several food products, and their increased concentration is considered an indicator
of the depleted level of freshness. Histamine intoxication is the most prevalent
foodborne infection among humans who consume fish products. If histamine con-
centration exceeds 500 ppm in fish products induces histamine poisoning (Gonzaga
et al., 2009). Bacterial contamination in fish gut and skin induces histidine decar-
boxylase activity and results in histamine production. Histamine levels were
screened by immobilized histidine decarboxylase and horseradish peroxide loaded
on the electrodes using the bovine serum albumin and glutaraldehyde complex.
Another study used the histamine oxidase enzyme from the bacterial
sp. Arthrobacter crystallopoietes (HOD) to build a biosensor. HOD biosensor was
effectively employed to measure histamine levels in fish and meat products, com-
paratively yielding a good agreement with the results obtained from HPLC analyses
(Rosini et al., 2014).

11.14.8 Biosensors to Quantify D and L Amino Acid

Amino acids have been extensively researched due to their nutritional importance
and speed up metabolic processes. D-amino acids (D-AAs) are, on the other hand,
primarily found in microbiological, aqueous, soil, and other environmental
pollutants. In general, the presence of D-AAs reduces protein digestion in the
body, resulting in decreased bioavailability of essential amino acids and nutritional
value. D-AAs are also utilized to evaluate the adverse effects of heat and alkaline
treatments on dairy products. D-AA biosensors based on the D-amino acid oxidase
(DAAO) enzyme were frequently employed to evaluate milk samples (Sarkar et al.,
1999; Rosini et al., 2008). The FAD domain is a cofactor expressed in humans, not
bacteria, in this peroxisomal enzyme.

11.15 Biosensors and Antioxidants’ Concentration

The antioxidant activity of phenolic chemicals found in berries has been shown to
help prevent cardiovascular disease and aging and have anti-cancer properties. Since
the TYN enzyme converts hydroquinone in foods like red wine to p-quinone, it then
reduces to hydroquinone at the electrode and generates a current. The TYR enzyme
from Agaricus bisporus is an oxidase enzyme that catalyzes the conversion of
290 P. Sudhakara et al.

phenolic substances to quinone derivatives, with amperometric techniques detecting

the end products. The TYR biosensor can detect the “total phenolic content” and
“total antioxidant capacity” of samples with low non-polyphenolic antioxidant levels
in general. The reduced lifespan of the Tyr enzyme reduces its stability. The tert-
butyl hydroquinone chemical quantities in standard salad dressing items were also
measured using the Tyr biosensor.

11.15.1 Biosensors for Heavy Metals

In recent days, heavy metal-induced toxicity among the food has been observed and
studied extensively. Heavy metals like Fe, Zn, Cd, Ni, Mn, Cr, Co, Pb, Cu, and Al
were leached from the eating utensils as well as cookware made up of iron, old
stainless steel, new stainless steel, old and new aluminum, and clay pots lead to
metallic contamination of food and water (Ghorai et al., 2009). In practice, the usage
of enzyme-based biosensors is used to detect these toxic substances like heavy
metals. Since these heavy metals react with the thiol groups in the enzymes like
glucose oxidase, urease, glutathione S-transferase, alkaline phosphatase, lactate
dehydrogenase, acid phosphatase, and invertase and biosensors, the function is
based on enzymatic inhibition. Nevertheless, limited enzymes have an affinity to
heavy metals.

11.15.2 Biosensors and Ethanol Concentration

Assessing the ethanol concentration and control is vital in brewing and winemaking
and also needs the exact data about the ethanol content in the beverages.
Immobilized alcohol oxidase catalyzes the reaction on the ethanol biosensor. Earlier
studies on ethanol biosensors used the combination of enzymes of alcohol oxidase
(AOx) along with horseradish peroxidase (HRP) and a mediator ferrocene
immobilized on an electrode. Later, Shkotova et al. established an amperometric
transducer and AOx-based biosensor immobilized on resydrol polymer and used to
detect ethanol concentration in alcoholic beverages. Another enzyme,
pyrroloquinoline quinone alcohol dehydrogenase (PQQ-ADH), is used in ampero-
metric biosensors to detect ethanol. PQQ-ADH has advantages like oxygen inde-
pendence and does not require a soluble cofactor. Certain studies employed the
bi-enzymatic biosensor to detect the ethanol concentration in alcoholic beverages.

11.15.3 Fructose Dehydrogenase-Based Biosensors

Because fructose is harmless, it is commonly employed in artificial sweeteners.

Fructose dehydrogenase (FDH) is a human enzyme that produces fructose in the
intestinal mucosa. This enzymatic process is also used to make corn syrup in corn
plants (Siepenkoetter et al., 2017). Fructose corn syrup was thought to be a better
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 291

substitute for natural sugars. As a result, fructose has been used to manufacture
beverages and foods (Antiochia et al., 2013).
On the other hand, overuse of fructose has been associated with high blood
pressure, decreased glucose tolerance, insulin resistance, and hepatic steatosis. For
many years, biosensors based on FDH have been used to detect fructose content in
food and beverages. To immobilize the NDFDH-modified nanoporous gold
electrodes, FDH was coupled with a thiol and diazonium-bound carboxylic acid
functional group. The fructose level in beverages may be accurately measured using
this manufactured electrode.

11.15.4 Xanthine Oxidase-Based Biosensors on Assessing the Fish

Freshness

Xanthine is a thoroughly studied fish freshness indicator. Adenosine triphosphate

(ATP) concentration in stored fish steadily degraded to generate xanthine as the
storage period extended beyond the permissible interval (Venugopal, 2002). On the
other hand, manufacturing needs fresh fish for human consumption and fish
products. Thus, a few decades ago, investigations began using electrochemical
xanthine biosensors as a biomarker to assess the freshness of fish. However, the
stability of this biosensor is restricted; thus, Devi et al. devised a xanthine oxidase–
chitosan–gold-coated nano-iron modified pencil graphite electrode with a detection
limit of 0.1 μM to increase the stability. This biosensor’s xanthine detection and
storage precision have been increased. Dervisevic et al. developed a CHIT–
polypyrrole–AuNP-modified glassy carbon electrode (GCE) with XOD as an
electrocatalyst that converts xanthine to uric acid in a follow-up study. With a
detection limit of 0.25 μM, this modified amperometric sensor could efficiently
detect xanthine.

11.15.5 Lactate Dehydrogenase-Based Biosensors on Quantifying

the Lactate Concentration

Lactate concentrations in red wine, white wine, beer, yogurt, curd, and milk samples
were used as biomarkers to determine the food quality. The food industry utilized
lactate biosensors to detect the desired level of lactate content in fermentation. Lactic
acid fermentation is induced by Lactobacillus spp. in fermented food products, and
the lactate content suggests an enhanced Lactobacillus population. Batra et al.
created an electrochemical lactate sensor using a PGE modified with graphene
oxide (GO) nanoparticles and LDH in 2016. This upgraded LDH–GO–PGE lactate
biosensor is currently being utilized to assess lactate levels in fermentation industries
(Batra et al., 2016). Lactose-related biosensors were also frequently employed, as
lactose intolerance affects roughly 15% of Northern Europeans, 80% of black and
Latino persons, and almost 100% of American Indians and Asians. These
individuals lack the digestive enzyme galactosidase, which converts lactose to
292 P. Sudhakara et al.

galactose and simple glucose. Electrochemical biosensors containing co-

immobilized-galactosidase and glucose oxidase enzymes were created to assess
the trace amount of lactose in lactose-free dairy products.

11.16 Biosensors in Mycotoxins Detection

Fungal species Aspergillus, Fusarium, and Penicillium produce specific secondary

metabolites that cause food poisoning among humans and animals after ingesting
contaminated food products (Turner et al., 2009). Contaminated fruits and cereals
induce mycotoxin in certain beverages like beer and wine. Sometimes contamination
of livestock animals’ feed during the processing also introduced the mycotoxin in
food products (Becker-Algeri et al., 2016; Iqbal & Selamat, 2016). Thus, residual
mycotoxin presence needs to be evaluated to ensure the food product quality.
Mycotoxins were categorized into distinct groups, namely aflatoxins, ochratoxin A
(OTA), zearalenone (ZEN), fumonisins, ergopeptine alkaloids, and trichothecenes
based on their impact on the economy and associated health risk. Enzymatic
electrochemical sensors were developed by “Grupo de Electroanalítica” GEANA
(Argentina) using enzyme horseradish peroxidase to assess the CIT in rice.

11.16.1 Epidemiology of Foodborne Illnesses

Foodborne infections that are infectious and toxigenic have been known for more
than a century. Salmonella, Staphylococcus aureus, and Clostridium perfringens
were the most common pathogens of concern worldwide by the mid-1900s. Botu-
lism was a fatal disease rarely linked to commercially canned foods, home canning
of vegetables, or traditional Arctic marine mammal products.
There was little interest in or research on acute foodborne illness agents during
those days. According to what was learned during outbreaks, most outbreaks might
have been averted if adequate time and temperature handling and storage protocols
had been followed, especially with poultry and beef. Clostridium, Salmonella, and
Staphylococcus issues were mainly resolved after the population was educated.
However, additional agents like Campylobacter, E. coli O157:H7, Listeria, and
Yersinia species emerged in the 1980s. It took many more years to realize that
these disorders might lead to life-threatening complications or even death and that
several goods could transmit them. Only a few of these diseases are detectable by
existing monitoring systems (Kothary & Babu, 2001; Wang et al., 2010; Fukushima
et al., 2011; Lindström et al., 2011). However, more and more international
outbreaks are being recognized thanks to DNA typing and next-generation sequenc-
ing technology.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 293

11.16.2 Worldwide Status of Foodborne Illnesses

Despite the adoption of new regulatory concepts, know-how, and initiatives on a

national and international level, there has been a broad plateau in the number of
instances of foodborne disease. The fundamental reason for this is that surveillance
of waterborne and foodborne illnesses has been limited in detecting instances other
than small clusters of patients. For decades, the traditional system of making
outbreak reports available to a central authority in a small number of countries has
been the source of our knowledge, but it is far from adequate.
Only significant outbreaks are reported, whereas home infections are never
reported. As a result, epidemics in mass catering and restaurants are widely
highlighted. Another issue that raises the risk of foodborne disease is the global
food market. Changes in lifestyle and activities, more extensive operations, and
higher throughput lead to increased profit, increasing the risk of contaminated
animal products. Even if the livestock is not ill, larger-scale animal rearing allows
for more microbial diseases. Antibiotics are mainly used to aid in the growth of
animals. However, widespread use has led to growing antibiotic resistance and
inappropriate use for self-treatment of human illnesses, posing a more significant
threat to the global population. Other contamination sources, including rats and
rodents in warehouses, have been identified as critical connections in the transmis-
sion chains of zoonoses.
Newer strains in humans are derived from environmental sources, where interge-
neric gene transfer may occur. Larger markets result in a more significant geographic
dispersion worldwide, resulting in massive outbreaks with millions of patients if
problems arise. This is an example of how tracing a product’s origins can be
challenging, mainly if the originating company is located in another country. People
are introduced to new meals by immigrants, and the public requires a diverse range
of products to consume. This can lead to new goods or modifying current ones, risk
introducing a new pathogen at each stage (Alvarez-Ordóñez et al., 2013).

11.17 Bacterial Pathogens

11.17.1 Staphylococcus aureus

Because of contamination from humans, cattle, and the environment, several Staph-
ylococcus genus species and subspecies are potentially harmful to foods. Both
coagulase-negative and coagulase-positive strains can produce enterotoxins that
cause human gastroenteritis. S. aureus is most commonly associated with food
poisoning. Enterotoxins are the most dangerous of the several toxins produced by
staphylococci. Enterotoxins are proteins generated by other Staphylococci strains
that, if allowed to flourish in foods, form enterotoxin. S. aureus is the primary
producer of structurally related, toxicologically comparable proteins (Lancette &
Bennett, 2001).
294 P. Sudhakara et al.

Meat and beef products, poultry and egg products, salads such as bacon, fish, rice,
potato, and macaroni, baking products such as cream-filled pastries, and cream pies
are the most common foods associated with staphylococcal food poisoning. Some of
these products are polluted and mismanaged as a result. Chemical, animal, or
environmental contaminants can contaminate pre-processed foods. At the same
time, foods exposed to temperatures favorable to S. aureus growth have a signifi-
cantly greater capability to form enterotoxin. This is also true of meat and milk
products that have been fermented. Secreted enterotoxins are a structurally and
functionally related family of virulence factors with critical super-antigenic qualities
that disrupt adaptive immunity. Standard enterotoxins (SEA-SEE) and modern
enterotoxins are two types of enterotoxins (SEG-SElY).
The pathophysiology of several human diseases, such as toxic shock syndrome
(TSS), sepsis-related infections, and pneumonia, is determined mainly by members
of these classes. These have emetic properties and are frequently linked to food
poisoning outbreaks. The enterotoxins are primarily responsible for the disease’s
consequences when the patient lacks denaturation resistance. The enterotoxin genes
are frequently found on many mobile genetic elements. The prevalence of
enterotoxins varies greatly among S. aureus isolates, and it is regulated by numerous
overlapping regulatory mechanisms (Lancette & Bennett, 2001).

11.17.2 Listeria monocytogenes

Listeria monocytogenes causes occasional outbreaks and ingestion associated with

infected food products as another significant cause of death due to foodborne
disease. Over the last few decades, Listeria monocytogenes have emerged as a
foodborne pathogen of considerable importance. Most of our knowledge about
foodborne Listeria transmission routes has been acquired through the study of
worldwide epidemiological evidence. Such outbreaks were related to food from
hospitals patients who had consumed spinach, carrots, and radishes.
The use of manure from infected sheep has been blamed for several outbreaks.
Most listeriosis infections in pregnant women have resulted in miscarriages,
stillbirths, and sporadic or live deliveries of poorly infants. Meningitis, aspiration
pneumonia, and sepsis symptoms were seen in many non-pregnant people who
showed no evidence of immunosuppression. These outbreaks accounted for around
41% of the overall death rate. Serotype 4b was assigned to some of these isolates,
and L. monocytogenes isolates from coleslaw packets were known as Serotype 4b.
Over the past few years, whole-genome sequencing has proven to be a valuable
tool in L. monocytogenes surveillance and outbreak investigations. WGS has
allowed rapid and reliable identification of infection clusters, helped trackback to
sources of infection, and ultimately removed the origin of infection. Various projects
have been initiated to encourage the typing of L. monocytogenes based on WGS and
many other foodborne pathogens. Nevertheless, there remains a need to create a
transition to an international standard. (Camargo et al., 2017; Colagiorgi et al.,
2017).
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 295

11.17.3 Bacillus cereus

Many extracellular compounds involved in pathogenesis are found in Bacillus

cereus, and many strains are employed as probiotics in humans. On the other
hand, others are highly poisonous and linked to dietary deaths. There are two
forms of foodborne sickness caused by these genera: diarrheal and emetic.
Enterotoxins produced in the small intestine during pathogen vegetative growth
cause the former, while the emetic toxin is pre-formed in the meal before ingestion
causes the latter. The disease is spread via food because of the surviving spores in
both forms. B. cereus, in addition to being an opportunistic pathogen, can cause a
variety of systemic and local illnesses, including septicemia and endophthalmitis.
The diarrheal disease is typically self-limiting, and the number of B. cereus
outbreaks is widely underestimated due to the relatively short duration of the disease
(typically less than 24 h). The total recovery is swift after the symptoms resolve.
The B. cereus community comprises many species whose members are capable of
causing foodborne emetic or diarrheal diseases. With the lack of modernity in this
pathogen’s diagnostic and characterization aspects, tens of thousands of diseases
have been documented in the world each year. Still, the issues are far from
conquered. Today, methods such as whole-genome sequencing are not widely
used to classify B. cereus isolates from foodborne diseases. In addition to using
state-of-the-art genomic methodologies, it is possible to use WGS techniques to
address future microbiological and epidemiological problems raised by B. cereus
outbreaks in the group. One specific obstacle is that, unlike other pathogens, cases of
disease caused by B. cereus are usually not reportable, while foodborne diseases are
widely documented, regardless of pathology. When mixing this with a usually
moderate course of B. cereus disease, human isolates of the B. cereus are rarely
available for techniques such as WGS.
In addition, even if clinical isolates of the B. cereus group are obtainable, WGS
should not be used to describe the isolate in cases where the disease or infection is
mild. The data and methods available from laboratories around the world not just
enable further detection of other outbreaks of B. cereus.

11.17.4 Clostridium perfringens

Clostridium perfringens produces two diseases transmitted through food: a common

foodborne illness and necrotic enteritis (pig-bel). Ingestion of infected foods causes
C. perfringens, one of the most frequent foodborne infections. While the link
between C. perfringens and foodborne sickness was first proposed over a century
ago, definitive evidence demonstrating that an enterotoxin is linked to the organism’s
sporulation in diseased intestines did not develop until the 1960s and 1970s. The
bacteria have many characteristics that contribute to its capacity to cause foodborne
illness: (1) an omnipresent distribution throughout the natural environment, allowing
it to contaminate food; (2) the ability to develop rapidly in food, causing high levels
296 P. Sudhakara et al.

of food poisoning; and (3) the ability to produce an intestinally active enterotoxin,
which is responsible for C. perfringens’ characteristic gastrointestinal symptoms.
C. perfringens-related food poisoning is one of the most common causes of food
contamination. The association between C. perfringens and foodborne sickness was
originally postulated around 100 years ago, it was not until the 1960s and 1970s that
conclusive evidence emerged demonstrating that an enterotoxin is linked to the
organism’s sporulation in the intestines. The bacteria have several characteristics
that contribute to its capacity to cause foodborne illness:

1. It has a wide distribution in the natural environment, allowing plenty of

opportunities to contaminate food.
2. Its ability to generate heat-resistant spores allows it to survive incomplete food
cooking or insufficient sterilization.
3. The ability to produce an intestinally active enterotoxin is responsible for
C. perfringens food poisoning’s typical gastrointestinal symptoms.

However, unlike other foodborne illness agents, it is frequently underreported,

owing to the anaerobic conditions required for its development and the sickness’s
apparent mildness. Meat and poultry-based products, as previously stated, are the
most prevalent carriers via which intestinal contents spread after slaughter. The
presence of this organism in the spore condition on herbs and spices (as well as
meat and poultry) poses a hazard since it can survive the chilling cycle and restart
growth in temperature-abused or inadequately cooled items, resulting in the enor-
mous number of cells needed for an outbreak. The organism’s specific development
requirements are easily met in meat and poultry products.
Temperature exploitation of cooked meals is often linked to outbreaks, showing
that the organism’s optimum development temperature is relatively high [43–46  C].
This, together with its ability to form spores, are two remarkable traits contributing to
its etiological role in foodborne disease. The sickness is more likely to be purchased
in restaurants, hospitals, jails, schools, and caterers (Lindström et al., 2011).

11.17.5 Clostridium botulinum

Botulism comes in a variety of types. Symptoms of foodborne botulism can appear

within 12–36 h and can take up to a week or longer after consuming botulinum
neurotoxin-containing food. Initial symptoms include nausea and vomiting, though
it is unclear whether the neurotoxin or other Clostridium botulinum compounds are
to blame.
The organism may get established in the intestines of young infants, most likely
because the normal intestinal flora has not yet developed sufficiently to prevent
pathogen colonization. Chronic constipation, severe exhaustion, and increasing
paralysis, as well as other neurological symptoms, may be present in the patient.
In some instances, spores of C. botulinum have been discovered in specimens from
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 297

children who died of sudden infant death syndrome (SIDS) (Health Protection
Agency, 2011).

11.17.6 Salmonella Species

Serotypes of Salmonella can cause localized gastroenteritis in humans, but the

severity of Salmonella infections in different hosts is determined by pathogen
virulence, host resistance, and immunity. Many serotypes have limited host ranges,
such as Salmonella typhi and Salmonella paratyphi A, linked to human infections,
and Salmonella gallinarum, linked to poultry infections. Many serotypes have
limited host ranges, such as S. typhi and S. paratyphi A, linked with human
infections, and S. gallinarum, associated with poultry infections.
Salmonella serotypes Typhimurium and Heidelberg have a broad host range and
can colonize and infect various animal hosts. Human infections of S. typhimurium
are caused primarily by transmission-contaminated foods such as poultry items.
Chicken eggs and egg products have been closely connected to Salmonella
enteritidis. This could be linked to its apparent proclivity for infection and reproduc-
tive tissue invasion in sensitive chickens, as well as contamination of internal
shell eggs.
Nonetheless, because there are so many cases of salmonellosis from non-poultry
and non-food sources, such as domestic mammalian and reptile pets, any attempt to
assign quantifiable rates to specific sources is futile. Bacteria may colonize and
develop in the small intestine, penetrate and dwell in intestinal tissues, and cause an
inflammatory response in the host when people eat a Salmonella infectious dose.
Salmonella can enter the body through macrophages or other immune cells and go to
lymph nodes in the liver, spleen, or mesenteric arteries.

11.17.7 Shigella Species

Shigella is spread through fecal–oral, feces, direct touch with people (fingers), food,
flies, and inanimate items (fomites). More than two-thirds of all shigellosis cases
occur in youngsters aged one to five who appear to use their lips to explore their
surroundings. Intake of contaminated water is frequently cited as a method of
shigella transmission. When people are crammed together, disease transmission
and bacillary dysentery epidemics are intensified. Inadequate sanitation creates an
environment for direct fecal–oral contamination, such as in daycare centers, custo-
dial institutions, psychiatric hospitals, nursing homes, and mass displacement and
gathering, such as in wartime refugee camps or political unrest.
In addition, those infected with the human immunodeficiency virus (HIV) might
develop more severe and chronic types of shigellosis. Secondary attack rates among
household contacts could be as high as 40% following primary case exposure.
Shigellosis outbreaks appear to follow seasonal patterns in some areas, with
298 P. Sudhakara et al.

transmission peaking in arid regions like Egypt’s hot, dry season. During times of
water scarcity, it is primarily ascribed to dirty water use and poor personal
cleanliness.
On the other hand, peak events occur in China and Thailand during the rainy
season because of water-washed-based transmission after heavy rains. These repre-
sent the link between shigellosis and the unclean settings that encourage fecal
transmission. Shigellosis is most common in developing countries during the sum-
mer months, when raw foods, fresh fruits and vegetables, and recreational facilities
are most plentiful. Years later, the proportion of S. flexneri, S. sunny, S. boydi, and
S. dysenteriae cases in developing countries was estimated to be 60% (mainly
serotype 2a), 15%, 6%, and 6% (30% of S. dysenteriae cases were type 1), respec-
tively; and 16% (predominantly serotype 3a), 77%, 2%, and 1% in developed
countries, with nearly half of travelers registering incidents and returning travelers
from (Kothary & Babu, 2001).

11.18 The Foodborne Vibrios

11.18.1 Vibrio vulnificus

Vibrio vulnificus is the opportunistic bacteria that cause foodborne illness and
devastating infections in humans. Ingestion of raw or undercooked seafood, con-
tamination of an open wound, or a sore contaminated with seawater or seafood
drippings are all possible entrance points. V. vulnificus produces wound infections
by entering existing or parallel wounds, ulcers, and insect bites, in addition to being
foodborne. Exposure to seawater or shellfish leaking from a cut is nearly always the
cause of these wounds. Puncture wounds from a bite from a marine animal (e.g.,
crabs, stingrays), stabbing or laceration wounds from the use of shellfish/fish
cleaning equipment, lacerations from commercial or recreational use of fishing
gear, rocks, or debris in or near seawater, or contamination of an existing wound
or insect bite with seawater or shellfish drippings are all reported wound scenarios.
Puncture wounds from a bite from a marine animal (e.g., crabs, stingrays),
stabbing or laceration wounds from the use of shellfish/fish cleaning equipment,
lacerations from commercial or recreational use of fishing gear, rocks, or debris in or
near seawater, or contamination of an existing wound or insect bite with seawater or
shellfish drippings are all reported wound scenarios. Because V. vulnificus is
associated with various seafood species, the health status is unknown. The human
sickness produced by this organism and other marine vibrios could become a
growing food safety concern. This problem is exacerbated by the fact that demand
for seafood is increasing, and it can now only be fulfilled by aquaculture and marine
fisheries sources harvesting seafood. These issues highlight the importance of
monitoring systems to track the safety of our seafood (Kothary & Babu, 2001).
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 299

11.18.2 Vibrio parahaemolyticus

Most foods implicated with V. parahaemolyticus infections are seafood or similar

foods, such as raw seafood, cooked seafood, fine seafood, and seafood. Tuna,
shellfish (oysters, clams, etc.), crab, lobster, and shrimp were among the seafood
used. In Japan, a majority of cases are caused by eating raw fish. However, this is not
often the case in other countries, where cases are frequently caused by poor prepara-
tion, recontamination, or storage. In cases of food poisoning, foods that do not
include marine elements may be contaminated by secondary contamination.
In Japan and the United States, V. parahaemolyticus food poisoning incidents are
only reported during the summer. However, in Thailand, cases are reported all year.
The seasonal change in the prevalence of infection may be linked to the epidemic
strain’s seasonal occurrence in the marine environment. While several reports
demonstrate V. parahaemolyticus distribution in the aquatic environment in various
world regions, epidemiologic research assessing the occurrence of virulent strains in
both clinical specimens and the marine environment in an endemic context is rare
(Parveen et al., 2008).

11.18.3 Vibrio cholerae

According to epidemiological data, fecally infected water is the predominant mode

of cholera transmission. Food has recently been recognized as a life-threatening
factor in cholera transmission. Seafood, particularly mussels, oysters, and shrimp,
has been implicated when consumed uncooked, undercooked, or prepared with dirty
water. While V. cholerae can be found in rice, fish, eggs, and milk, environmental
studies in Bangladesh have found that most foods are not infected with the bacteria.
Cholera expanded through seaports and river ports during the seventh pandemic,
particularly in Africa during the 1970s epidemics. Patients with active disease are
regarded to be the predominant source of infection. Asymptomatic carriers may play
a role in cholera transmission. However, this is not required. El Tor vibrios appear to
live longer in the carrier form than Classical vibrios, but this difference is not
epidemiologically significant. It has been demonstrated that V. cholerae O1 can
survive in the aquatic environment without the help of zooplanktons and that
V. cholerae is connected with a variety of aquatic flora, including water hyacinth
duckweed and green algae. The blue–green algae have been discovered. V. cholerae
is found in Anabaena variabilis, which serves as a reservoir. It has been
demonstrated that V. cholerae O1 can survive in the aquatic environment without
the help of zooplanktons and that V. cholerae is connected with a variety of aquatic
flora, including water hyacinth duckweed and green algae. The blue–green algae
have been discovered. V. cholerae is found in Anabaena variabilis, which serves as a
reservoir.
Similarly, the recent isolation of toxigenic V. cholerae O1 from coastal waters in
the United States and Australia, where cholera cases were unknown, suggests that
these species have adapted to the aquatic environment as a natural microhabitat.
300 P. Sudhakara et al.

Although cholera is a watery illness, new evidence suggests that food, particularly
seafood, plays an essential role in cholera transmission. Foods are likely to be fecally
contaminated during preparation, primarily if the diseased food handlers work in an
unsanitary setting. High moisture content, neutral or alkaline pH, low temperature,
high organic content, and lack of other competing bacteria are physicochemical
features of foods that promote V. cholerae O1 survival and development. Seafood,
such as fish, shrimp, crabs, oysters, and clams, have been blamed for cholera
epidemics in various nations, including the United States and Australia.
Dirty rice, millet gruel, and vegetables have been linked to epidemics. Cholera
can be transmitted through various foods, including fruit (excluding sour fruit),
chicken, meat, and dairy products. Food must be made and consumed in a sanitary
environment free of feces to prevent foodborne cholera transmission hazards. To
reduce foodborne cholera transmission, proper food preparation, storage, and
reheating before eating and hand washing with clean water before and after defeca-
tion are beneficial health interventions (Sack et al., 2004; Parveen et al., 2008).

11.18.4 Yersinia enterocolitica

Effective health practices preventing foodborne cholera transmission include ade-

quate food preparation, storage, reheating before eating, and hand washing with
clean water before and after defecation. According to studies, the potential of
Yersinia enterocolitica to thrive and develop in chemically contaminated foods
under varied storage circumstances has been proven to be better at room temperature
and refrigeration than at moderate temperatures. Cooked meals allow
Y. enterocolitica to survive longer than raw foods, probably due to better nutrient
availability and the presence of certain psychrotrophic bacteria, including
non-pathogenic Y. enterocolitica strains. Bacterial growth in uncooked food can
be inhibited. Within 24 h or 10 days, a viable number of Y. enterocolitica will
increase by more than one million on cooked beef or pork at 25  C, or 7  C. Raw
beef and pork grow at a slower rate. Y. enterocolitica can grow at refrigerator
temperatures (soybean curd). Pork, cattle, lamb, chicken, and dairy products, partic-
ularly milk, butter, and ice cream, are all potential hosts for
Y. enterocolitica. Y. enterocolitica can be found in various terrestrial and freshwater
habitats, including soil, wetlands, lakes, rivers, wells, and streams, and can persist
for long periods in soil, vegetation, streams, reservoirs, wells, and spring water.
Infections with Yersinia pseudotuberculosis have been observed in various
domesticated or caged animals, including horses, goats, deer, and monkeys. In two
well-known Yersinia outbreaks, infection with human pseudotuberculosis has no
known etiology. Untreated drinking water infected with wild animal feces was
illegal (Fukushima et al., 2011).
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 301

11.18.5 Campylobacter

C. jejuni, C. colli, and C. lari have also polluted natural water surfaces. Consuming
unchlorinated, contaminated water or eating food prepared with untreated or
inadequately treated water might lead to infection.
In every corner of the globe, Campylobacter is one of the most common human
bacterial causes of diarrhea. Approximately 80% of Campylobacter infections are
thought to be caused by diet. Infection among young children is hyperendemic in
developed countries. Domesticated animals (chickens, pigs, goats, horses, dogs,
cats, and birds) are likely to get infected. Ingesting undercooked poultry, pork, and
beef, unpasteurized milk, contaminated drinking water, and infected animal excre-
ment can spread this virus to humans.
Campylobacter jejuni and C. coli invade domestic fowl with incredible ease. At
least 60% of chickens sold in supermarkets are contaminated with Campylobacter,
and broiler chickens are responsible for 50–70% of human infections in underdevel-
oped nations. The most common way to contract Campylobacter jejuni is by eating
undercooked chicken. Red meat, on the other hand, can be contaminated. Infection
can also be spread by eating barbecued pork or sausages. Infection can also be spread
by eating raw or undercooked fish, shellfish, or mushrooms.
In the recent past, raw and unpasteurized milk was a common source of Cam-
pylobacter and was responsible for large foodborne outbreaks. However, outbreaks
caused by raw milk have dropped significantly in developing nations due to the
regular pasteurization of milk and improved public awareness (Acheson & Allos,
2001).

11.18.6 Escherichia coli

Foodborne infections, or infectious diseases spread by food, are a bothersome and

sometimes life-threatening concern for millions of individuals. Drinking infected
foods or beverages causes foodborne illness. Several different microorganisms, or
disease-causing pathogens, can contaminate foods, resulting in various foodborne
diseases. More than 250 distinct foodborne diseases have been documented, with the
majority of them being infections caused by bacteria, viruses, and parasites.
The epidemiology of foodborne microbial illnesses has improved in the previous
10 years. It is ascribed not only to an increase in the human population’s vulnerabil-
ity to diseases or changes in lifestyle, such as more adventurous eating, fast food, and
less time spent preparing food, but also to the introduction of newly known
foodborne pathogens. Bowel infections are among the most prevalent infectious
disorders in humans (Kaper et al., 2004). The gastrointestinal tract is a specific target
for many foodborne viruses.
302 P. Sudhakara et al.

11.19 Aflatoxins and Aspergillus flavus

11.19.1 Aflatoxins

The aflatoxins are a collection of poisonous chemicals produced by structurally

related Aspergillus flavus, A. parasiticus, and A. nomius. Some strains of these
fungi can thrive on specific crops in the field and during storage under favorable
temperature and humidity circumstances, resulting in aflatoxins formation.
Approximately 18 aflatoxins: aflatoxins (AF) B1, B2, G1, and G2 are naturally
occurring aflatoxins, whereas the other 14 are animal-system metabolic products.
Naturally occurring aflatoxins are found in various foods; AFB1 is usually the most
prevalent in quantity and toxicity. A. flavus is the most frequent corn contaminant in
temperate zones around the world, and it produces both AFB1 and AFB2.
A. parasiticus is the most common peanut contaminant in tropical and subtropical
areas, producing all four naturally occurring poisons. Aflatoxin M1 (AFM1) is a
poisonous metabolite of AFB1 formed in mammals’ livers. Because aflatoxins are
very stable compounds, they can be detected in varying levels in finished foods
depending on the manufacturing methods or procedures utilized. Aflatoxin levels in
foods have been examined concerning various processing methods.
The occurrence of aflatoxins in food and feed is extensively documented in the
literature, with the most widespread exposure happening in humidified and hot
environments. Aflatoxins (except AFM1) have been discovered in corn and maize
products, peanuts and peanut products, cotton, various tree nuts (e.g., Brazil, pista-
chio, almonds, pecans, and walnuts), copra and its products, edible seeds (e.g.,
watermelon, sunflower), wheat, sorghum, spices, rice, unrefined vegetable oils,
figs, and dried fruits). They were sporadically detected in other materials at very
low or negligible rates (Richard et al., 2003).

11.20 Fusarium and Fumonisins: Toxigenic Fusarium Species

11.20.1 Toxins Produced by Fusarium Species

Fusarium is a fungus that causes plant disease and is commonly found in soil.
Because they infect plants in the field rather than in grain storage, Fusarium species
infect cereal grains, known as “field fungus.” As of 1996, more than 60 species of
Fusarium have been identified in raw and cooked foods worldwide, with more than
100 secondary toxigenic metabolites characterized. Variations in mycotoxin-
producing potential have been identified between strains within the same species.
Detailed assessments of the various presence of the numerous mycotoxins produced
by the Fusarium species have recently appeared in the literature. The most potent
poisons to Fusarium include deoxynivalenol (vomitoxin), fumonisins, and
zearalenone from human exposure and regulatory concern.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 303

11.20.2 Other Molds and Mycotoxins

Most fungal species found in soil are susceptible to invasion by field crops. Many
fungal species can infect growing plants and produce secondary poisonous
compounds termed mycotoxins under ideal temperature and humidity conditions.
Some fungal species can multiply as plants and grain kernels age, producing
poisonous compounds that are not eliminated or destroyed during harvesting.
Assume that harvested grains (crops) are not stored properly. In that instance,
some fungus on the crops brought in from the field may continue to thrive and
create mycotoxins, or they may eventually be replaced by fungi that may multiply
under different storage circumstances and produce new mycotoxins. As a result,
mycotoxins can infiltrate the food supply throughout preharvest and postharvest
periods. Small amounts of these compounds in foods and feeds can be permissible,
as long as the amounts are not considered detrimental to human and animal health.
Part of the goal of this chapter is to provide an overview of the significant
mycotoxins that are causing significant public health problems at both the national
and worldwide levels. Aflatoxin A, deoxynivalenol, fumonisins, patulin, and
zearalenone are the most commonly regulated mycotoxins (Zinedine et al., 2007).

11.21 Foodborne Viruses

11.21.1 Caliciviruses

Caliciviruses are an enteric virus family that infects a variety of marine and terrestrial
organisms. While there are three genogroups of human caliciviruses, there are two
distinct physical and biological forms of human viruses: classic human caliciviruses
and Norwalk-like viruses. These viruses are known as small round standardized
viruses (SRSVs) in the literature. While classic calicivirus strains have been shown
to induce gastroenteritis in adults, classical caliciviruses are primarily associated
with self-limiting gastroenteritis in newborns and babies. Traditional caliciviruses
account for just a small percentage of instances of infantile gastroenteritis. As a
result, traditional caliciviruses are generally considered less medical and foodborne
concern than the NLV.
Multiple genotypes are occasionally discovered during outbreak investigations.
This common group infection goes undiscovered until an infected person
contaminates a typical food or water source or fosters rapid human-to-human
transmission through intimate contact with people in a closed or institutional setting.
Contact with dirty linens, vomitus, excrement, aerosols, or fomites can direct person-
to-person transmission in these conditions. NL disease outbreaks have been reported
at banquets, geriatric institutions, mental units, emergency rooms, cafeterias, recrea-
tional lakes, swimming pools, dormitories, campgrounds, hotels, schools,
restaurants, and cruise and navy ships (Scharff, 2012).
Although mussels are well known for spreading NL illness, other foods have also
played a role in the disease’s transmission. Viruses are commonly transmitted
304 P. Sudhakara et al.

through raw fruits and salads. Surface contamination of fruits and vegetables can
occur due to irrigation or fertilization, harvesters or transporters, or food preparation
contamination. Sick food handlers have contributed significantly to the spread of NL
illness by contaminating foods with unwashed hands or exposing objects to unsani-
tary surfaces. One illness outbreak in Newfoundland was caused by mixing potato
salad in a sink where a restaurant worker had vomited the day before. According to
epidemiological studies, many NL disease outbreaks are caused by a general lack of
awareness among some food handlers due to inadequate sanitation and hygiene
training.

11.21.2 Hepatitis

Hepatitis A and E are the only hepatitis viruses spread through the feces–oral route;
the others are primarily transmitted through parenteral routes. Hepatitis A virus
(HAV) is transmitted chiefly through fecal–oral, direct person-to-person contact,
and contaminated food and drink. For example, HAV is sexually transmitted,
particularly among homosexuals, and has been documented on multiple occasions
via blood products, particularly factor VIII. The hepatitis E virus is spread through
contaminated food and infected water. Except for vertical transmission, there is no
known parenteral transmission of the hepatitis E virus.

11.21.3 Hepatitis A Virus

Virus-related disease burden varies substantially around the globe and is directly
influenced by socioeconomic growth and sanitation rates. The disease is more
prevalent in small and high endemic countries; in the former, infection and disease
are uncommon due to low virus circulation in the population due to adequate
cleanliness and sanitation. In countries with high endemicity, on the other hand,
the virus circulates widely. As a result, infection is common in young children, often
asymptomatic and unnoticed, contributing to long-term safety. This is true in areas
of intermediate endemicity, or countries with the highest disease rates, where
developed and developing people coexist (Mesquita et al., 2011).

11.21.4 Hepatitis E Virus

The development of hepatitis A and B viral serological tests revealed that neither
virus was responsible for most hepatitis cases. This resulted in the coining of the
term non-A, non-B hepatitis, which had two epidemiologically distinct variants: a
parenteral type with transmission routes similar to parenterally transmitted hepatitis
B virus infection and an enterically distributed non-A, non-B hepatitis with trans-
mission routes similar to hepatitis A virus infection.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 305

On the Asian continent, enterically non-A transmitted non-B hepatitis was

responsible for enormous epidemics of watery hepatitis. Serological investigations
revealed that the etiologic culprit was a new virus unrelated to hepatitis A. The
infection was significantly more widespread than the geographic location of the
significant waterborne epidemics suggested. In addition to outbreaks, the virus,
commonly known as the hepatitis E virus (HEV), caused sporadic instances of
hepatitis among indigenous populations and imported cases from travelers in devel-
oping countries.
The virus is present at lower concentrations in the blood, up to 100,000 particles
per mL, and the viraemic stage is mainly associated with the time when the virus is
expelled in the feces. It can now explain why the number of blood-borne hepatitis A
infections is increasing. It has not been identified whether the virus secreted in saliva
has a function in transmitting the hepatitis A virus. No evidence is found that sharing
dining utensils, cigarettes, or kissing can spread hepatitis A infection.
The fecal–oral route is the most common hepatitis A virus transmission, with
person-to-person transmission being the most fundamental mode of virus transmis-
sion. The findings of infection rates among patient household contacts and children
in the surroundings of daycare centers exemplified this point.
The virus is hardy, and it can survive in water and food for up to 10 months. As a
result, foodborne outbreaks (common-source epidemics) are becoming more wide-
spread. Food handler contamination—generally a food handler in the disease’s
asymptomatic pre-icteric stage—can cause foodborne outbreaks. It will mostly
show up in those who do not wash their hands properly and handle raw foods like
salads, sandwiches, and cold meats. Foods may be polluted, particularly shellfish
from water near sewage outlets or vegetables fertilized with untreated human night
soil. Drinking or bathing in filthy fecal water can potentially result in severe
waterborne outbreaks.
There is no doubt that improvements in clean water and sanitation and personal
and public hygiene have considerably impacted the global epidemiology of hepatitis
A infection. Endemic transmission has ceased in several highly developed countries,
such as Scandinavia, Germany, Switzerland, and Japan. Imports or travelers
returning from underdeveloped nations account for most illnesses in these countries.
HAV antibodies are present in less than 10% of blood donors in these countries. In
most other affluent countries, endemic viral transmission is also growing, resulting
in increased vulnerability in adults and higher morbidity and mortality (Scharff,
2012).

11.22 New Technologies for Microbial Detection

Some of the new techniques for microbial detection are ELISA, Lateral Flow
Immunoassay (LFIA), colloidal gold label-paper-based dipstick, and quantum dots
labels. ELISA has better sensitivity and lesser time consuming compared to culture-
based technique. The LFIA technique is a simple, rapid, feasible, and potent
foodborne pathogen detector. Other novel techniques to detect the antibodies are
306 P. Sudhakara et al.

optical strategies—gold nanoparticles combined with Raman spectrum molecules,

label-free method for pathogen detection, and electrochemical strategies—high
sensitivity, feasible, and fast compared to optical strategies (Jyoti et al., 2011).

11.23 Microbial Biofilms and Food Safety

Biofilm is formed by surplus microbial communities based on food fabricating

conditions and the colonizing species. Combined biofilms possess higher resistance
to the anti-biofilm compounds like disinfectants and biocides (Giaouris et al., 2015).
Biofilms are made of extracellular matrices, and those matrices are formed of
polysaccharides. These matrices can attach to hard surfaces like equipment in food
industries. Overall, biofilm formation helps the microorganism associated with the
food industry resists chemical protection and physical and mechanical resistance
(Wingender et al., 2016). The most crucial part of biofilm in the food industry is that
most microbes that form biofilms in the food industry are human pathogens, and they
get attached to the surfaces of food industry equipment (Colagiorgi et al., 2017).

11.24 Biofilm in the Food Industry and Health Defects

Foodborne illnesses depend on the biofilm formation on the surface of the food
industry. The occurrence of biofilm changes the type of the food industry, such as
pipes, water, animal carcasses, dispensing tubes, and packing materials (Camargo
et al., 2017). The five critical foodborne pathogens that can form biofilm are Bacillus
cereus, Escherichia coli, Listeria monocytogenes, Salmonella enterica, and Staphy-
lococcus aureus.

11.25 Techniques for Controlling the Formation of Biofilm

in the Food Industry

Biofilm detection is incompatible with the classic culture-based approach. It takes

time, and few bacteria are viable but not culturable (VBNC) because they have
limited metabolic activity. The biofilm produced by VBNC-PCR amplification is
used to detect it. Biofilms can also be detected via metagenomics and meta-
transcriptomics. The previous approach of biofilm detection is being replaced with
an alternate method for improved efficiency due to the time consuming and higher
reactive equipment.
Furthermore, these old technologies are unsuitable for use in the food business.
As a result, online monitoring is employed. Electrochemical detection and vibra-
tional detection are two different approaches. Controlling biofilm in the food busi-
ness can be done in various ways. These approaches include cleaning and
disinfection, clean-in-place, chemical-based controls, ultrasonication, enzymatic
disruption, phage, and hurdle technology.
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 307

11.26 Food Chain—Safety and Quality

As the distance between the producer and the consumer has grown due to globaliza-
tion, one of the significant issues is supplying quality food without contamination.
Food traceability is an essential criterion in ensuring food safety and quality.
Globalization has posed a challenge not only to food safety but also to food quality.
Consumers want to know everything there is to know about retailing history.
Traceability is crucial in this case. Traceability is based on unit identification,
product tracking, and data storage principles. The importance of food in the country
cannot be overstated. Food production is a global phenomenon that influences social,
economic, and environmental factors. Assume that any food-related difficulties will
impact the country’s socioeconomic condition. It has an impact on the environment
because of increased transportation carbon labeling. Food providers have recently
relied on two techniques to assure global food safety: managing the food chain by
adhering to regulations and standards and regulating logistics through traceability.
FAO and WHO have a significant role in food safety, FDA legislation, and Interna-
tional Standardization Organizations (ISO) participating in the international food
trade. Analyzing the risk Hazard Analysis and Critical Control Points (HACCP) is
essential for maintaining food safety standards. After HACCP, good manufacturing
procedures are implemented. Many rules and regulations were in place to assure
food quality and safety across the food supply chain (Aung & Chang, 2014).

11.27 Methodologies for Improved Quality Control Assessment

of Food Products

In these decades of higher technological development, the improvement in the food

analysis techniques will have greater insight. Analysts and chemists will have greater
value in using novel analytical tools. Traditional tools are more time consuming and
require skilled labor, so the demand for novel analytical tools has increased. Tradi-
tional high-performance liquid chromatography system analyzes the hydrophobic
and hydrophilic constituents in reversed phase and the normal phase. However, the
conventional method is less susceptible and time consuming. So, to analyze the
higher and lower molecular weight analytes, novel polymer-based stationary phase
is introduced. Capillary electrophoresis that discrete the analyte based on the molec-
ular weight and ionic moiety is introduced as an alternative. There is a higher
technique named capillary electrochromatography which is better than HPLC and
capillary electrophoresis (Huck et al., 2000). Spectroscopic strategies at this phase of
the systematic technique either incorporate mass spectrometry (MS) or vibrational
spectroscopy individually. Laser desorption ionization determines higher molecular
weight samples (Feuerstein et al., 2006). Vibrational spectroscopy is used for
detection.
308 P. Sudhakara et al.

11.28 New Developments in Food Quality Assurance and Safety

Control

The essential aspects of food are maintaining food safety and food quality. There
were numerous techniques to detect food safety primarily. We rely upon the
traditional method, which is time consuming and high cost. Then after several
findings in the 1960s, an electronic nose was introduced, which will mimic the
human olfactory system with its sensors. There were many factors for accepting and
rejecting the food and one among the important factor is the aroma, the smell of the
food. Aroma plays a crucial role in food. Aroma acts as a biomarker to test food
contamination. Few biosensors are listed in Table 11.6.
The food industry is benefiting from significant progress in developing enzymatic
biosensors with various transduction systems that can be used in food safety, quality,
and process control; studies are primarily focused on determining the composition
and contamination of raw materials and processed foods. The food industry is
benefiting from significant progress in developing enzymatic biosensors with vari-
ous transduction systems that can be used in food safety, quality, and process
control; studies are primarily focused on determining the composition and contami-
nation of raw materials and processed foods.

11.29 Conclusion

Intense research is ongoing to find a solution to the global food crisis. Enzyme
assimilation is well known in the food processing industry. This chapter addresses
the fundamentals of enzymes and the sources of various enzymes and their
applications in the food industry. The intrinsic features of microbial enzymes have
rocketed them to renown due to their simplicity of synthesis and yield manipulation
in laboratory settings. Enzymes in food processing convert physiochemical pro-
cesses to environmentally friendly processes, improve product biodegradability, and
reduce energy usage. The food processing industry could benefit from a new
approach for enzyme uses. Foodborne infections also harm human health and create

Table 11.6 E-nose in accordance with food (Rayappan et al., 2017)

Food E-nose
Meat NST 3210 emission analyzer, along with the ANN prediction algorithm,
Heracles II electronic nose, Artificial Olfactory System (AOS) ISE Nose
2000, Total Volatile Basic Nitrogen (TVB-N), Cyranose-320
Milk BH-114: Bloodhound Sensors Ltd., Applied Sensor 3320
Fish and Colorimetric array of sensors
seafood
Fruits and FOX 4000
vegetables
Adulterants Pen 2
11 Enzymes as a Tool in Food Analysis and Foodborne Pathogen Detection 309

financial losses. As a result, rapid diagnosis of foodborne pathogens and the appli-
cation of countermeasures are critical. Preventing foodborne illness requires quick
detection of the pathogen. Enzymes can be used as biosensors to detect food
adulteration and contamination for faster food product screening. The dietary sub-
strate determines the enzyme biosensor’s characteristics. Molecular diagnostics and
serological methods are two new technologies for food sector monitoring.
While there are various methods for detecting food safety, the most common is
the time consuming and expensive method. The electronic nose stimulates the
olfactory system of humans using sensors, which is a breakthrough. Many aspects
influence whether or not a person accepts or rejects food. One of the most crucial
factors is the aroma of the dish. In food, the aroma is significant. The aroma as a
biomarker can detect the contamination in food. With the expanding dimensions of
the food industry, several critical procedures such as food preservation, monitoring,
adulteration, and foodborne diseases are critical to the industry’s overall perfor-
mance. Furthermore, enzymes perform multiple roles in all processes, one of which
is the constant monitoring of food and food products.

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Enzymes in Flavor Development and Food
Preservation 12
Fataneh Hashempour-Baltork and Parastou Farshi

Abstract

Flavors and fragrances have extensive application in the pharmaceutical, cos-

metic, feed, chemical, and food sectors. Most of the flavor compounds on the
market are formed from animal and plant sources, through chemical synthesis or
extraction; however, a prompt alteration for the bio-production and use of flavor
compounds with (micro) biological origin—bioflavors—is occurring. Enzymatic
flavor production is commonly preferred over fermentation due to their high
yields of production. On the other hand, it is undeniable that food is important
for mankind and there is not any way of living without eating. Although the need
to feed has not changed during the years, the way of food preservation has seen a
lot of changes in today’s global market which is highly competitive, and it is
desirable to use the cheapest method of food preservation, such as food additives
which can be a good choice. Among food additives, enzymatic process for
longitude shelf life is one of the suitable choices. Using of bioprocesses involving
enzymes in food processing can decrease the antinutritive factors and toxins of
the by-products, besides solving the environmental issues, and increasing their
consumer acceptance by enhancing their nutritive value. Although, it is important
to select the proper fermentative processing technologies.

F. Hashempour-Baltork
Halal Research Center of IRI, Iran Food and Drug Administration, Ministry of Health and Medical
Education, Tehran, Iran
P. Farshi (*)
Food Science Institute, Kansas State University, Manhattan, KS, USA
e-mail: pfarshi@ksu.edu

# The Author(s), under exclusive license to Springer Nature Singapore Pte 317
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_12
318 F. Hashempour-Baltork and P. Farshi

Keywords

Enzyme · Flavor · Food preservation · Shelf life · Fermentation · Bioflavor ·

Ecofriendly

12.1 Enzyme in Flavor Development

12.1.1 Introduction

In the modern food industry, different types of enzymes have been applied for the
synthesis of important food additives, food properties, and development of food
production processes. Flavor and fragrances have found wide application in the
food, feed, cosmetic, chemical and pharmaceutical sectors. Most of the flavor
compounds in the market are formed from animal and plant sources, which takes
place through chemical synthesis or extraction; however, there is a quick alteration
for the bio-production and use of flavor compounds with (micro) biological origin,
namely, bioflavors. Chemical synthesis usually results in an unfavorable and
environmentally unfriendly production process of racemic mixture compounds.
Moreover, the consumer has established a “chemophobia”-approach for synthetic
or chemical compounds, specifically for food and products being used in the home.
The definition of natural flavors by EC Flavor Directive (88/388/EEC), is “flavoring
ingredients or formulations which are achieved by suitable enzymatic,
microbiological, or physical processes from plant or animal origin materials”
(Vandamme & Soetaert, 2002). Until now, animal and plant sources were considered
to be essential sources of bioflavors; however, the amount of these compounds is
usually very low in these sources or they are only present in exotic (plant) species,
which results in their expensive formulation and isolation (Carocho et al., 2015).
However, nowadays biotechnology is considered as a preferred method with the
advantage of “natural” labeling that can draw consumers’ attention. De novo micro-
bial production (fermentation) and enzymatic synthesis are two biotechnological
ways to obtain bioflavors. Enzymatic flavor production is commonly preferred over
fermentation due to their high yields of production (Asunción Longo & Sanromán,
2005; Vandamme & Soetaert, 2002).
Enzyme immobilization and coenzyme regeneration techniques can lead to an
effective and exclusive biocatalytic processes for flavor synthesis (Kragl et al.,
1996). The benzaldehyde with almond and cherry taste can be made from the
cyanogenic glycoside amygdalin, which is present in almond meal and cherry
kernels, by mandelonitrile lyase and b-glucosidase enzymes (Fig. 12.1). An indus-
trial example for this is L-menthol production which is the major ingredient present
in peppermint oil (Fig. 12.2) (Menzel & Schreier, 2007).
Recently, synthesis of flavor compounds by biotechnological processes has
gained growing importance in the food industry. By considering the growing market,
and the growing public concern for the total wholesomeness and chemical safety of
food ingredients, researches on bioprocesses and inventing new products are also
12 Enzymes in Flavor Development and Food Preservation 319

Fig. 12.1 Enzymatic route to benzaldehyde

Fig. 12.2 Stereoselective bioconversion of DL-methyl acetate into L-menthol

increasing (Basset, 1990). In 2017, the total global flavors and fragrances market got
to US$28.2 billion, which means an increase of 4.6% rather than the previous year,
and it is estimated to increase at 4.9% average annual rate to get to approximately
US$36 billion in 2022 (Consultants, 2018). A wide range of options are provided by
enzymes, for food flavor production. The specificity of the enzymes and whether
they are being used through whole-cell or cell-free systems support the production of
chemicals that are hard to synthesize; also their stereoselectivity is considered as a
principal advantage for the food industry where a particular optical conformation can
affect flavor properties. Application of enzymes as food additives can produce or
liberate flavor from precursors, as well as correcting off-flavors produced by specific
naturally occurring compounds, or those that are produced during processing
(Bigelis, 1992). Besides intentional addition of enzymes to the food, they can also
be inherent to the food or may originate from microbial sources, or may come from
contamination.
In this chapter, several ways that enzymes can have effect on flavor are reviewed,
showing examples of actual research, their potential applications, as well as
examples of the fundamentals of biosynthesis and the market potential of some
products. In some instances, processes and reactions known for two decades are
stated and they are updated with recent developments, such as reaction in organic
media or whole-cell biocatalysts.
320 F. Hashempour-Baltork and P. Farshi

12.1.2 Enzymatic Synthesis of Flavor Compounds

12.1.2.1 Lyases

Alliinases
Alliinases [S-alk(en)yl-L-cysteine sulfoxide lyase; alliin lyase; EC 4.4.1.4] remove
S-alk(en)yl-L-cysteine sulfoxides, and in this way they produce pyruvate, ammonia,
and thiosulfinates, (Fig. 12.3). Pyridoxal-50 -phosphate (PALP) is needed by this
enzyme as a cofactor.
The strong aroma and flavor of the most of the species of the Amaryllidaceae
family are because of the activity of this enzyme. Among other lyases, this enzyme is
detached from the substrate in the intact tissue, and the reaction occurs upon
breakdown of cells. The first alliinase to be explained was from garlic bulbs (Tishel
& Mazelis, 1966). Until now alliinases have been purified from onion, Welsh onion,
Chinese chive, garlic, and leek (Whitaker et al., 2002). Despite that they are from the
same genus, their specific alliinases are different in physicochemical properties. It is
many years that garlic and onion are being used by several cultures for different
purposes. In spite of having therapeutic and nutritional importance in diets, they are
being consumed around the world due to their unique flavor and their properties,
such as enhancing the flavors of other foods. Diallylthiosulfinate (allicin) is the
major sulfur compound in garlic that provides its distinctive aroma. This
thiosulfinate results from the elimination reaction by alliinase which is done on
S-2-propenyl-L-cysteine sulfoxide (alliin), via the intermediate 2-propenesulfenic
acid. There are three flavor precursors in onions, such as S-methyl-L-cysteine
sulfoxide, S-propyl-L-cysteine sulfoxide, and S-1-propenyl-L-cysteine sulfoxide.
Different thiosulfinates made by the activity of alliinase on the mentioned substrates
impart distinctive onion flavors, based on their concentrations (Masuda et al., 2012).
Other Allium spp. have diverse combinations of these substrates in addition to S-2-
propenyl-L-cysteine sulfoxide; this compound is not present in onion (Masuda et al.,
2012). It is stated that the lachrymatory factor in onion (syn-propanethial-S-oxide) is
originated from S-1-propenyl-L-cysteine sulfoxide (Jayathilaka et al., 2014).
Alliinases do the primary reaction which is responsible of producing distinctive
aromas and flavors in vegetables of the Allium genus. The maintenance of full
enzymatic flavor is the principal challenge of food processors who deals with

Fig. 12.3 Alliinase general reaction

12 Enzymes in Flavor Development and Food Preservation 321

onion and garlic. Some preharvest and postharvest practices, such as environmental
factors influencing the flavor strength, including growing temperature, water supply,
and sulfur content of soil, are monitored in alliaceous foods to enhance their flavor
potential (Kamenetsky, 2007). Flavor potential of onions goes up with storage of its
bulb for a few months (Petropoulos et al., 2017). Nearly, half of the S-1-propenyl-L-
cysteine sulfoxide is bound as a peptide, L-glutamyl-S-allyl-L-cysteine sulfoxide, in
nature. It has been stated that the substrate is liberated by the activity of glutamyl
transferase. The most part of the flavor potential of the onion is lost during dehydra-
tion, which is due to the loss of the substrate more than the enzyme. In the other word
30% of the alliinase substrate is changed to cyclo alliin during the primary phases of
dehydration.
Cyclo alliin and the S-alkyl-L-cysteines are not substrates for alliinase. The
altered onion powder includes only one-third of the onion aroma of fresh onion
juice (Ramírez & Whitaker, 1999). Meticulous consideration of the speed of drying
is critical throughout the design of the dehydration process to diminish the substrate
loss; process with longer time normally leads to better yields. The activity of
alliinase can also lead to the development of unnecessary pigments in alliaceous
vegetables, such as pink pigments in onions and leeks, and green pigment in garlics
(Whitaker et al., 2002). Despite the ability of alliinase to catalyze the preliminary
reaction and thiopropanal-S-oxide formation (in onion), the formation of pigment is
dependent on the concentration of carbonyl and may differ from pink to red based on
the composition.

Cystine Lyases in Plants

Cystine lyases (cystathionine L-homocysteine lyase; EC 4.4.1.8) is able to cleave
L-cystine through generating pyruvate, ammonia, and thiocysteine or cysteine
persulfide (Fig. 12.4). The substrate for this enzyme is not exactly determined.
Cysteine lyase and cystathionase are both found in fungi and bacteria. However,
cystine lyase does not consider cystathionine as a substrate, in plants. The activity of
this enzyme was first identified in plants by Tishel and Mazelis (1966). They studied
the capability of homogenates of cabbage leaves for degrading L-cystine (not
L-cystathionine), to pyruvate. Also it is stated that this enzyme needs pyridoxal-50
phosphate (PALP) as a cofactor.
Cystine lyases have importance for food quality. These enzymes catalyze the
preliminary reaction in important vegetables of the Brassica genus, producing
distinctive aromas and flavors. Cruciferous vegetables, such as cabbage, cauliflower,
Brussels sprouts, and broccoli, have specific sulfurous flavors and aromas which

Fig. 12.4 General reaction of cysteine lyase

322 F. Hashempour-Baltork and P. Farshi

results from the activity of the mentioned enzymes and subsequently tissue softening
happens in these vegetables. Cystine lyase was identified to be the key enzyme in
broccoli, and its activity results in off-aroma decay (Barrett et al., 2000).
In a study, addition of the purified cystine to blanched broccoli was observed and
the enzyme-added broccoli could produce the aroma characteristic of the unblanched
broccoli. Chin and Lindsay (1993) determined the sulfur compounds produced in
damaged tissues of cabbage as dimethyl disulfide (DMDS), dimethyl trisulfide
(DMTS), and methanethiol (MT). Although these compounds are specific to brassica
vegetables, they are unfavorable for the consumer. These unpleasant sulfurous odors
make people to avoid consuming them, especially in the use of modified
atmospheres. The mechanisms of the formation of these compounds in cabbage,
which results from the activity of cystine or cystine sulfoxide lyase, were clarified
(Chin & Lindsay, 1994a). Researchers could not find significant differences between
the DMDS formation in anaerobic and aerobic circumstances; however, DMTS was
only identified in anaerobic conditions. Different treatments, such as soaking the
vegetable in caraway (Carum carvi) seed extract, ascorbic acid (500 ppm), phospho-
ric acid (0.1 M), sodium hydroxide (0.01 M), and tertiary butylhydroquinone
(TBHQ), have been done to decrease the levels of these undesirable volatiles in
broccoli stored in modified atmosphere condition (Chin & Lindsay, 1994b). All of
the mentioned treatments could decrease the intensity of sulfurous aromas, in which
alkaline treatment had the highest effect that could suppress the formation of DMDS.
Obenland and Aung (1996) discovered that the formation of MT and DMDS in an
intact broccoli flowerets can be nearly removed by infiltration of hydroxylamine
which is an inhibitor of cystine lyase, and can be increased 2.8 times by infiltration of
S-methyl cysteine sulfoxide before the anaerobiosis.

12.1.2.2 Isomerase

Xylose (Glucose) Isomerase

High-Fructose Corn Syrup (HFCS) is a combination of fructose and glucose made
from cornstarch (Parker et al., 2010). HFCSs have extensive use in food industry and
the most substantial application is their addition to soft drinks as a sweetener where
they substitute cane and/or beet sugar. HFCs have 10–20% lower price than sucrose,
in the same levels, and have less calorie due to the lower absorption of fructose.
Glucose and fructose in HFCS have better solubility rather than sucrose and this
accounts for their higher advantage to sucrose. Therefore, they have lesser affinity
for crystallization in a broad range of food products. This feature has made them
suitable compounds for application in different products, such as meat products, ice
cream, canned products, confectionery, bakery, jam and jellies, sauces, and pickles
(Parker et al., 2010).
The process for producing HFCSs includes three continuous enzymatic steps.
Starch which is primarily originated from corn is used as the raw material in this
process. Starch is consisted of amylose (linear) and amylopectin (branched) which
are both polymers of D-glucose. The glucose moieties are linked by 1,4-glycosidic
12 Enzymes in Flavor Development and Food Preservation 323

bonds in amylose and the main chain of amylopectin. However, the branches are
linked by 1,6-glycosidic bonds.
The starch which is produced from corn is exposed to liquefaction, in the first step
of enzymatic process, by application of a thermostable bacterial amylase
(EC 3.2.1.1) from Bacillus spp, such as B. stearothermophilus and
B. licheniformis. In this step, enzyme is able to produce a dextrin solution with a
dextrose equivalent (DE) of 10 by cleaving the 1,4 linkages in an endo fashion.
In the second step, other enzyme named as glucoamylase (amyloglucosidase; EC
3.2.1.3) releases single glucose moieties from the nonreducing end by hydrolyzing
the dextrins in an exo fashion.
Glucoamylase shows a higher activity for the 1,4-glycosidic bonds rather than the
1,6-branch bonds. Moreover, in some situations a debranching enzyme, such as
pullulanase (EC 3.2.1.41), is also used and the produced glucose solution has a DE
of 95.
In the next step of enzymatic process, other enzyme named as glucose isomerase
catalyzes the conversion of glucose into fructose until a concentration of 42% (molar
fraction based on total sugars). This reaction is exclusive because it is taking place in
a column reactor using immobilized form of the enzyme. Glucose isomerase was one
of the earliest enzymes used for this purpose on an industrial scale. This process is
done by encapsulating the enzyme in a packed-bed column reactor with 1- to 2-mm
particles (Ruiz-Matute et al., 2010). The diameter and height of the columns may be
1.5 m in and 4–5 m, respectively. This system has a high yield which produces
500,000 kg fructose (dry weight) per day by 4000 kg enzyme. High-glucose syrup
with DE ¼ 95, resulted from previous glucoamylase reaction with 45% solids and
pH adjusted to 7.5, is continuously pumped through the column. Several factors
have effect on the temperature and flow rate of the system. The flow rate should be
suitable to provide enough residence time for production of 42% fructose. Low flow
rates give rise to the risk of microbial contamination, also extremely high rates do not
provide enough conversion and result in channeling through the support material.
Temperatures below 55–60  C increase the syrup’s viscosity and increase the risk of
microbial contamination. However, higher temperatures decrease the stability of the
enzyme and results in the production of unfavorable by-products via chemical
reactions such as Maillard reactions.
Practically, temperatures of 55–60  C are being used. Regulation of the glucose
stream’s flow rate can compensate for the enzyme’s inactivation to maintain the
required conversion. These processes can continue for several weeks, under the
mentioned circumstances until the activity of the enzyme has become too low. After
getting to that point, the columns are disassembled and packed again with a batch of
fresh biocatalyst. The final HFCS is consisted of sugars, such as oligosaccharides
(up to 4%), fructose (42%), and glucose (54%) (Parker et al., 2010; Ruiz-Matute
et al., 2010).

12.1.2.3 Oxidoreductases
Oxidative and reductive processes play a significant role in foods. They affect many
aspects of food stuffs, such as taste, shelf life, texture, nutritional value, and
324 F. Hashempour-Baltork and P. Farshi

appearance. Both nonenzymatic and enzymatic redox processes are included. Some-
times processes lead to detrimental effects, such as textural problems, off-flavor, and
decreased shelf life. In some cases, also, they provide positive effects to the final
aroma, a better texture, a more desired appearance, or an improved shelf life. It is
very important to control redox behavior throughout all stages of processing and
storage, in food systems. Redox reactions in foods were controlled primarily by
addition of antioxidants or chemicals, designing air-tight packaging materials, cau-
tiously choosing raw materials, or by adjusting process situations, until now. How-
ever, there is not much paid attention to redox reactions in foods by the addition of
oxidoreductases or by modifying the profile or changing the content of
oxidoreductases in raw materials of foods by genetic implements. Apparently, the
main reason for this little attention for using oxidoreductases in foods is that it is still
not possible to produce most of the enzymes, cost-effectively. Moreover, general
concerns about the application of recombinant enzymes in foodstuffs are indicating
their commercial introduction. Various oxidoreductases are included in the in situ or
in vivo biogenesis of appropriate aroma compounds, in vitro production of flavors
and top notes, and in the endogenous development of off-flavors. The present
applications of oxidoreductases for controlling the taste of food products are
provided below:

Lipoxygenases
Lipoxygenase (LOX), which was previously known as carotene oxidase or
lipoxidase, is a dioxygenase containing iron, which catalyzes the oxidation of
polyunsaturated fatty acids comprising of cis,cis-1,4-pentadiene groups
(arachidonic, linolenic, and linoleic acids) to the equivalent conjugated cis-, trans-
dienoic monohydroperoxides. Moreover, LOX has a broad range of substrates which
are phenolic compounds (Markus et al., 1991); also it is able to oxidize other
substrates rather than the actual substrate. This process is identified as
co-oxidation and involves compounds, such as polyphenols and carotenoids. LOX
is one of the endogenous enzymes that plays a significant role in off-flavor formation
and flavor generation and in almost all food products it is originated from raw
materials of plant. Based on the type of food product and final concentration, a
flavor compound can be a desirable aroma component or an off-flavor at higher
concentrations. For instance, alcohols and C6-aldehydes derived from oxidation of
(poly)unsaturated fatty acids, which are catalyzed by the LOX, mostly have a
positive impact on the aroma profile (such as in wines and juices), but in other
products, such as beverages (e.g., beer), does not have desirable effect on flavor.
Similarly, endogenous LOX is known to have impact on the bread flavor due to
generating carbonyl compounds in dough systems (Pico et al., 2015).
The C6 compounds’ formation involves the consecutive action of four enzymes,
including two redox enzymes, such as a LOX, and an acylhydrolase, a yeast-derived
alcohol dehydrogenase, and a hydroperoxide lyase (Whitaker et al., 2002).
Normally, the formation of off-flavor is inhibited by the means of LOX
isoenzyme-deficient crop variants, by genetic tools or by screening or controlling
the oxygen levels during processing, in addition to eliminating the putrid oxidation
12 Enzymes in Flavor Development and Food Preservation 325

products subsequently (Mandal, 2012). Physical techniques, such as adsorption, or

enzymatically altering the undesired alcohols or aldehydes to their equivalent car-
boxylic acid which are less flavorful, can be good ways to deodorize the off-flavors.
This can be done by aldehyde dehydrogenase/oxidase or alcohol oxidase. The
application of redox enzymes for this purpose has been proved for different food
products, such as fish oil, cream, noodles, soybean products, cooked rice, and
margarine (Raghuvanshi & Bisht, 2010; Antrim & Taylor, 1990).
Additionally, LOX is mostly applied to dairy products and beverages to modify
their flavor profile for in vitro production of numerous natural top note flavors.
Examples of these applications can be the conversion of polyunsaturated fatty acids
to delta-decalactone (butter flavor) (Tunick, 2007) or to several short to medium-
chain aldehydes/alcohols (Schwab et al., 2008; Scrimgeour et al., 2005). A well-
known flavoring derived from fatty acid (FA), amdaldehydes/alcohols, comprises
the above-mentioned C5 and C6 compounds, (E3, Z5, Z8)-undecatetraene (sea-
weed), 1-octen-3-one (field mushroom), (E3, E5)-undecatriene (balsamic), (E2,
E6)-nonadienal (cucumber), and (Z5)-octadien-3-one (geranium leaves).
The regioselectivity and unsaturation degree of the LOX determines the forma-
tion of various hydroperoxy compounds, from which consequently the mentioned
compounds can be derived by enzymatic reactions. Linolenic acid is being used as a
substrate for (Z3)-hexenol; however, for most of the other alcohols/aldehydes,
higher unsaturated fatty acids are needed. (Z3)-hexenol production has lately been
industrialized by means of plant homogenates, such as green peppers and alfalfa
sprouts, which are pretty rich in hydroperoxide lyase enzyme which is needed to
cleave the hydroperoxide fatty acid to more little fragments.
Reductive enzymatic power of baker’s yeast was responsible to transform (Z3)-
hexenal to (Z3)-hexenol (Ricard, 1991). After the linoleic acid hydroperoxide
formation, a unique strategy is needed to be followed for the production of lactones.
It includes the fermentative oxidation of the hydroperoxide intermediate by the yeast
Pichia etchellsii, and the consequent cyclization of 5-hydroxydecanoic acid to the
equivalent S(-)-decalactone (Laane et al., 1997).
Moreover, there are relatively extensive spectrum of phenolic compounds that
LOXs accept as substrates. Conversions of coniferyl benzoate and isoeugenol from
Siam resin into vanillin is within the interest of flavor industry. Presently, the
industrialization of these biotransformations is hindered due to the fact that
isoeugenol is not easily accessible and that coniferyl benzoate is hard to work within
a reactor (Priefert et al., 2001). Other reactions of LOX which are well known
involve the co-oxidation reaction of carotenoids to ionone. Also it is stated that
LOX and alpha-amylase combination can generate a toasted or cookie-like flavor in
starchy materials (Desjardins et al., 1997).

Alcohol Oxidases and Dehydrogenases

Generally, aldehydes are considered as stronger flavor compounds rather than their
alcoholic equivalents. Thus, alcohol oxidases are the enzymes which are of interest
for the in vitro production of flavoring compounds in beverages. Enzymes, such as
methanol oxidase from Candida, Pichia, and Hansenula, are good examples of
326 F. Hashempour-Baltork and P. Farshi

alcohol oxidases that are used for the formation of natural acetaldehyde from ethanol
(Lopez-Gallego et al., 2007). This enzyme is produced throughout growth on
methanol. Phase cells are collected and incubated with ethanol, at the final stage of
the logarithmic growth. Therefore, 1.5% natural acetaldehyde concentrations can be
reached, that can be further concentrated to the preferred application level. The
substrate specificity of the alcohol oxidase is different, in various yeasts. Hence, this
method can also be used to transform other alcohols, to their equivalent aldehyde.
Therefore, dehydrogenases can be used as substitutes to alcohol oxidases. However,
there is a limitation to use these dehydrogenases, because they need the costly
cofactor NAD(P)+instead of oxygen which is cheap, as an electron acceptor.
Although there are several designed complex NAD(P) cofactor stimulating systems
and significant cost reductions, it is obvious that oxidases are preferred in industrial
applications to their dehydrogenase equivalents. The application of dehydrogenases
for food purposes is limited to whole-cell transformations. There is a special kind of
alcohol oxidase, namely, vanillyl alcohol oxidase (VAO) which is derived from
Penicillium simplicissimum. Recently, it has been indicated that this enzyme, which
contains flavin and is relatively stable, has an extremely wide substrate specificity
and can convert para-substituted phenols into flavoring compounds or flavor
precursors (Ewing et al., 2018). In addition to the natural coniferyl alcohol and
vanillin, allylphenols and various vinylphenols, such as para-vinylguaiacol, can be
generated from cheap raw materials and oxygen (as an electron acceptor). VAO can
also be used for the production of building blocks of flavor. VAO can also be applied
to a natural mixture of phenolic compounds to enrich foods or flavor preparations
with a variety of aldehydic compounds and vinylic/allylic.

Sulfhydryl Oxidases
Formation of disulfide bonds from (protein) thiols is catalyzed by sulfhydryl oxi-
dase. This enzyme is a glycoprotein which contains Cu/Fe and has been detected in
goat, pig, human rabbit, bovine, and rat milks (Thorpe et al., 2002). The enzyme is
able to oxidize the cysteine and sulfhydryl groups of glutathione, and milk proteins
using molecular oxygen as electron acceptor, and transform them to their equivalent
disulfides (Faccio et al., 2011). SOX of bovine milk might be added to UHT milk for
reducing the cooked flavors. The enzyme has been immobilized on porous glass, and
its efficacy in improving the cooked flavor has been showed on a pilot scale through
the immobilized enzyme columns.

Peroxidases
Peroxidase (POX) happens extensively in nature and is the name which is generally
applied to a group of both nonspecific and specific enzymes which use hydrogen
peroxide as an electron acceptor. POXs, particularly the enzymes containing heme,
are able to catalyze a majority of various reactions, including N-demethylation,
oxidation, hydroxylation, and sulfoxidation; therefore, there is a special interest for
these enzymes in the production of specific flavoring top notes. Demethylation of
methyl N-methylanthranilate (ex Citrus) to mono-methylanthranilate is an example
of the application of these enzymes (Van Haandel et al., 2000). The mentioned
12 Enzymes in Flavor Development and Food Preservation 327

produced compound is the main top note flavor in Concord grapes. Horseradish,
microperoxidase, and soybean were found to be appropriate catalysts for this
reaction. Moreover, POX increases the fresh flavor profile when it is used in tomato
paste (Wilding & Woolner, 1997).

Polyphenol Oxidases
Polyphenol oxidase (PPO) enzymes are a group, including various enzymes with
different activities. Tyrosinase, a monophenol monooxygenase, is able to transform
a phenol into a catechol group. The other enzymes of this group, catechol oxidase or
1,2-diphenol oxidase (EC 1.10.3.1), converts catechol into an O-quinone, and
laccase is able to produce p-quinone from 1,4-diphenol. Generally, the first two
activities are related, since catechol is much more oxidized rather than a phenol. Also
most of the enzymes, that are able to catalyze the oxidation of 1,4-diphenol, can act
on 1,2-diphenols. In addition, guaiacols which are polyphenolic compounds impart
the bitter taste in several food products. PPOs are able to oxidize these compounds;
therefore, they can be used to decrease bitterness. There are several studies about the
application of these enzymes for this purpose, such as debittering of cacao beans
(Takemori et al., 1992), Adzuki beans (Okazawa et al., 1993), and coffee beans
(Small & Asquith, 1989). The other example of their application is the use of laccase
for debittering of olives (Charoenprasert & Mitchell, 2012).

12.1.2.4 Hydrolayses

Lipase and Esterases

Lipases and esterases are lipolytic enzymes that are able to catalyze the hydrolysis of
triglycerides which are primarily different in the activities on substrates. These
enzymes are different in substrates which can be in emulsion or solution forms.
Lipases show extremely greater activity on emulsified substrates and prefer long-
chain fatty acids; however, esterases indicate their activity on aqueous solutions of
short acyl chain esters (Arpigny & Jaeger, 1999). Ester synthesis is generally
catalyzed by enzymes, such as esterases and lipases, which can be expressed in
many food microorganisms. These enzymes are serine hydrolases and are able of
hydrolyzing or synthesizing esters based on the environmental conditions. Microbial
lipases are commercially important due to their better stability, availability, and
affordability rather than lipases from other sources (Molimard & Spinnler, 1996).
Generally there are following common types of lipases according to (Waldmann &
Parhofer, 2019): milk lipases; lipoprotein lipase; pancreatic lipases; hepatic lipases;
hormone-sensitive botanical lipases; and microbial lipases, including Candida and
Torulopsis yeast species, Geotrichum, Aspergillus, Rhizopus, Penicillium, and
Mucor mold species, Staphylococcus, Pseudomonas, and Achromobacter bacterial
species. Esters synthesized by lipase play a significant role in food industry as flavor
and aroma constituents. Synthesizing esters by lipase can be conducted in vessels in
batch or continuous condition and esters achieved from these sprouts have more
purity and low cost, rather than other chemical methods which require special
conditions and lead to redundant by-products production. However, low conversion
328 F. Hashempour-Baltork and P. Farshi

rate of these systems is their main drawback (Aravindan et al., 2007). Commercial
lipases are mostly used in dairy products, meat industry, baked foods, fruit,
vegetables, beer, and wine (Nagodawithana & Reed, 1993). Immobilized lipase on
silica and microemulsion-based organogels are extensively being applied for syn-
thesis of ester (Ghosh et al., 1996; Sharma et al., 2001). Lipases are typically highly
specific. For instance, in a study Larios et al. (2004) in which the synthesis of esters
in n-hexane was considered, it was found that lipase (fraction B) from Candida
antarctica indicates substrate specificity. This specificity involves both acids and
alcohols. It was shown that it has activity on unsaturated fatty acids and short-chain
fatty acids with linear and branched chain structures, and 2-phenylethyl, n-butyl,
isopentyl, and geraniol as alcohols. Generally enantiomeric selectivity of enzymes is
among their important properties for food aroma synthesizing. As an example, only
(S)-form of 2-methylbutanoic acid methyl ester has the fruity flavor which is the
main flavor compound of apple and strawberry. It is found that lipases from
Aspergillus niger, Rhizomucor miehei, and Aspergillus javanicus have the selectivity
for this flavor compound (Kwon et al., 2000). Moreover, in a medium without
solvent, lipase from Candida rugosa was selected for L-menthol esterification
with long-chain unsaturated fatty acids to regulate the strong flavor related to it,
whereas it was acted weakly on D-menthol (Shimada et al., 1999). However, some
lipases indicate only restrained enantioselectivity, like Candida antarctica lipase
(fraction B) selectivity for chiral short-chain carboxylic acids (Larios et al., 2004).
Generally, lower water activity medium is preferred for lipase activity, so solvents
with high hydrophobic activity are more favorable due to their insolubility in water
layer around the enzyme surface which is important for protection of their active
stereoconfiguration (Huang et al., 1998; Langrand et al., 1990). Additionally, enzy-
matic synthesis of esters can take place in systems without solvent. In these systems,
reactant itself (i.e., an alcohol) acts as a solvent. This method is desirable in food
industry, but because of the heterogeneity of the reaction, there are limitations, such
as mass transfer and consequently low reaction rate in these systems. Researchers
could find a new thermostable esterase derived from a marine mud metagenomic
library, with potential use in industry, which is named as EST4. It is reported that this
enzyme can efficiently produce several short-chain flavor esters under high
concentrations of substrate and in media which is nonaqueous (Gao et al., 2016).
Researchers found that thermo-tolerant strain, such as Bacillus licheniformis
(cloned in E. coli), has good ability to produce thermophilic lipolytic enzymes.
These enzymes can tolerate high temperatures and organic solvents, which indicates
their promising feature to use them for ester synthesis (Alvarez-Macarie & Baratti,
2000; Dominguez et al., 2004; Fuciños et al., 2005). In a study by Chang et al.
(2001) the capability of mutant and wild-type lipases from Staphylococcus
epidermidis to catalyze the formation of flavor esters was investigated in aqueous
phase. This lipase was shown to have strong specificity for geranyl esters, unsatu-
rated esters, and medium-chain esters. Seitz has stated that Debaryomyces klockeri
and Candida mycoderma yeasts can produce lipases that have the best activity at
pH 4.5 (Seitz, 1974). In another study it is shown that lipases from Penicillium,
Aspergillus, and Paecilomyces, which are responsible for the fatty acid profile of the
12 Enzymes in Flavor Development and Food Preservation 329

cocoa bean, show different activities and specificities which are useful when they are
applied to milk fat (Hansen et al., 1973).
Applications of lipolytic enzymes require good interdependence between the total
substrate composition and the enzyme activity. For instance, the activity of milk
lipase was as a result of the effect of the both stimulatory and inhibitory activity of
several milk components on the enzyme (Hansen et al., 1973). In a study by Baianu
et al. (2003), the free lipase derived from M. miehei could conduct the direct
esterification of geraniol and citronellol with short-chain fatty acids, which had
high yields in n-hexane. According to Sánchez et al. (2002), flavor esters, such as
hexyl esters, produced by lipases are green note flavor compounds that are widely
used as flavor and fragrance in different industries, including food industry.
Recently, there is an increasing requirement for natural flavors, including “green
note” characterized by hexanol derivatives. For instance, hexyl butyrate is of
particular significance as it characterizes a model of flavor ester. Shieh and Chang
(2001) have investigated the capability of immobilized lipase from R. miehei to
catalyze the transesterification of tributyrin and hexanol. They determined that
decreasing the amounts of tributyrin as a co-substrate would decrease the cost for
the production of the hexyl butyrate. However, for transesterification of hexanol with
triacetin with the same lipase, results indicated that substrate molar ratio and
temperature of the reaction were the factors with the paramount importance and
the added water content had less impact on the reaction.
In dairy industry lipases and esterases are regularly used to enhance the buttery
flavor of the final product. Lipases are mostly used to hydrolyze milk fat. By
modifying fatty acid chain lengths, lipases can enhance flavor of different kinds of
cheeses. Short-chain fatty acids, specifically butanoic acid, play an essential role in
dairy products’ flavor and different commercial enzymes which have different
specificities towards short-chain fatty acids are considered as flavoring tools
(Saerens et al., 2008). There are several studies about the cheese ripening accelera-
tion and fat, cream, and butter lipolysis by lipases (Ghosh et al., 1996; Sharma et al.,
2001). Lipase from pregastric glands, such as bovine and porcine, and pregastric
tissues of ruminants were initially used for enhancing cheese flavor.
Various kinds of cheeses can be produced by using a specific kind of microbial
source or mixture of them. To produce enzyme-modified cheese (EMC) usually
cheese is incubated with enzymes at an increased temperature and in this way a
concentrated flavor is produced which can be used in sauces, snacks, soups, and
dips. In these cheeses the concentration of fat is tenfold higher rather than normal
cheeses (Ghosh et al., 1996; Sharma et al., 2001). Indigenous or exogenous milk
enzymes can contribute to lipolysis. Exogenous enzymes can be either microbial
lipases and/or mammalian pregastric esterase lipases. This process is an essential
factor in flavor development of cheese. From different reviews on lipolysis in cheese,
it can be derived that lipolysis results in the FFAs formation which are considered as
major flavor compounds of cheeses and can be transformed to other flavor
compounds, such as acetoacetate, aldehydes, β-keto acids, lactones, methyl ketones,
esters, and secondary alcohols which are more effective and can directly affect flavor
in different kinds of cheeses (Collins et al., 2003). Lactic acid bacteria (LAB) are
330 F. Hashempour-Baltork and P. Farshi

used in cheese fermentation as starter adjuncts, starter cultures, or as secondary

microbial flora (Beresford et al., 2001; Crow et al., 2001). Moreover, Custry et al.
(1987) reported that methyl ketones are main group of compounds contributing to
cheese flavor and are originated from the FAs released by lipolysis reaction. A
secondary flora is needed for enzymatically oxidization of these FAs into methyl
ketones via β-oxidation. Additionally, the oxy fatty acids in milk fat can also be the
source of methyl ketones.
Development of cheese flavor is a complex process in which enzymes from
different sources are responsible for the degradation of proteins, carbohydrates,
and milk fat. Straight-chain carbons of C2 to C18 are the main FFAs in cheese
which have quite high flavor perception thresholds, leading to different flavor notes,
such as pungent, goaty, rancid, soapy, waxy, or cheesy notes (Collins et al., 2003).
However, the flavor perception thresholds of esters which are resulted from FFAs
with carbon chain of less than C10 is much lower, revealing a fruity flavor notes
which are pleasant for some people. Esters are also able to mask effect of off-flavors,
such as pungent and sharp off-flavors. On the other hand, high levels of ethyl esters
of short-chain FFAs can cause a fruity flavor defect in cheeses, such as Cheddar
(Bills et al., 1965; McGugan et al., 1975).
Esterases of LAB can hydrolyze aromatic esters, thioesters, and alkyl esters, as
well as hydrolysis of b-naphthyl or p-nitrophenyl esters of FAs, partially hydrolyzed
milk fat, and synthetic glycerides. It has been stated that esterases of Lb. casei,
Lc. Lactis, and Lb. helveticus are able to hydrolyze ethyl esters of FAs of C2 to C6,
acetate esters of hexanol, propanol, and butanol, as well as aromatic esters, such as
phenyl acetate and phenyl thioacetate. Researchers could increase ester production
by lowering water activity (0.8 5 and 0.9) and increasing the levels of substrates
(up to 1000 ppm of FFA and alcohol) in model system of cheese containing esterases
from several LAB. According to studies, each esterase results in hydrolysis of a
different flavor-active ester (Fenster et al., 2000, 2003a, 2003b). Candida lipolytica
was used in the manufacture of Blue cheese (Price et al., 2014). Moschopoulou
(2011) successfully indicated the flavor of Pecorino cheese by using lipolytic
enzymes derived from microorganisms obtained from the abomasum of lambs.
Rhizopus lipase has been applied to development of artificial butter flavors and in
yogurt manufacture (Singh & Banerjee, 2017).
Generally, it is known that esterification of FFAs with ethanol results in ester
formation and these esters impart aromatic molecules in foods, specifically in
cheeses. Ethyl esters or methyl esters derived from short-chain fatty acids mostly
carry significant fruity flavors, whereas thioesters from thiols have sulfur and
cabbage aromas (Collins et al., 2003; Fox & Wallace, 1997).
It is known that LAB have little contribution to lipolysis, but additional cultures,
such as molds usually have high activities in fat conversion in the case of surface-
ripened cheeses (Molimard & Spinnler, 1996). The derived flavors from the fat
conversion are predominantly important in Camembert and Roquefort (soft cheeses).
Esterases of LAB are also known as alcohol acyltransferases due to their ability in
transferring fatty acyl groups from a glyceride to either water, which is called
hydrolysis, or alcohols, which is called alcoholysis, in aqueous systems. Moreover,
12 Enzymes in Flavor Development and Food Preservation 331

esters synthesizing in aqueous systems can be done through alcoholysis from other
alcohols and glycerides. For instance, it is found that St. thermophilus is able to
synthesize aromatic esters (2-phenyl ethyl hexanoate and 2-phenyl ethyl octanoate)
from an aromatic alcohol (2-phenyl ethanol) and two glycerides [monooctanoin
(mono-caprylin, C8) and dihexanoin (dicaproin, C6)](Liu et al., 2003a). Generally,
di- and monoglyceride substrates are preferred for alcoholysis by LAB esterases
which have the same specificity in hydrolysis (Liu et al., 2003a).
In cheese medium ester synthesizing can also done by selecting specific starters
that have high esterase or transferase activity, by changing the mono-, di-, and
triglyceride composition of the cheese fat glycerides, or by controlling ethanol
availability. For instance, in cheese made with mesophilic starters, by increasing
the ethanol level, ethyl hexanoate and ethyl butanoate concentrations are signifi-
cantly increased (Crow et al., 2002). In other study by spiking 1 M ethanol in
Cheddar-type cheese curd, during an incubation of 2 months, ethyl butanoate and
ethyl hexanoate are synthesized by the recombinant esterase from Lb. rhamnosus.
Availability of ethanol is possibly an essential factor in controlling ester formation in
cheese. Also primed cheeses with prehydrolyzed milk fat contain more esterases and
this is because of the presence of more di- and monoglycerides and it is found that
without addition of ethanol there is no ester formation in these cheeses. Moreover,
higher ester levels in cheese curd result in fruity aroma. In addition, some commer-
cial fungal lipases also found to synthesize esters from glycerides and ethanol in
cheese medium; also it is shown that they also have similar mechanism to LAB (Liu
et al., 2003b). Moreover, yeasts and molds have effect on ester formation in cheese
by producing the alcohols needed for ester synthesizing in cheese.
Salvadori (1961) could double the flavor of Pecorino cheese prepared with ewe’s
milk by addition of lipases taken from bacteria which were isolated from the
abomasum of lambs. In addition, it is found that esterase from Mucor miehei also
shows lipolytic activity in cheese similar to that achieved by pregastric oral esterases
(Richardson & Nelson, 1968).
In a study by comparing flavor characteristics of Romano cheese and Fontina
cheese containing Mucor miehei esterase to that of cheeses prepared with pregastric
esterase, it was found that by using fungal esterase and pregastric calf esterase at the
same lipolytic activity in the Fontina cheese, the desired flavor develops in both
cheeses. However, based on lipolytic activity, in Romano cheese it was necessary to
add a five time greater of fungal esterase, rather than pregastric kid esterase.
According to Baillargeon (1990), lipase from Geotrichum candidum which is a
dairy yeast has selectivity towards unsaturated fatty acids with cis-9 double bonds,
such as oleic, linoleic, and linolenic acids. Also, Yarrowia lipolytica (a dairy yeast)
could majorly release oleic and palmitic, from milk fat (Suzzi et al., 2001). It was
also previously found that G. candidum and Y. lipolytica can selectively release
unsaturated long-chain fatty acids from milk fat, in the cheese environment (Das
et al., 2005). However, these FAs have not favorable flavor and they impart a soapy
flavor to cheese, but they can be converted to conjugated linoleic acid (CLA) by
Propionibacteria which are usually used in Swiss cheese production (Jiang et al.,
1998). However, in another study researchers used three strains of
332 F. Hashempour-Baltork and P. Farshi

Propionibacterium freudenreichii ssp. Shermanii as adjunct strains which are able to

convert linoleic acid to CLA in laboratory media, with strains of Yarrowia lipolytica,
Geotrichum candidum, and Lactobacillus fermentum (to produce ethanol from
lactose) for preparation of washed-curd, dry-salted cheese, but the results showed
the failure of the Propionibacteria strains to form CLA from linoleic acid, in the
cheese media (Das et al., 2005). CLA introduction in dairy products been also done
by lipase immobilization method (Baianu et al., 2003).
Based on different cheese varieties and production conditions, nonstarter LAB
and the derived products can be varied. Psychrotrophic bacteria produce heat-stable
lipases which can adsorb onto the milk fat globules and resist pasteurization. They
may contribute to lipolysis in cheese made from milk prior to pasteurization
(Cousins et al., 1977). Penicillium sp. (a Psychrotrophic bacteria) can produce
extracellular lipases which are predominantly responsible for lipolysis in mold-
ripened cheeses (Cousins et al., 1977; Smit et al., 2005). Facultative hetero-
fermentative lactobacilli, such as Lb. casei, Lb. paracasei and Lb. plantarum,
which are dominant in cheese varieties as nonstarter LAB, are weakly lipolytic.
However, obligatory homo-fermentative lactobacilli, such as Lb. delbrueckii subsp.
lactis, Lb. helveticus, and Lb. delbrueckii subsp. bulgaricus, which are used as
starters, also produce esterases (El Soda et al., 1986). According to Bhowmik and
Marth (1990) Pediococcus and Micrococcus are also weakly lipolytic. The effect of
FFA on the flavor of hard Italian cheese varieties is more than Blue mold-ripened
cheeses, which is probably due to neutralization by increasing the pH during
ripening, and also the principal effect of methyl ketones on the flavor of Blue
mold cheeses. Actually alkan-2-ones (2-methyl ketones) is the dominant flavor of
Blue mold cheeses which is derived from FFA. β-oxidation, which is the pathway of
alkan-2-onesformation, involves the liberation of FAs by lipolysis, and their oxida-
tion to β-keto acids. After decarboxylation of them, alkan-2-ones with one less C
atom are formed. This compound can be reduced (which is reversible in aerobic
conditions) and leads to the formation of corresponding secondary alcohols (alkan-
2-ols).
Intramolecular esterification of hydroxy fatty acids results in the formation of
lactones. For instance, γ- and δ-lactones, which are strongly flavored, stable, and the
main lactones present in cheese, are formed from δ- or γ-hydroxy fatty acids.
According to Urbach (1993) in full-fat cheeses, δ-decalactone increases to a maxi-
mum concentration (in 14 weeks) and then its concentration decreases; however, in
low-fat cheeses, the δ-decalactone level remains constant during ripening. However,
Cheddar cheese flavor is improved by decreasing the δ-decalactone level, indicating
that δ-decalactone plays an insignificant role in flavor of Cheddar cheese (Dimos
et al., 1996). In a study, lipase from a Sporidiobolus pararoseus strain was derived
and it was found that the specificity of the enzyme for triglyceride was comparable to
pregastric esterase. Using this microbial lipase during the process of mozzarella
cheese imparted a strong flavor to the cheese. Hence, the lipase taken from
S. pararoseus can be used instead of pregastric esterase in dairy industry (Mase
et al., 2010). In a study six lipases/esterases, related to buttery flavor production,
were compared. It was shown that Candida cylindracea lipase has the most
12 Enzymes in Flavor Development and Food Preservation 333

specificity to butanoic acid and production of buttery flavor in cream. Moreover, this
enzyme led to the highest concentration of free fatty acids (FFAs) (Saerens et al.,
2008).
Based on literatures milk lipases have insignificant importance in cheeses
prepared with pasteurized milk, and milk bacterial lipases, which are resistant to
thermal condition, can act better in cheese production from pasteurized milk.
Moreover, both bacterial and milk lipases have importance in milk fat hydrolysis,
whereas bacterial lipases and esterases (from Streptococcus cremoris) have signifi-
cant role in cheeses made from pasteurized milk (Chambers et al., 2010).
Using pregastric esterase in pasteurized milk for Cheddar cheese production
results in more FFA and flavor improvement; however, enzyme’s activity is lower
than its activity in raw milk. Also it was shown that the flavor of Cheddar cheese is
associated to a balance between acetate and FFAs and the ratio of 1.0 to 0.55 of
acetate: FFA results in the most favorable flavor. Microbial lipases increase FFAs
which affect the flavor of cheese (Collins et al., 2003). Kosikowski (1975) also
studied the prospective application of microbial lipases in continuous cheese
processing, and suggested good flavor development in Cheddar cheese from differ-
ent fungal lipases and in Blue cheese from Aspergillus sp lipase. However, organo-
leptic properties of Cheddar cheeses are increased in the presence of gastric lipase in
their production (Wolf et al., 2009). Moreover, there are studies about the effect of
lipase application in Feta (Karami et al., 2009) and Samsoe cheese (Jensen, 1970) in
their flavor development.
Lane and Hammer (1936) were the first people suggesting that homogenization of
cows’ raw milk or cream which is used in Blue cheese production can activate the
milk lipase in it; therefore, they concluded that homogenization has a great impact in
cheese flavor development and concentrations of volatile acids are two to fourfold
greater in cheese produced by homogenized milk. Studies showed that P. roqueforti
lipase acts specifically on triglycerides with smaller molecular weight. Secondary
alcohols and methyl ketones are fundamental compounds for the Blue cheese flavor
characteristics and it is known that they are derived from FFAs which are abundant
in Blue cheese (Tunick, 2007). Other researchers found out that methyl-n-amyl
ketone (2-heptanone) has a significant role in Blue cheese flavor characteristics,
which is suggested to be derived through the P. roqueforti activity on caprylic acid.
The methyl ketones’ formation and their metabolism by fungi in mold-ripened
cheeses act through different enzymatic mechanisms, such as release of FFAs
from triglycerides of milk fat by lipases (Walker & Mills, 2014). Dwivedi and
KINSELLA (1974) studied the continuous addition of lipolyzed milk fat and gradual
release of FFAs, in Blue cheese through the action of milk lipase. As a result, a
product was formed which was suitable for application in salad appetizers, dressings.
Its amount was 7- to 12-fold more than the ketone amount in a decent Blue cheese,
and its flavor efficacy was 4 times higher. Also these researchers reported the
production of methyl ketone by P. roqueforti from lipolyzed milk fat and suggested
that higher concentrations of FA had an impermanent inhibitory effect on the FAs
metabolization by enzyme.
334 F. Hashempour-Baltork and P. Farshi

Researchers, who studied the effect of pregastric esterases from lamb, kid, and
calf on bovine milk fat substrate, showed that these lipases have preference for
releasing short-chain FFAs. Each esterase had specificity to release butyric and other
short-chain FFAs and they were different in the release ratio of short-chain to long-
chain FFAs producing specific flavor characteristic while using cheese. It was found
that kid esterase has the highest specificity for butyric and other short-chain FFAs.
Studies on Provolone cheeses showed a direct relationship between FFAs (such
as butyric acid) and flavor quality and acceptance in Provolone and Romano cheeses
(Christensen, 1964; Collins et al., 2003). According to application of both lamb
pregastric esterase with lamb gastric extracts, results in more suitable “Provolone-
like” flavors, showing that addition of gastric lipase makes Provolone cheeses
organoleptically more acceptable. Overall, the extensive use of lipolytic enzymes
and commercial rennet paste in Italian cheese manufacture indicates the importance
of controlled lipolysis in this industry (Richardson & Nelson, 1968). It is found that
gastric lipase also has application for accelerating the cheese ripening and its flavor
development in different cheeses, such as Provolone, Ras, and Cheddar cheeses.
Addition of calf lipase increases the release of FAs (C4–C6) rate and accelerates the
development of flavor. The drawback of this method is that the lipase remains active
after ripening and can lead to development of an intense rancid flavor. By addition of
the mixture of fungal protease and lipase, relatively high-soluble proteins and FFAs
will develop in various cheeses which show better development of flavor in 3 months
of ripening. Also the amount of enzyme addition is of high importance and high level
of it results in unfavorable characteristics and decrease in the yield (Jahadi et al.,
2016).
Literatures indicated that using liposome technology for cheese ripening
decreases the losses in yield and bitterness (Jahadi et al., 2015; Vafabakhsh et al.,
2013). In cheese made from unpasteurized milk, the lipase which is inherent in the
cheese has significant effect in lipolytic action (Jahadi et al., 2020). P. roqueforti and
P. camemberti in Camembert and Blue-veined cheeses are considered as lipolytic
cultures and are responsible for lipases production, that accounts for lipolysis.
Moreover, lipases are often added to Italian cheese, such as Romano, Provolone,
and Paramesan to increase their flavor (Ghosh et al., 1996). Through ripening, there
is a constant rise in the concentration of released FAs and total soluble nitrogen
(Jahadi et al., 2012). Lipases are able to liberate the FAs from triglycerides, and in
this way they can trigger cheese flavor development (Maia et al., 1999; Mohammadi
et al., 2015).
In Gouda and Cheddar cheeses, esters, such as ethyl butyrate, have high contri-
bution to their flavor. Moreover, extra esters in Cheddar can impart fruity defect
(Caspia et al., 2006). Furthermore, there are compounds in Cheddar and Camembert
cheese, resulting from degradation of phenylalanine (such as phenylacetaldehyde,
2-phenylethanol, and the ester phenylethyl acetate), playing role in its floral rose-like
aroma and desirable floral note (Carunchia Whetstine et al., 2005).
There is an increasing interest for lipolytic enzymes in baking industry. Findings
suggested (phospho) lipases application as alternatives for producing emulsifying
lipids due to the enzymes ability to degrade polar wheat lipids to produce
12 Enzymes in Flavor Development and Food Preservation 335

emulsifying lipids in situ (Collar et al., 2000; Kirk et al., 2002). Lipase was mainly
used to increase the flavor in bakery products through esterification (releasing
short-chain fatty acids). In addition to flavor development, it also extended the
shelf life of most of the bakery products. Also softness and texture can be developed
by lipase catalyzation (Laboret & Perraud, 1999). Lipase from A. oryzae was also
used in baking industry, as processing aid (Greenough et al., 1996). Hydrolytic
enzymes, such as lipase, were discovered to be effective in decreasing the initial
firmness and enhancing the specific volume of breads (Laboret & Perraud, 1999).
According to Sánchez et al. (2002), yeasts with LIP A (bacterial lipase gene) result in
enzyme with high productivity which can be used in bread as an additive. Butter
flavor increases, by hydrolyzing the butterfat with an appropriate lipase in baked
foods.
Recent studies have investigated the possibility of application of various enzyme
systems for milk fat modification to incorporate them into bakery products. Gener-
ally, lipases of animal origin were found to be most suitable for milk fat modification
and including milk fat that is modified by these enzymes in bakery products led to
the formation of better flavors in them rather than milk fats modified by other
enzymes. Penicillium roqueforti, Geotrichum candidum, and Acromobacter
lipolyticurn lipases were mostly inappropriate due to the liberation of a large
concentrations of medium- or long-chain fatty acids and production of musty or
soapy flavors. Also lamb and kid esterases were not suitable due to the production of
excessive quantities of short-chain FAs imparting rancid flavors in bread
formulations (Arnold et al., 1975).
Vanillin (4-hydroxy-3-methoxybenzaldehyde), which is a generally desirable
flavor, mostly is present in Vanilla Planifolia beans. This flavor is extensively used
in foods (Priefert et al., 2001). Ferulic acid is precursor of vanillin and feruloyl
esterase has been recognized as the critical enzyme in the biosynthesis of it. This
enzyme can be produced in microbial cultures of several fungi grown on various
pretreated cereal brans (Mathew & Abraham, 2005). The metabolism of ferulic acid
in some microorganisms has also been investigated (Falconnier et al., 1994; Narbad
& Gasson, 1998).
Carboxyl esterases (carboxyl ester hydrolases) are enzymes extensively used in
different industries (Ewis et al., 2004). These enzymes prefer to catalyze the
hydrolysis of esters of short-chain FAs, but they also are able to catalyze the
synthesis of ester and transesterification reactions (Bornscheuer, 2002). Amid
these esters, flavor acetates from primary alcohols have a great application due to
their characteristic flavor and fragrance (Romero et al., 2005). Isoamyl acetate is also
one of the most significant flavor compounds used in the food industries and is
produced using lipases. In a research, the ability of type II esterase enzyme to
catalyze the esterification of isoamyl alcohol to isoamyl acetate which is less toxic
compound was studied. This compound is one of the most substantial flavor
compounds used in the food industries due to its distinctive banana flavor (Krishna
et al., 2001). This ester is widely used as a flavoring compound in several foods and
drinks, such as artificial coffee, honey, butterscotch, and alcoholic beverages. Pro-
duction of isoamyl acetate is typically doable through Fischer esterification
336 F. Hashempour-Baltork and P. Farshi

mechanism (Welsh et al., 1989) and all of the enzymatic synthesis reactions of this
compound were taken place using lipases (Krishna et al., 2001). Also there are
applications of lipases in ice cream and single cell protein in which C. rugosa lipase
has significant role in their flavor characteristics (Sánchez et al., 2002).
Tomato flavor, which is composed of a complex mixture of volatiles containing
multiple acetate esters, is resulted from esterases activity. It is believed that
red-fruited species of the tomato clade collects a quite low content of acetate esters
rather than the green-fruited species and the difference is related to the insertion of a
retrotransposon adjacent to the most enzymatically active member of family of
esterases, which results in higher expression of the esterase and decrease in the
levels of multiple esters that have negative impact on human preferences for tomato
(Goulet et al., 2012).
Studies on the flavor and odor characteristics of Suanyu (a traditional Chinese
fermented freshwater fish), in the presence of the esterase activity of Saccharomyces
cerevisiae 31, Lactobacillus plantarum 120, and Staphylococcus xylosus
135, indicated that all strains displayed esterase activities in both intracellular and
extracellular fractions against p-NP-butyrate, p-NP-octanoate, and p-nitrophenyl
(p-NP)-acetate. Moreover, it was found out that a low Aw (water activity), a low
pH, and also a high temperature would be more suitable for Suanyu processing,
which results in more flavor compounds which are favorable for consumers (Gao
et al., 2017).
Also in wine industry high levels of 4-vinylguaiacol and 4-vinylphenol were
identified from grape juice primarily treated with two enzymes. Firstly, cinnamoyl
esterase activity releases cinnamic acids from the related tartaric acid esters. Sec-
ondly, decarboxylase activity by the yeasts transforms cinnamic acids into 4-vinyl
phenol and 4-vinyl guaiacol (Dugelay et al., 1993).
In oil industry Cooney and Emerson strains of Mucor miehei esterase has some
functions. This enzyme have impact on natural fats such as lard oil, beef tallow, and
vegetable oils and some synthetic substrates, such as sorbitol esters of FAs. Also
fatty acid profiles which are resulted from the hydrolysis of beef tallow and soy oil at
pH 8.0 with both pancreatic lipase and M. miehei esterase, are comparable
(Moskowitz et al., 1977).

Protease
Proteases are enzymes able to break down the peptide bonds of proteins; they are
categorized as alkaline, neutral, and acid proteases. Animals, plants, and
microorganisms can be potential sources for these enzymes, in different conditions,
such as high salt concentrations. Wouters et al. (2016) explained that the flavor and
odor of the protein constituents have significant impact in food industry. In fact,
peptides and amino acids, besides other molecules, such as salts or sugars, define the
taste of foods. The human gustatory system is able to detect 5 basic flavors, such as
sweetness, bitterness, saltiness, sourness, and savoriness or umami. Sweetness,
bitterness, and savoriness are the major tastes associated with peptides (Iwaniak
et al., 2016). Bitterness needs a particular care, due to its cause for product rejection.
The peptides’ flavor can change based on their amino acid sequence. Studies have
12 Enzymes in Flavor Development and Food Preservation 337

indicated a correlation between the chain length of the peptides and their bitterness.
Their bitterness increases when they are made by up to 8 amino acid groups.
Furthermore, this is affected by its general hydrophobicity and the particular
amino acid positioned at the N- and C-terminus. For instance, the terminus position
of tyrosine or phenylalanine can determine the bitter taste (Iwaniak et al., 2016;
Maehashi & Huang, 2009). Therefore, selecting the right protease to accomplish the
hydrolysis of protein extract can reduce the unpleasant flavor in the final product and
also produce peptides with suitable tastes. For instance, during cheese production
and ripening, a continuous proteolysis is normally considered to be a required
condition for producing right flavor of cheeses (particularly Camembert or Brie)
(Singh et al., 2016). Moreover, proteolysis occurs during the main biochemical
modifications in cheese making, considering that in most circumstances, the first
step is an enzymatic coagulation. In the next steps, during the cheese ripening, a
progressive transformation of flavor and texture occurs. In fact, proteolysis is not
taking place only by the activity of external added enzymes but also it can be done by
enzymes of microorganisms. In Blue-veined cheeses, Blue molds generate a typical
odor and flavor through the conversions catalyzed by their enzymes. The same
happens at the moldy surfaces of cheeses, such as Camembert and Brie (Seratlić
et al., 2011).

Glutaminase
L-Glutamic acid is a verified flavor-enhancing amino acid which is extensively used
as a fundamental seasoning in food service, home cooking, and processed food
industries around the world. On the other hand, L-glutamic acid is originated from
proteins present in raw materials in fermented foods. L-Glutamic acid can also be
produced by the hydrolysis of L-glutamine in the protein hydrolysate; however, in
the absence of glutaminase, most of the released L-glutamine is transformed to
pyroglutamic acid which is tasteless (Fig. 12.5).
Soy sauce is a product known for its unique and strong umami taste, which is
created through its hydrolysis and fermentation. Umami taste induced by
monosodium L-glutamate has been widely accepted as one of the basic kinds of
tastes which is also named as savory, broth-like, or meaty taste. In fact, this taste is
linked to the taste characteristics and the chemical structure of L-glutamyl
oligopeptides. The presence of glutamic acid in di- or tripeptides is strongly
correlated with umami taste, because of the fact that the anionic L-glutamyl
oligopeptides can be present as umami taste (Zhang et al., 2017). Glutaminase
derived from Aspergillus oryzae is often used for the soy sauce fermentation, and

Fig. 12.5 Formation of pyroglutamic acid in the absence of glutaminase (tasteless)

338 F. Hashempour-Baltork and P. Farshi

high salt concentrations in the soy sauce fermentation process can inhibit its activity.
Thus, salt-tolerant type of this enzyme have been studied for application in soy sauce
fermentation. Glutaminase enzymes from Micrococcus luteus, Bacillus subtilis, and
Aspergillus oryzae have been widely studied (Masuo et al., 2005). The glutaminase
of B. subtilis is commercially available. Zhuang and others (Zhuang et al., 2016)
indicated the significance of peptides’ effect on the umami taste of soy sauce. They
showed that the amino acid sequences of the peptides (Glu-Ala-Gly-Ile-Gln,
Ala-Leu-Pro-Glu-Glu-Val, Ala-Gln-Ala-Leu-Gln-Ala-Gln-Ala, Glu-Gln-Gln-Gln-
Gln, and Leu-Pro-Glu-Glu-Val), with the presence of glutamic acid/glutamine
persistent, are responsible for the umami taste in soy sauce.
Theanine (γ-glutamylethylamide) is an amino acid derived from a nonprotein
source that initially was isolated from green tea leaves (Sakato, 1949). This amino
acid is considered to be a distinctive amino acid in nature due to its limited
occurrence to the Camellia genus, especially the tea-producing plant, C. sinensis
(Nathan et al., 2006). Theanine is acknowledged for its unique umami taste. There-
fore, teas containing high theanine are normally considered to have higher quality.
Theanine can be formed from ethylamine and glutamate using sugar fermentation
reaction of baker’s yeast as an ATP regenerating system and bacterial glutamine
synthetase reaction (Nandakumar et al., 2003). There are some beneficial effects for
theanine, including improvement of learning ability, memory, and relaxation,
strengthening the immune system vascular diseases, prevention of cancer, and
weight gain (Kimura et al., 2007; Owen et al., 2008). In a mixture of γ-glutamyl
donor and γ-glutamyl acceptor, glutaminase from Pseudomonas nitroreducens has
been found to catalyze a γ-glutamyl transfer reaction. This enzymatic method has
been industrialized to produce theanine using glutaminase enzyme from
P. nitroreducens. For the enzymatic synthesis of theanine, GGT (γ-glutamyl
transpeptidase) catalyzes the hydrolysis of γ-glutamyl compounds and transmissions
of γ-glutamyl moiety from γ-glutamyl compounds, such as glutathione, to amino
acids and peptides. Also GGT was responsible for synthesizing other
physiologically significant γ-glutamyl compounds, including γ-glutamyl taurine
and γ-Glutamyl-L-3,4-dihydroxyphenylalanine (γ-glutamyl DOPA). Additionally,
bitter taste of some amino acids can be decreased by the mean of γ-glutamylization
by GGT (Suzuki et al., 2002).

Thermolysin
Synthetic sweeteners, involving anticariogenic and noncaloric sweeteners, have
been suggested to be a substitute to natural, calorie-containing sweeteners to
decrease the risk of cardiovascular diseases, diabetes, and obesity. For instance,
saccharin, aspartame, acesulfame potassium, and sucralose are examples of sugar
substitutes regularly used alone or with other natural sweeteners as food additives, in
several foods and soft drinks. Aspartame (α-aspartyl-phenylalanine methyl ester),
which is a peptide-type artificial sweetener, has been broadly used in processed and
pet foods, and beverages and drinks, such as tea, coffee, and sports drinks. The
sweetness degree of this sweetener is around 200 times higher than that of sucrose.
Aspartame thermolysin is a neutral protease from Bacillus thermoproteolyticus and
12 Enzymes in Flavor Development and Food Preservation 339

it is revealed that its enzymatic activity enhances by increasing NaCl concentration.

The reverse reaction of hydrolysis of peptide bonds in the mixture of
N-carbobenzoxy-L-aspartic acid and DL-phenylalanine methyl ester can be
catalyzed by this enzyme (Inouye et al., 2007; Ogino et al., 2010). At the end, the
N-carbobenzoxy-protecting group is detached chemically, and unreacted
D-phenylalanine methyl ester is altered to DL-phenylalanine methyl ester which is
its racemic form.

β-Galactosidase
D-glucoside glucohydrolase, also called lactase or β-Galactosidase, is able to cata-
lyze the hydrolysis of the galactosyl moiety from the nonreducing end of several
galactosyl-oligosaccharides. This enzyme is usually used in the dairy industry for
hydrolyzing lactose, Gal-(β1,4)-Glc, which is the main saccharide naturally present
in milk (Dutra Rosolen et al., 2015). Moreover, this reaction (hydrolyzing lactose to
galactose and glucose by β-galactosidase) inhibits the lactose crystallization in
condensed and frozen milk products besides strengthening the sweetness. Moreover,
β-galactosidase is capable of catalyzing transgalactosylation, in which a galactose
moiety is transmitted to the saccharides, emerging different galacto-oligosaccharides
(GOs). GOs have some advantageous effects, such as decreasing blood serum
cholesterol level, suppressing the colon cancer progress, and improving the mineral
absorption (Vulevic et al., 2013). The initial application of glycosidases was in wines
which was used to liberate flavor compounds from glycosidic precursors. The first
reason for this is that significant flavor compounds in wines from Vitis vinifera
cultivars are present as flavorless glycoconjugates in grapes, and the second reason is
that the glucose inhibitory effect of -glucosidases restricts their use to the media
containing low levels of glucose (<1 g/L), such as wine. However, due to the
presence of flavor glycoconjugates in various fruits, enzyme applications have also
been reported for fruit juices.
A. Wine Flavor Enhancement: During grape juice fermentation, there is only a
slight decrease in the amount of glycosidic flavor precursors. This can be described
by the low levels of glycosidase activities of enological yeast strains and grapes
(Hernandez-Orte et al., 2008). Therefore, there is a special interest for the use of
enzymes, such as pectinases and hemicellulases, which are commercially available
fungal enzymes (mainly derived from Aspergillus spp.) that also have glycosidase
activities. However, recent regulations do not accept including the enzymes in the
wine. The hydrolysis of monoterpenyl glycosides in muscat cultivars releases
odorous alcohols that can be identified by sensory analysis and can be analyzed by
GC/MS. Generally, the addition of glycosidase during wine making increases in the
concentration of monoterpenes, shikimate-derived compound, and
C13-norisoprenoids (Hernandez-Orte et al., 2008).
B. Fruit Juices: There are few studies about the use of glucosidases in fruit juices
which is due to the inhibition of them by glucose of the “key enzyme” glucosidase in
enzyme derived from fungi. According to a study curing passion and mango fruit
juices with a glucosidase derived from Candida cacaoi resulted in a rise in mono-
terpene concentrations (Chanprasartsuk & Prakitchaiwattana, 2015). Nevertheless,
340 F. Hashempour-Baltork and P. Farshi

this increase was pretty low rather than acid hydrolysis. Moreover, immobilization
of the enzyme on sodium alginate beads, released less terpenols rather than the free
enzyme. The addition of immobilized C. molischiana-derived glucosidase (onto
Duolite A-568 resin) to apple juices, mango, strawberry, apricot, and peach, caused
a rise in the concentration of monoterpene alcohols, such as 2-phenylethanoal,
geraniol, terpineol, benzyl alcohol, and linalool (Pogorzelski & Wilkowska, 2007).
However, there were no differences in the flavor liberations using immobilized and
free glucosidase from C. molischiana. The immobilized enzyme was stated to be
much more stable in fruit juice settings. In passion fruit juice, an immobilized (onto
cellulose or acrylic beads) endo glucosidase derived from A. niger increased the
concentration of volatile compounds, such as benzyl alcohol, benzaldehyde, and
linalool. Generally, an aroma improvement was determined in the treated juices
(Pogorzelski & Wilkowska, 2007).
Although by application of glucosidase, the levels of volatile compounds in fruit
juices have increased, there were two significant parameters restricting the effective-
ness of the process: firstly, it is known that there are lots of flavor compounds in fruit
juice, such as diglycosides, that in general, fungal glucosidases are not able to
hydrolyze them; secondly, the examined fungal glucosidases are prevented by
glucose, which might then constraint their action in fruit juices. There is a report
about the process that the amount of glycosides alters in a sweet wine, with 30 g/L
glucose, in the presence of fungal glycosidases. It is shown that the monoterpene
diglycosides’ level decreases remarkably in the enzyme-treated wine, which can be
described by the activity of exoglycosidases. The produced monoterpenyl-D
glucosides were not hydrolyzed and consequently they were accumulated in the
wine. This clearly shows that the fungal glucosidase activity is inhibited by glucose,
which significantly limits the release of volatiles as it is approved by their analysis.
In contrary, the hydrolysis of monoglucosides was very efficient though a dry wine
was made using the same conditions, such as the same enzyme dosage and grape
juice (Pogorzelski & Wilkowska, 2007).

Naringinase(α-L-Rhamnosidase)
Naringinase (EC 3.2.1.40) is mostly used for the naringin’ breakdown which is the
main bitter flavanone glycoside present in citrus fruits (Puri, 2012). Through the
activity of α-rhamnosidase and β-glucosidase, naringin is converted to a naringenin
and rhamnose. The sources for naringinase are mostly fungal isolates, such as
Circinella, Eurotium, Aspergillus niger, Rhizopus, Trichoderma, Fusarium, and
Penicillium, and bacteria, such as Pseudomonas paucimobilis, Bacillus sp.,
Thermomicrobium roseum, Bacteriodes distasonis, and Burkholderia cenocepacia
(Puri, 2012). Naringinase derived from fungi has more application rather than the
bacterial ones due to increased yield. Naringinase, as a debittering enzyme, has a
principle role in food processing when it is added to fruit juices, in both immobilized
and free forms. There are several food additives, such as sweeteners and
biopolymers, which can be produced by naringinase or rhamnosidase. Moreover,
there is another application for naringinase in addition to arabinosidase and
β-glucosidase to develop the aroma of wine (Custodio et al., 1996). It is also stated
12 Enzymes in Flavor Development and Food Preservation 341

that naringinase can be used in preparation of tomato pulp and prunin, and treatment
of kinnow peel waste (Puri, 2012).
Citrus juice is considered as one of the most commonly used juices in the world.
However, mostly the taste of it is too bitter and this bitterness is mainly because of
the presence of the naringin in the citrus fruits. Naringinase (α-L-rhamnosidase) is
used to diminish the bitterness by breaking naringin into rhamnose and prunin and it
is regularly used in the industrial production process of citrus juice (Puri et al., 2005).
β-glucosidase, which is responsible for hydrolyzing the prunin to naringenin
(a flavorless flavanone) and glucose, is usually used with naringinase, for a success-
ful debittering (Table 12.1).

12.2 Preservation

12.2.1 Introduction

It is undeniable that food is important for mankind and there is not any way of living
without eating. Therefore, it is highly important for the health of everyone to eat
food. Although the need to feed did not change during the years, the way of eating
has seen a lot of changes. Nowadays, food additives are needed to make various
foods that are meeting the progressively challenging market and legal demands
(Saltmarsh & Saltmarsh, 2013). Foods can be made in different facilities and then
can be transported to local markets that are inside the same country or even in distant
ones (Atkins & Bowler, 2016). Food transportation in suitable conditions needs
special equipment and considerations, such as controlled packaging, refrigeration, or
using some additives to prevent food spoilage and diminish food alterations. In
today’s global market which is highly competitive, it is desirable to use the cheapest
method of food preservation and food additives can be a good choice.

12.2.2 Lysozyme

Antimicrobials with animal origin are compounds, such as proteins and enzymes that
are isolated from animals. Currently, the only certified natural antimicrobial with
animal origin that is used in US and the EU is lysozyme (E-1105). This enzyme is
derived from eggs (Carocho et al., 2015), and it has high antimicrobial activity
against gram-negative bacteria. The reason for this higher activity is that gram-
negative bacteria are mostly (90%) composed of peptidoglycan, and lysozyme’s
antimicrobial activity is dependent on the hydrolysis of the β-1,4 linkage site of the
peptidoglycan in the bacterial walls. This enzyme has only a moderate effect against
gram-positive bacteria that have much less peptidoglycan in their wall, and appar-
ently it has no activity against fungi or yeasts (Barbiroli et al., 2012). It is reported
that the principal commercial use of this natural antimicrobial is in the cheese
industry, that is used for prevention of “late blowing” of cheese; also there are
studies about its application in eggs, beef (200 mg/90 mg), and milk (2 mg/mL in
342 F. Hashempour-Baltork and P. Farshi

Table 12.1 Enzyme role related to food flavor

Enzyme
role Enzyme Bound volatiles Flavor like Ref
B-glucosidase Benzaldehyde, benzyl Papaya, apple, Eriksson
alcohol, 2-henylethanol, apricot, peach, (2012)
CI3-norisoprenoid, yellow plum
monoterpenes,
CI3-nonsoprenoids,
Marmelactones, hexanol,
2-henylethanol, linalool,
dienediols, nerol
Endo-glucosidase Terpenes (linalool) Fruity Martinez
Ortega
(2015)
ß-glucosidase Vanillin Vanilla Esparan
(2015);
Lim
(2012)
Enhance or Tannase Terpenes (linalool) Fruity Beniwal
produce et al.
flavor (2013)
Pectinase 2-phenyl ethanol, benzyl Tomato de
alcohol, benzoic acid Andrade
Júnior and
Andrade
(2015)
Anthocyanase Terpenes (linalool) Fruity Yang et al.
(2020)
Glucosidase Norisoprenoids from Grape Williams
lutein, violaxanthin, et al.
neoxanthin (1992)
Lactoperoxidase, Complex (methyl esters, Milk and milk Campbell
xanthine oxidase, ketones, aldehydes, and products and Drake
proteinase, lipase FFA) (2013)
Sulfhydryl Oxidase the volatile thiol Removal of Faccio
oxidases compounds cooked flavor et al.
in UHT milk (2011)
Limonoate Oxidize limonin (a bitter Debittering Kola et al.
dehydrogenase compound of grapefruit (2010)
and orange) to nonbitter
limonoate A-ring lactone
Diacetyl Reduce diacetyl Removal of Krogerus
reductase (responsible of unpleasant diacetyl from and
butter-like note) to 2,3 beer Gibson
butylene glycol (2013)
Elimination Naringinase Hydrolyzes the naringin Debitterize Kiefl et al.
of (responsible for the bitter kumquat, (2017);
off-flavors taste) to prunin and grapefruit Zeng and
rhamnose juice or Huang
(2011)
(continued)
12 Enzymes in Flavor Development and Food Preservation 343

Table 12.1 (continued)

Enzyme
role Enzyme Bound volatiles Flavor like Ref
grapefruit
concentrate
Laccase Significant reduction of Elimination of Schroeder
the concentrations of musty, earthy, et al.
guaiacol and or like roots (2008)
2,6-dibromophenol
(poly)phenol Oxidation of poly phenols Debittering of Kongor
oxidases coffee, cacao, et al.
and olives (2016)
Lipoxygenase Hexanal, nonanal, Beany-grassy Iassonova
nonenal, and 1-octen-3-ol flavor in soy et al.
bean (2009)
Production Lipoxygenase 2-methoxy-3-isopropyl- Off-flavor in Zhang
of off-flavor (5 or 6)-methyl pyrazine, pea milk et al.
hexanal, (E,E)-2,4- (2020)
nonadienal, and (E,E)-
2,4-decadienal
Lipoxygenase 1-octen-3-one, hexanal, Off-flavor in Zhang
(E,E)-2,4-nonadienal, and soy milk et al.
(E,E)-2,4-decadienal (2020)

25 mL milk) (Sung et al., 2011). Lysozyme has also been assessed for making edible
coatings and biofilms (Barbiroli et al., 2012) and its synergistic effects were studied
in corporation with other natural antimicrobials (Bayarri et al., 2014).

12.2.3 Oxidoreductase

Redox enzymes have applications for extending the shelf life of food products.
These enzymes include those that able to remove oxygen or reactive oxygen species,
such as H2O2 and superoxide anion, and those that are capable of generating
antimicrobial agents. Therefore, the stability of foods can considerably be improved
with regard to their appearance, taste, and microbial spoilage.

12.2.3.1 Lactoperoxidases
Lactoperoxidase which belongs to the superfamily of peroxidase-cyclooxygenase
enzymes is the most important enzyme present in the bovine milk in concentrations
of approximately 30 mg/L. This enzyme shows its antimicrobial activity through the
lactoperoxidase system, including lactoperoxidase, hydrogen peroxide, and thiocya-
nate, which are referred to as the lactoperoxidase system (LPS). The intermediate
compounds formed through the oxidation of thiocyanate have antimicrobial activity
(Silva et al., 2014). The main application of lactoperoxidase enzyme is to preserve
raw milk, specifically in places that it is not readily available to refrigerate it. By
344 F. Hashempour-Baltork and P. Farshi

addition of thiocyanate to the milk, the LP system will start showing its antimicrobial
activity. It is necessary to add thiocyanate, because of the low amount of thiocyanate
in the milk which is not enough to initiate the antimicrobial activity. The antimicro-
bial activity of the LP system is activated by production of thiocyanate (SCN)
oxidation products, primarily hypothiocyanate ions (OSCN), that are able to attack
the sulfhydryl groups of major metabolic enzymes of the microorganisms. More-
over, the LP system does not affect the mammalian cells. Usually barely 10–20 ppm
of lactoperoxidase is necessary for an efficient system. Also the required amount of
thiocyanate and H2O2 is pretty low which are 10–25 ppm and 10–15 ppm, respec-
tively. H2O2 is also bactericidal, but it shows its antimicrobial activity at higher
concentrations (300–900 ppm) (Martin et al., 2014). Hence, lactoperoxidase (LPO)
is usually used in combination with enzymes that generate H2O2. Moreover, in the
LP system the amounts of cofactors and also the oxidation products are toxicologi-
cally harmless. LP system has applications in dental products, veterinarian products
such as antidiarrheal, milk replacers, and antimastitis, and food products such as
functional foods, cheese, fish, meat, poultry products, and liquid milk. There are
claimed applications of this system alone or in combination with other systems for
pickled foods, fish products, Lactobacillus fermented milk products, and white mold
cheeses (Laane & Bruggeman, 2002). Moreover, there is an interest for its effect on
yogurt. By addition of LPO to yogurt, the production of unnecessary acid of LAB in
it is stifled (Masud et al., 2010). The mentioned applications are becoming accessible
now that isolating the lactoperoxidase of milk is doable with high purity on an
industrial scale. This system is commercially available now, at considerably low
costs. Latest applications recommended for lactoperoxidase are preservation of fruit
juices, and also using as coating of foodstuffs (Cissé et al., 2015).

12.2.3.2 Xanthine Oxidases

Xanthine oxidase (XO, EC 1.2.3.2) is broadly spread in microorganisms, plants, and
animals. It is able to catalyze the oxidation of hypoxanthine to xanthine and
consequently conversion of xanthine to uric acid. Moreover, XO is capable of
oxidizing a wide range of pteridines, purines, and aldehydes, with simultaneous
reduction of O2 to H2O2. Under definite circumstances, XO also is able to produce
the highly reactive superoxide anion. Bovine milk is a good source of XO containing
35 mg. L of it. (Lin et al., 2015). XO also produces superoxide anion during
oxidation of its substrates and in this way it involves the oxidative deterioration
dairy products (Zarepour et al., 2010). This enzyme can also make a bactericidal or
bacteriostatic agent in milk by producing H2O2 which is used in the lactoperoxidase
system of milk (Whitaker et al., 2002).

12.2.3.3 Superoxide Dismutases and Catalases

Superoxide dismutase (SOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) are the
enzymes in milk that are capable of removing reactive oxygen species formed by
other chemical or biochemical processes (Koh & Kim, 2001). Application of SOD is
for catalyzing the superoxide anion (produced by XO) reduction, to O2 and H2O2.
Consequently, catalase is able to convert H2O2 to oxygen and water. So application
12 Enzymes in Flavor Development and Food Preservation 345

of exogenous SOD, together with catalase, is a very efficient antioxidant in dairy

products (Koh & Kim, 2001). Also it was shown that SOD can be a protection
against free radical damage in beer (G Yao et al., 1987). Clearly, the commercial
application of SOD as an antioxidant depends on the cost, especially rather than
chemical antioxidants. However, it is known that SOD has not commercial applica-
tion as an antioxidant in food systems.
Catalase have application for cold sterilization of milk in areas without refrigera-
tion and also it is used for the treatment of cheese milk in developed countries
(Czyzewska & Trusek, 2018). Sweet potato, Aspergillus niger, liver, and beef are
good sources of catalase. There is also growing interest in using immobilized
catalase reactors for milk pasteurization or glucose oxidase–catalase reactions
(McSweeney, 2016). In addition to the application of SOD and catalase for elimina-
tion of reactive oxygen species, there are other enzymes, such as ascorbic acid
oxidase, alcohol oxidase, glucose oxidase, and D-amino acid oxidase, that can be
used to remove them but in lower amounts. The drawback of these enzymes is that
they produce H2O2, which is a potent oxidant. Addition of catalase can be a way for
removing H2O2, but then oxygen is produced again. Lately, new enzymes (such as
laccase from PPOs) have been stated to use for deoxygenation of juices and beer
(Nunes & Kunamneni, 2018). These enzymes remove oxygen more efficiently and
do not produce H2O2 which is a benefit of using them; therefore, it is not needed to
combine them with catalase. Catalase is usually used in combination with glucose
oxidases for food preservation. Ough (1975) used a catalase with glucose oxidase for
removing the oxygen from wine prior to bottling and assessed the acetaldehydes
formation. Results indicated that proper treatment of the wine with the enzyme
makes the amount and color of acetaldehyde stable (Röcker et al., 2016). Catalase
is also used in milk processing industry to remove peroxide of the milk; it is used in
bakery industry for removing glucose from egg white. Also in food wrappers this
enzyme is used to inhibit and control oxidation and perishability of food (Röcker
et al., 2016). However, this enzyme has limitations for using in cheese industry
(Sindhu Raveendran et al., 2018).

12.2.3.4 Glucose Oxidase

Glucose oxidase is usually used in baking industry for making better dough due to its
oxidizing effects. Moreover, this enzyme increases the aroma, flavor, and stability of
food products by eliminating oxygen and glucose from egg white and diabetic
drinks. Glucose oxidase modifies the flavor, texture, color, and shelf life of food
products and inhibits decay and spoilage. Glucose oxidase is used to extend storage
life of food products during packaging, by eliminating oxygen (Hanft & Koehler,
2006).
346 F. Hashempour-Baltork and P. Farshi

12.3 Conclusion and Future Prospect

Recently, there is a developing attention for food research by the international

scientific community, and the principal areas of their focus include the increased
production of food products, modification of foods with improved nutritive value,
flavor development, increasing shelf life, strengthening food protection with
procedures to preserve food from food pathogens, packaging technology and
materials, and quality control. Food industry researchers are directing serious
researches about the improvement of modified foods with additives
(Chandrasekaran, 2012). The World Health Organization (WHO) food safety unit
has stated the role of microorganisms and derived enzymes from them for getting
safe, secure, and nutritive foods. Therefore, this can be applied to food processing
industries. It is important to determine and select the proper fermentative processing
technologies and then to improve, and apply them in the food industries. These
technologies can be used for preparation and storage of food, in addition to
transforming the vast amount of waste materials, resulting in the environmental
damage.
Using of bioprocesses involving enzymes in food processing can decrease the
antinutritive factors and toxins of the by-products, besides solving the environmental
issues and increasing their consumer acceptance by enhancing their nutritive value.
Thus, food industries are changing their functions toward green processes. However,
widespread research and control on the stability of bioprocessed products, food-
borne illness, development of fermentation process, and risk of food contamination
are needed (Chandrasekaran, 2012). Currently, industry is considering the enzymatic
catalyzes as a way to produce high amount of its products in environmentally
friendly green processes, also as a tool to make additional value to the final product
and increase the economical production. Therefore, the increased need for enzymes
and enzymatic processes is making a new movement in enzyme technology,
supported by improvements in chemical sciences biotechnology.

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Enzymes from Genetically Modified
Organisms and Their Current Applications 13
in Food Development and Food Chain

Senthilkumar Muthusamy, Shilpa Ajit, Asha V. Nath,

J. Anupama Sekar, and T. S. Ramyaa Lakshmi

Abstract

The fulfillment of nutrition needs is in high demand due to the increase in

population, especially in developing countries with a reduction in arable land.
Though the use of natural enzymes from plants, animals, and microbes is high,
the demand and modern food processing conditions require improved enzymes
both qualitative and quantitatively. Current recombinant DNA technologies such
as altering genetic code and introduction of a gene allow genetic modification in
an organism, which gives desired products. Some of the genetically modified
foods are on the market with rigorous safety assessment and many of them are
under development. Genetically modified enzymes that ameliorate the processing
of carbohydrates, protein, and lipid are in pipeline to use in various food
industries. This chapter emphasizes the enzymes derived from genetically
modified organisms; the methods used to generate genetically modified
organisms and the use of these enzymes in food industries for the application of
various food development and food chain. The chapter will also highlight
people’s thoughts and safety concerns about the use of enzymes from genetically
modified organisms.

Keywords

Enzymes · Recombinant DNA technology · GM foods · Applications

S. Muthusamy · S. Ajit · A. V. Nath · J. Anupama Sekar

Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology,
Thiruvananthapuram, Kerala, India
T. S. Ramyaa Lakshmi (*)
Department of Zoology, Thiagarajar College, Madurai, India

# The Author(s), under exclusive license to Springer Nature Singapore Pte 357
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_13
358 S. Muthusamy et al.

13.1 Introduction

Enzymes are proteins (also referred to as biocatalysts) that can be extracted from
cells, which catalyze the biochemical reactions in living organisms. They can be
used to catalyze a good range of commercially important processes in paper and
pulp, textile, chemical and pharmaceutical, agriculture, and food industries. Among
food, enzymes represent a major share in the enzyme industry. For many years,
enzymes are utilized in food processes, like for the clarification and filtering of wines
and beers, for baking, the assembly of cheeses, and far more (Robinson, 2015).
Global population is accelerating and is estimated to reach ten billion by 2057. The
use of industrial enzymes is also estimated to increase in correlation with the
population from 5.8 billion USD in 2020 to 20 billion USD in 2057 (Fig. 13.1).
The preferred source of enzymes are plant and animal, which constitutes around 15%
and the remaining 85% of industrial enzymes are from microorganisms such as fungi
and bacteria. The economic and technical ease put microorganisms forefront of
enzyme synthesis.
The development of recombinant technology brought considerable improvements
in the enzyme industries by generating Genetically Modified Organisms (GMOs). In
today’s market, around 50% of the enzymes are derived from these GMOs, which
are intended to increase their yield, purity, specificity, and stability (Petersen, 2005;
Poulsen & Bucholz, 2003). As per FAO (Food and Agriculture Organization of the
United Nations). GMOs are “the organisms those not occur by natural reproduction
or natural recombination.” GMOs are the major source of enzyme production in food
industries that requires a comprehensive understanding of the integration between
enzymology, molecular biology, and bioinformatics. The enzymes from GMOs
especially from the microorganisms have several safety concerns such as contami-
nation with toxins, allergens, and other impurities (de Santis et al., 2018; Srivastava,
2019). Thus, before getting into the marketing, enzymes from GMOs need approval
from regulatory bodies such as the Food and Drug Administration (FDA; USA) and
European Food Safety Authority (EFSA; Europe) with rigorous assessment in
addition to regular ethical concerns. This chapter deals with the enzymes from
GMOs; applications in food industries and regulatory concerns for their use.

Fig. 13.1 Projected global population and enzyme production

13 Enzymes from Genetically Modified Organisms and Their Current. . . 359

13.2 Use of Genetically Modified Organisms for Enzyme

Synthesis

The biological variants form very often due to natural recombination and mutation
by changes in the surrounding microenvironment. More than a century, we also
exploiting the genetic materials that are naturally present in animals, plants, and
microbes. Initially, the geographic distribution of genetic variants occurred unknow-
ingly from one part of the world to another with distinct climate conditions. For
example, Asian soybean to America and American potato throughout the world. The
history of genetic engineering begins with Charles Darwin’s notions of species
variation and selection. The evolution of biotechnology leads to the development
of biological techniques that help to overcome unbeatable physiological barriers and
to exchange genetic materials among all living organisms. In late 1950s and 1960s,
the scientists discovered the double helix structure, central dogma, and genetic code
which led to the development of DNA recombination technology in 1973 by Cohen
et al. The development in the DNA recombination technology showed that the
genetically engineered DNA molecules can be transferred among same species
and in different species.

13.2.1 Genetically Modified Organisms

According to World Health Organization (WHO), GMOs are the organisms (i.e.,
plants, animals, or microorganisms) in which the genetic material (Deoxyribonucleic
acid) has been altered in a way that does not occur naturally by mating and/or natural
recombination. The genetic modification occurs in two ways; either mutation or
recombination, which may be spontaneous/natural or induced. The evolution of
biotechnology leads to the development of biological techniques that help to over-
come unbeatable physiological barriers and to exchange genetic materials among all
living organisms. The technology is often called modern biotechnology, recombi-
nant DNA technology, or genetic engineering.
Initially, the first-generation GMOs were developed for improving the quality and
yield by increasing their resistance to certain diseases and unfavorable environments.
In 1983, the first genetically modified plants antibiotic-resistant tobacco and petunias
were produced by three independent research teams (Bevan & Chilton, 1982; Fraley,
1983; Herrera-Estrella et al., 1983). In 1990, China first commercialized genetically
modified tobacco and in 1994 USA marketed the genetically modified tomato which
is approved by the Food and Drug Administration (FDA). Many transgenic crops,
such as Canola with modified oil composition, cotton and soybeans resistant to
herbicides, etc. have received FDA approvals. Genetically modified potatoes,
eggplants, strawberries, carrots, and many more are available in the market (Bawa
& Anilakumar, 2013). The second-generation GMOs were aimed to transfer genetic
information from one organism to the other to improve specific product quality and
quantity.
360 S. Muthusamy et al.

13.2.1.1 GM Plants
The Food and Agriculture Organization (FAO)/International Atomic Energy Agency
(IAEA) listed more than 3000 plants that have been developed using genetic
modification with physical and chemical mutagenic agents. These GM foods are
either used for the human consumption or used as feed for livestock and poultry.
The first commercialized food crop is papaya, which was genetically modified for
its resistance against papaya ringspot virus. Later, some fruit ripening characteristics
have been improved using genetic modification. Polyphenol oxidase is responsible
for the production of melanin in apples and potatoes, which cause a brown tint when
exposed to air. The restriction of polyphenol oxidase synthesis below 10% by
genetic modification allowed the apple and potato remain same after slicing. The
herbicide-tolerant varieties of alfalfa, soybean, sugar beet, sweet corn (maize), and
cotton have been developed using genetic engineering. GM Golden rice, which
contains beta-carotene (precursor of vitamin A) along with enhanced iron content
has been approved in various countries. At present, more than 170 million hectares
are under cultivation of GM crops worldwide (James, 2006).

13.2.1.2 GM Animals
Though GM animals are in the use of biomedical and pharmaceutical applications to
produce certain recombinant proteins and to study selective gene functions, the
generation of GM animals for food from a variety of target species traits is still on
bench side due to the inefficacy of the production and ethical issues. The major
causes to attain genetically modified organisms are; to increase animal health, (Tong
et al., 2011; Wheeler et al., 2001), to make them disease resistant (Lyall et al., 2011;
Richt et al., 2007), to improve growth rate (Devlin et al., 2009), to increase meat
quantity (Lai et al., 2006), to increase milk production and alter composition
(Wu et al., 2012), and to increase wool production (Damak et al., 1996). The fast-
growing Atlantic salmon is the only GM animal that has been approved for food by
health Canada and USA-FDA, in which the growth hormone gene from Chinook
salmon was transferred into the Atlantic salmon genome. The use of this fast-
growing GM fish for the food in future is still uncertain.

13.2.1.3 GM Microorganisms
Microorganisms, produce endogenous enzymes responsible for several food
products like yoghurt, cheese, beer, wine, vinegar, and many more. Humans used
these microorganisms even without knowing they are responsible for the fermenta-
tion process in various food products (Jay et al., 2005; Zhang et al., 2017). During
the eighteenth century, the discovery of the microorganisms responsible for a certain
process in food industries began to research efficient ways to use microorganisms
(Barnett, 2003). The discovery of DNA followed by recombinant technology
allowed genetic alteration to optimize the use of microorganisms in effective way
for food production. These genetic modifications either improve the production of
existing proteins or increase their potential or produce a new protein with different
characteristic features. The advancement in molecular biology led to widespread use
of GM microorganisms in food industries and is recognized as an environmentally
13 Enzymes from Genetically Modified Organisms and Their Current. . . 361

friendly and cost-effective method for food production. The yeast Saccharomyces
cerevisiae was the first GM microorganism used for enzymes synthesis in the dairy
and brewing industries. The trypsin and chymosin from the GM microorganisms
served as an alternate for animal sources such as pigs and cattle. Replacement of
certain plant-based products by GM microorganisms could meet the demand with
reduced land usage and waste generation. The GM yeast-based production of
vanillin, an alternate for vanilla extract; GM bacteria that produce riboflavin are
certain examples of the use of GM microbes in food industries (Brochado et al.,
2010; Schwechheimer et al., 2016). Other food ingredients known to be produced by
microbes are includes vitamins, amino acids, nutritional proteins, oligosaccharides,
flavoring agents, and sweeteners (Adrio & Demain, 2010).

13.2.2 Enzyme Synthesis from GM Microbes

According to legal authorities such as FDA, the European Food Safety Authority
(EFSA), the enzymes are considered nutritional additives. The major industrial
enzyme market relies on the food and beverage industry, which is expected to
reach 2.3 billion USD in 2021 (Raveendran et al., 2018). Although the majority of
the organisms including plants and animals produce enzymes, microbial sources
become more valuable. Table 13.1 shows various industrial enzymes used in the
food and animal feed industries.

13.2.2.1 Advantages of Using Microbes for Enzyme Synthesis

(a) Many MO produce enzymes and release into the medium which simplifies the
extraction method during the purification. It is easy to extract intracellular
enzymes as well with the disruption of microbial cells compared to the extrac-
tion process from animal and plant sources (Robinson, 2015).
(b) Specific microbial strains can be used for the yield of well-characterized
enzymes with specific properties (Chang et al., 2016).
(c) Cost-effective production due to shorter time and simple facility requirements.
The whole production can be achieved in one location where animals and plants
need to be transported to extraction facilities (Raveendran et al., 2018).
(d) High yield can be obtained for microbial enzymes compared to animal and plant
sources where the availability depends on a specific period, which could limit
the continuous production (Singh et al., 2016).
(e) Microbial enzymes usually have a higher activity and stability (Vieille &
Zeikus, 2001).
(f) Genetically modified microbial strains can be easily produced in order to obtain
higher yields, optimized enzyme products, and better characteristics.
Modifications of animals and plants are difficult and ethically more sensitive
(Lievens et al., 2015).

The wild-type microbes are unstable and may not produce desired quality enzyme,
especially in fermenter conditions. Sometimes the feedback repression occurs that
362 S. Muthusamy et al.

Table 13.1 Industrial enzymes used in food and animal feed industries
Industry Enzyme Function
Dairy Lactase (beta Lactose reduction in milk, coagulation, faster cheese ripening,
galactosidase) debittering, flavored cheese, protein crosslinking
Aminopeptidase
catalase
Proteinase (acid
and neutral)
Lipase
Transglutaminase
Baking Amylase Bread softening, enhance lifespan, dough conditioning,
Transglutaminase dough stability, and strengthening
Lipase
Glucose oxidase
Xylanase
Beverage Pectinase Pectin digestion, starch hydrolysis fruit liquefaction, protein
Cellulase breakdown, oxygen removal, debittering, restrict mist
Amylase (Alpha formation
and Beta)
Beta Glucanase
Protease
Glucose oxidase
Pullulanase
Naringinase
Limoninase
Aminopeptidases
Animal Beta glucanase Enhance digestion
feed Phytase
Xylanase

Fig. 13.2 Major steps involved in the enzyme production from genetically modified microbes

ultimately affect the enzyme production. Considering the advantages of using

microbial strain for the food enzyme production, the genetic modifications can be
easily done in the microbial strains which improve the industrial enzyme production.
GM microbes could overcome these drawbacks and enhance the production of
quality enzymes. The GM microbes mainly certain bacterial and fungal strains
have been optimized for increased enzyme production with a limited synthesis of
unwanted secondary metabolites (Deckers et al., 2020). The GM enzymes are mostly
expressed in well-established standard microbial model systems like Escherichia
coli, Bacillus, lactic acid bacteria, filamentous fungi, and yeast. These microbes have
advantageous characteristics over other microbes, which resulted in the use of a
number of biotechnological applications. The major steps involved in industrial
enzyme production are given in Fig. 13.2. Few strategies that are required to produce
genetically modified enzymes are;
13 Enzymes from Genetically Modified Organisms and Their Current. . . 363

1. Codon optimization for the heterologous expression and to limit mRNA folding
for optimal translation (Rosano & Ceccarelli, 2014).
2. In-depth understanding of enzyme sequence, corresponding amino acids, struc-
ture, and related activity. This will allow one to modify the sequence by mutation-
like site-directed mutagenesis to alter or increase the enzyme activity, specificity,
and structural stability (Hua et al., 2018).
3. Developing an efficient screening protocol for identifying effective variants
(Packer & Liu, 2015).
4. Knowledge about fusion and truncation of target sequence to produce an enzyme
with multiple functions and to eliminate the unwanted sequences those are not
important for the function (Dediu, 2015).

13.2.2.2 Selection of Host Organism

In order to obtain a successful enzyme synthesis, it is important to select a host
organism having suitable characteristics for the production and expression of the
specific enzyme and that is relatively cheap to culture. The type of expression
vectors, the transformation methods, and the selection markers used to select the
transformed strains need to be determined are also important for obtaining geneti-
cally modified organisms for use in food industries (Olempska-Beer et al., 2006).
Many expression systems are available for the recombination of bacteria resulting
in the easy integration of modifications. Bacteria are not able to perform posttransla-
tional modifications resulting in only simple proteins being produced. In the case of
fungal strains, correct folding and posttranslational modifications were done by the
subcellular organization and they secrete a limited amount of proteins into the
medium (Demain & Vaishnav, 2009). The commonly used bacterial host strains
are Escherichia coli, Bacillus subtilis, Bacillus licheniformis, and Bacillus
amyloliquefaciens. Availability of molecular techniques for the construction of an
expression system in E. coli results in the high-yield enzyme production in the
cytoplasm and additional purification steps are required. However, the Bacillus
species secrete higher concentrations of the enzymes into the medium (Puetz &
Wurm, 2019). Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei are
commonly used filamentous fungi. A. oryzae used for the production of fermented
food, which is also known to produce mycotoxin aflatoxin. The mutant strain was
constructed to overcome this issue, in which the production of these products was
reduced (Meyer, 2008).

13.2.2.3 Gene Transfer Methods

Expression Vector System

Once a suitable host has been selected, the selected host strain is modified by using
the expression vectors. A vector is a DNA molecule that is a vehicle to transfer
foreign genetic material into another cell. Plasmids, viral vectors, cosmids, and
artificial chromosomes are the different types of vectors. Plasmids are called expres-
sion vectors those express transgene in the target cell, and they generally have a
promoter sequence that drives the expression of the transgene. A typical expression
364 S. Muthusamy et al.

Fig. 13.3 Basic structure of expression vector used in recombinant enzyme synthesis

vector is shown in Fig. 13.3. An expression vector contains, at the least, an origin of
replication (ORI), a Multiple Cloning Site (MCS), in which the gene of interest will
be integrated, and a selection marker for the selection of the recombinant strain
(Fig. 13.1). Promoter and terminator regions are also present to control the expres-
sion of the gene of interest and the selection marker. In order to avoid an increase in
the metabolic load on the host organism and to avoid reduced plasmid stability, the
size of the vector is kept as small as possible (Rieder et al., 2019).
It is important to select the recombinant strain and the selection is based on the
selectable marker. A selectable marker is usually a gene that shows resistance to an
antibiotic that would otherwise kill the normal cells which lack these genes under
culture conditions with a specific antibiotic. Kanamycin (kanR), ampicillin (ampR),
and tetracycline (tetR) resistance genes are commonly used in the bacterial expres-
sion vector. Antibiotic resistance markers are associated with a public health con-
cern, which is replaced by auxotrophic strains, where it requires specific nutrients for
growth. Some auxotrophic markers are amdS, URA3, which are usually used as the
selectable marker in fungal strains (Hjort, 2007; Olempska-Beer et al., 2006).

Preparation of GM Microbes
The common method employed in the preparation of GM microbes is to cut open the
plasmid DNA; inserting the target DNA molecule (named molecular cloning) and
transform it into host microbes, which can efficiently express the target DNA.
Dissecting each part, (1) the plasmid expression vector has been chosen based on
the host organism; (2) the target DNA has been amplified from the target genome
which has restriction enzyme site overhanging on both the ends that matching to the
sites present in the multiple cloning site of the plasmid; (3) restriction digestion of
the plasmid vector and amplified target DNA molecule using restriction enzymes
(which can form either blunt or sticky ends); (4) Purify the restriction digested DNA
and plasmid; (5) Ligate the plasmid and target DNA using ligase; and (6) transform
the recombined plasmid to the host organism. The recombinant DNA molecule can
13 Enzymes from Genetically Modified Organisms and Their Current. . . 365

Fig. 13.4 General

recombinant technology
involved in the recombinant
enzyme synthesis

be transformed into cells of higher organisms (Fig. 13.4). Transformation of the

target DNA is done by transfection in which the introduction of nucleic acids into
cells by nonviral methods. Calcium phosphate, DEAE-dextran, or other substances
are nonviral methods of transformation. Physical methods used are electroporation
which cellular membranes of an organism are disrupted by using an electrical pulse,
direct microinjection into a cell, and biolistic particle disruption into cells using gene
guns. The plasmids are the most commonly used DNA carrier molecules and the
viral DNA molecule also can be used to stably express the target enzyme. Plasmids
are isolated from bacterial cells and specific sequences of DNA molecule are altered
in vitro by insertion or deletion. Antibiotic resistance, ß-galactosidase, luciferase,
etc. act as gene reporters for the screening of transformed cells.

Other Gene Editing Options

Several molecular tools are used to insert desired DNA sequence directly into the
genome or delete unwanted DNA bases from the genome. For example, the insertion
of functional genes such as active promoter sequence activates the production of the
desired enzyme and the insertion of transporter sequence can improve the transport
of enzymes. The source of DNA can be either endogenous or exogenous from
closely related/distinct organisms. Currently, the use of synthetic DNA allows fast
development of GM microbes rather than isolating and amplifying from the other
organisms. Recent techniques allow to insert DNA sequences to specific loci, which
can be confirmed by genome sequencing. Today, the discovery of CRISPR (Clus-
tered Regularly Interspaced Palindromic Repeats) facilitates insert or delete DNA
sequence at precise position using specifically designed guide RNA sequences that
can recognize specific DNA code.
366 S. Muthusamy et al.

13.2.3 Enzyme Extraction Process

The extraction and purification of enzymes from GM microbes is much easier

compared to plant and animal sources. Depending on the enzyme of interest and
the host-microbe, the growth environment, such as pH, temperature, gas exchange
rate, and substrate can be optimized during the extraction process while using GM
microbes in comparison with wild-type organisms. Current genetic engineering
techniques enabled the large-scale production of enzymes for use in food and
other industrial applications. The level of downstream processing mainly depends
on the application of the finished product (Headon & Walsh, 1994; Wheelwright,
1989). The overall enzyme extraction and purification process is illustrated in
Fig. 13.5. The enzyme extraction process at the industrial level involved two basics
bioreactor-based fermentation methods, which are (1) submerged and (2) solid-state
fermentation.

1. Submerged Fermentation
Submerged fermentation (SF) is the most preferred method for industrial-level
enzyme production, where the selected GM microbes (bacteria or Fungi) are
cultured in the liquid nutrient medium at a higher concentration of oxygen. In this
process, the microbes release the enzymes to break down the complex
carbohydrates, proteins, and lipids in the medium. A large fermenter with a
high capacity of around 1000 cubic meters is used to produce a large quantity
of enzymes. Simple batch reactors are used, where the reactor is filled with
selected microbe and nutrients and allowed for fermentation. After fermentation,
the contents are emptied for further enzyme purification. On the other way, the
batch-fed process is a continuous process, where the sterile nutrient mix is added

Fig. 13.5 Enzyme extraction

and purification process from
GM microbe
13 Enzymes from Genetically Modified Organisms and Their Current. . . 367

to the fermenter at the same level of fermented broth leaving for further enzyme
purification.
2. Solid-State Fermentation
Solid-state fermentation (SSF) is another method that involves the culture of the
GM microbe on a solid substrate, such as pulp, corn, wheat or rice bran, and
grains. The advantages of SSF over SF are; a high concentration of enzyme
production with less affluent in a simple fermenter. In SSF, the factors such as
particle size and moisture play a major role in enzyme production. For example,
smaller substrate particles make larger surface area microbial growth, whereas
larger particles provide efficient aeration. In SSF the water level should be
optimized for enzyme production, as moisture level greatly influences the micro-
bial activity.

The enzyme produced during fermentation should be further processed to purify

the enzyme from the biomass and other byproducts. First, the solid waste and the
biomass are removed from the medium and the solution is further concentrated using
filtration, evaporation, and crystallization. Chromatography method such as
ion-exchange chromatography is employed to remove impurities. If the enzyme is
localized intracellular, the biomass has to be collected and processed further for
enzyme purification. Depending on the nature and use of enzymes, the enzymes can
be further stored in lyophilized powder form, liquid form if crude, or granule form.
Some enzymes are immobilized on the surface of inner granules.

13.3 GMO-Derived Enzymes in Food Industries

From the ancient period onwards a wide variety of enzymes from a natural source
has been used for the production of numerous food products. Natural sources for
obtaining enzymes are animals, plants, and microorganism. There are about 3000
known enzymes produced by all animals, green plants, fungi, and bacteria that
catalyze about 4000 biochemical reactions (Patel et al., 2017). Among these sources,
microorganisms have been involved in the food industry for various applications like
the production of alcoholic beverages, ethyl alcohol, organic products, dairy
products, and drugs including antibiotics through fermentation (Verma et al.,
2018). For example, the usage of chymosin for cheese production and pectinases
for fruit and beverage processing (Chandrasekaran, 2015). In modern food
processing methods extreme conditions, such as high temperature, high pressure,
extreme pH, salinity, etc., are required to improve the commercialization of enzymes
and their products (Li et al., 2012). This condition may affect natural enzymes. So,
most of the enzymes produced by microbes are no longer native enzymes, but
instead engineered versions. Genetic modifications through genetic engineering
and recombinant DNA technology creates Genetically Modified Organisms
(GMOs) that have specific modifications introduced into genetic material which
enable the production of new protein or food ingredient, enhance the production, and
modify the characteristics of an existing enzyme for a new application (Maghari &
368 S. Muthusamy et al.

Table 13.2 Common GM Microorganism Species

Microorganisms involved
Bacteria Escherichia Coli
in food enzymes production
Bacillus megaterium
Bacillus brevis
Lactobacillus lactis
Pichia pastoris
Yeast Saccharomyces cerevisiae
Hansenulapolymorpha,
Schizosaccharomycespombe
Yarrowialipolytica
Filamentous fungi Aspergillus oryzae
Aspergillus Niger
Trichoderma reesei
Penicilliumpurpurogenum
Rhizopusoryzae

Ardekani, 2011). Thus, the quality and productivity of enzymes can be improved.
Enzymes obtained from genetically modified organisms are intended to improve or
alter the enzymological properties or to increase the purity and yield of enzymes.
Although there are challenges and safety concerns for using recombinant proteins in
food processing, genetically modified enzymes are promising because of their
potential benefits for the food industry, consumers, and the environment (Zhang
et al., 2017).
Food enzymes are commonly used to perform a wide variety of applications in
food production, which include; lactase that reduce lactose in food and dairy
products, amylases that strengthen dough in bakery, mannase that helps in coffee
production, proteases that hydrolyze protiens, chymosin that used to make cheese,
glucoamylase, and transglucosidase those reduces starch into simple sugar and
phospholipase that refine vegetable oil. Considering the safety and synthesis of
other metabolites, selected bacterial, yeast, and fungal species are commonly
involved in the recombinant enzyme synthesis (Table 13.2). The list of major
enzymes derived from GM microbes and their function are given in Table 13.3.
The main industrial enzymes can be classified into three groups: carbohydrases,
proteases, and lipases (Castro et al., 2018). Microorganisms secrete a variety of
enzymes that plays an important role in the food manufacturing industries (Hanlon &
Sewalt, 2020). With the growing research on genetically modified organisms,
production, and development of these enzymes are also getting better.
Carbohydrases: Carbohydrases belonging to glycosidase family are a set of
enzymes that catalyzes hydrolysis of complex carbohydrates (polysaccharides) into
simple sugars (monosaccharides). These enzymes are used in the food industry for
baking, brewing, sweetener production, etc. Carbohydrases include amylases,
cellulases, glucanases, pectinases, xylanases, invertase, galactosidase, and others
(Castro et al., 2018).
Amylase: Amylase catalyzes the hydrolysis of glucosidic linkages of large
polysaccharides (starches) into oligosaccharides. These enzymes are used in the
13 Enzymes from Genetically Modified Organisms and Their Current. . . 369

Table 13.3 List of major GM food enzymes and their functions

Food
enzymes Functions
Amylase Breaks down starches and sugars found in grains, fruits, and vegetables
Galactosidase Breaks down the polysaccharides and oligosaccharides in foods, such as
legumes and cruciferous vegetables
Beta- Breaks down plant cell walls (cellulose) beta-linked glucose polymers
glucanase associated with fibers, grains, and cereals
Cellulase Breaks down food fiber (cellulose) found in fruits and vegetables
Glucoamylase Breaks down off long chain carbohydrates or starches (corn, potatoes, wheat
and rice) into sugar
Invertase Breaks down simple sugar (fructose and fruit sugar)
Lactase Breaks down lactose found in milk
Lipase Breaks down fats and lipids
Pectinase Breaks down pectin, a polysaccharide found in plant cells in many fruits and
vegetables
Peptidase Breaks down proteins into amino acids
Phytase Breaks down carbohydrates; helpful in the breakdown of phytic acids in the
leaves of plants, grains, seeds
Protease Breaks down protein found in meats, fish, poultry, grains, nuts, gluten, milk
proteins (caseins)
Xylanase Breaks down plant nutrients from vegetables with high fibercontent (fibrous
veggies, grains, and legumes)

production of maltodextrin, modified starches, glucose and fructose syrups, produc-

tion of bread, cakes, and pastries (De Souza & Magalhaes, 2010). These enzymes
degrade starch in the flour used as dough of bread into smaller dextrins which
enhance the rate of subsequent fermentation by the yeast and reduce the viscosity
of dough which in turn improves the volume and texture of the product. Through the
action of enzyme additional sugar is produced in the dough, which improves the
quality of the bread, such as taste, crust color, and toasting qualities (van der Maarel
et al., 2002).
The conditions during the development of fermentation processes are very
important such as thermostability, pH profile, pH stability, and Ca-independency.
Industrial source of α-amylase is the bacteria Bacillus licheniformis. Usually, the
α-amylase produced by wild-type Bacillus licheniformis is thermostable but acid-
sensitive. Genetically modifying Bacillus licheniformis through directed evolution
by replacing active site domain His residues with Arg and Asp residues produces
α-amylases having higher activity at pH 4.5 than the wild type enzyme which
enhances the properties of an enzyme such as starch liquefaction (production of
nutritive sweeteners from starch), saccharification and fermentation processes where
the pH of the starch slurry is usually <6.0 (Liu et al., 2014). Genetically modifying
Rhizopus oryzae using site-saturation mutagenesis of His 286 produces α-amylases
having higher optimum temperature (60  C) and lower optimum pH (4.0–4.5) than
370 S. Muthusamy et al.

the wild-type enzyme which makes the enzyme more suitable for maltose syrup
production (Li et al., 2018).
Glucosidase: These enzymes are involved in the breaking down of starch and
glycogen into their monomers. It is used as a flavor enzyme to enhance the flavor of
wine, tea, and fruit juice. β-glucosidases play a critical role in plant biomass
deconstruction. β-Glucosidases cause flavor liberation by catalyzing the breakdown
of β-1, 4-glycosidic linkages and producing free fermentable glucose used for the
production of glucose syrups from fruits and other plant tissues. In modern industrial
conditions, wild-type fungal β-glucosidases show weak activity at high glucose
concentrations limiting enzymatic hydrolysis of plant biomass. Genetic modification
of Trichoderma harzianum by site-directed mutagenesis efficiently improved the
functional properties of a β-glucosidase where the enzyme displayed high glucose
tolerance levels by the substitution of two amino acids that act as gatekeepers,
changing active-site accessibility and preventing product inhibition. The efficiency
of the engineered enzyme was enhanced by the yield of glucose and ethanol (Santos
et al., 2019). Genetically modifying Aspergillus aculeatus using site-saturation
mutagenesis produces β-glucosidase having enhanced hydrolytic efficiency, espe-
cially to cellobiose, and improves saccharification of alkaline-pretreated bagasse
(Baba et al., 2016).
Xylanses: These enzymes are involved in breaking down the hemicellulose in the
plant cell wall, specifically hydrolyzes xylan and arabinoxylan. It is used in the
application of the food industry for bread making, the production of corn starch,
clarification of fruit juice and wine, and alcoholic fermentation. In bread-making
process, xylanases are added to the dough for stabilizing the dough by hydrolyzing
polysaccharides in the wheat and making it more flexible, and enhancing the gluten
strength. Treating with xylanase increases moisture retention and shelf life of the
dough (Ahmad et al., 2014). It is also used in fruit juice processing where it breaks
down the polysaccharide plant cell walls to intensify the juice extraction efficiency
and thus extracting more nutrients and aroma compounds. Also, it helps in juice
clarification by decreasing cloudiness (Bajaj & Singh, 2010).
Even though xylanases have potential applications there is a need for xylanases
that can endure relevant processing conditions and industrial settings. A variant of
this enzyme generated using site-directed mutagenesis exhibited higher specific
activity and was utilized in the production of xylooligosaccharides from wheat
straw under thermal and alkaline conditions (50–65  C, pH 7–10) (Faryar et al.,
2015). Endo-b-1,4-xylanases with modification in secondary binding sites also
showed increased specificity toward water-insoluble, but not water-soluble, wheat
arabinoxylan, and its application in bread making resulted in increased loaf volumes
(Leys et al., 2016).
Glucanase: The glucanase enzymes help in the breakdown of polysaccharide
glucan by hydrolyzing the β-glycosidic bonds between glucose molecules in
glucans. This enzyme is mainly employed in the brewing industry where it
hydrolyses barley gums. It helps in beer brewing by lowering the viscosity of
warts, improves clarification by breaking the turbidity system by hydrolyzing the
beer haze, and aid in production of clear wart. β-glucanase is usually found
13 Enzymes from Genetically Modified Organisms and Their Current. . . 371

endogenously in barley itself and is called endo-b1, 3-1, 4-glucanases having an

optimum pH and temperature values of 6.0 and 45–50  C; and denaturizes at 60  C.
So, industrial source β-glucanase from microbial organisms is used for the standard
production of light beers. Industrial sources for glucanases are Bacillus
amyloliquefaciens and fungi of the group In the brewing industry β-glucanase
hydrolyzes barley gums. Glucanases are also used during wine-making processes
where it hydrolyses Botrytis and yeast glucans and thus enhance the clarification and
wine filterability. β-1, 3-1, 4-glucanases used in malt extract production in the
brewing industry are obtained from Bacillus species. Genetically modified forms
of β-1, 3-1, and 4-glucanases were produced by replacing Lys with Ser and these
modified glucanases showed higher efficiency under industrial operating conditions
with improved optimal temperature and thermostability and halostability following
replacement of hydrophobic Lys 48 with Ala and Leu (Lee et al., 2017).
Invertases: Carbohydrases that hydrolyze sucrose and polysaccharides into
fructose and glucose as final products. They are used as a catalytic agent in obtaining
an artificial sweetener and so it is used in food industry for confectionery, syrups,
condensed milk, infant milk, and beverages (Kotwal & Shankar, 2009). When
sucrose is present in higher concentrations invertases are able to synthesize fructo-
oligosaccharides through fructotransferase. Though the natural sources of invertases
are plants, bees, and microorganisms, the most prominent organisms used for
invertase production are the filamentous fungi belonging to the Aspergillus genus
and yeast such as Saccharomyces cerevisiae and Candida utilis (Veana et al., 2018).
From Aspergillus genus A.niger, A.terreus, A.japonicus, A.versicolor A.tubingensis,
A.fumigatus, A.oryzae, A.aculeatinus, and A.homomorphus are the main source.
The most important characteristic required for the invertase application in the
industry are pH and thermostabilities (50–75  C). Fungal invertases have these
features, i.e., they have these optimal values and stabilities but low-specific activity.
This limitation had been overcome using gene modification in the host organism.
Invertase produced by genetically modified Saccharomyces cerevisiae where the
enzyme was modified by the substitution of hydrophilic residues in the active site
region or peripheral loops with hydrophobic amino acids have optimal pH and
stability ranges for sucrose breakdown into glucose and fructose (Sainz-Polo et al.,
2013) Genetically modified invertase with high transfructosylating activity is used
for simple and efficient production of prebiotic fructo-oligosaccharides.
β-galactosidase: commonly known as lactase, is an enzyme that hydrolyzes
lactose. This enzyme has wide applications in food processing industries. It helps
in the digestion of lactose present in dairy products and converts it to glucose and
galactose. This enzyme holds importance in food industries as it manufactures
lactose-hydrolyzed products which act as a “predigester” for people with milk
intolerance problems, milk destined for cheese and yogurt making, for the produc-
tion of sweeteners and hydrolyzed whey syrups (Dekker et al., 2019). Lactases are
used in the production of ice cream and sweetened flavoured and condensed milks.
The sources of β-galactosidases are microbial, plants, and animal origin. Among
these sources, microbial lactase shows higher productivity, and it is cost-–effective.
Bacteria, fungi, and yeasts are the microbial sources. On a commercial and industrial
372 S. Muthusamy et al.

scale, the most commonly used sources of β-galactosidase are Aspergillus and
Kluyveromyces (Saqib et al., 2017). The enzyme source is selected depending on
the required reaction conditions. Bacterial β-galactosidases work with optimal pH
between 2.5–5.4 and are mainly used for acidic whey hydrolysis whereas yeast
β-galactosidase shows maximum activity at pH 6.0–7.0 which is more suitable for
the hydrolysis of milk and sweet whey. The temperature at which lactose-free milk
product is processed is relatively low. The lactase derived from the dairy yeast
Saccharomyces lactis has optimal process conditions (35–40  C, pH 6.6–6.8) that
are close to the natural temperature and pH of the milk (O’connell & Walsh, 2006). It
shows considerable activity at lower temperatures, down to about 4  C, and slows
the growth of spoilage bacteria thus it is the most widely used commercial lactase
source. Another source for lactase is fungal lactase derived from Aspergillus niger.
Its optimal process conditions are around 50  C and pH 3.5–4.5 so its application is
limited to acid (or acidified) whey or lactose.
To satisfy the growing demand for lactase the development of genetic engineer-
ing technology makes the large-scale industrial production of lactase possible
through the introduction or modification of genes to promote characteristics such
as higher enzyme activity or higher production. Modification of lactase produced by
microbes through directed evolution had high activity in industrial type conditions
for milk processing, that is, substrate lactose, buffer pH 6.75, and 8  C. The
production of recombinant strains using the lacA gene from Aspergillus niger and
Saccharomyces cerevisiae can produce a high quantity of lactase (Domingues et al.,
2010).
Proteases/peptidases: The major use of proteases in the food industry is the
hydrolysis of protein matrices to enhance flavor, texture, or functional properties in
dairy, meat, and fish products. Commonly used food proteases are derived from
animals which include trypsin, chymotrypsin, chymosin/rennet, pepsin, etc., and
papain, bromelain, and ficin from plants and from microorganisms microbial acid
protease, proteinase A, alkaline proteases, flavourzyme, etc. (Mazorra-Manzano
et al., 2018). Microbial proteases had vital roles in the production of traditional
fermented foods.
Trypsin: Trypsin belongs to the serine protease superfamily that cleaves on the
carboxyl end (C-terminal) arginine (Arg) and lysine (Lys) on peptide chains. Trypsin
is used as a baking enzyme to improve the workability of dough, in the extraction of
flavorings from vegetable or animal proteins, to control aroma formation in cheese
and milk products, to improve the texture of fish products, to tenderize meat, in the
production of hypoallergic food, i.e., proteases can break down specific allergic
components present in cow’s milk into nonallergenic peptides and diminishes the
risk of babies developing milk allergies (Bu et al., 2013).
Usually, trypsin for food uses has been obtained from purified extracts of bovine
or porcine pancreatic tissue but the trypsin from mammal sources has several
limitations, including high costs, security, and ethical issues. So microbial trypsin
is employed for easy preparation and low costs. But there have been a few strains,
including those from the genera of Fusarium, Streptomyces, and Trichoderma
reported. The reported microbial trypsins show poor specific activity and operational
13 Enzymes from Genetically Modified Organisms and Their Current. . . 373

stability. Thus, trypsin is derived from genetically modified microorganisms for

example F.venenatum containing the gene for trypsin from F.oxysporum, which is
used as a food processing aid. The genetic modifications improve serine protease
enzyme yields. Studies also showed that trypsin produced by recombinant Strepto-
myces griseus showed higher enzyme activity.
Chymosin (rennin): is an aspartyl proteinase. It is found in the fourth stomach of
the unweaned calf. It leads to limited or partial proteolysis of K-casein in milk
resulting in clotting. The enzyme is used in the food industry as a processing aid for
the manufacture of cheese and curd and other milk coagulants. As an industrial
enzyme it is widely used in cheesemaking and it was mainly extracted from dried
calf stomachs. But the cheesemaking industry has expanded beyond the supply of
available calf stomachs and in order to meet the increasing global demand expres-
sion of chymosin has been achieved in many species such as Escherichia coli,
Bacillus species, Aspergillus species and yeast (Wei et al., 2016). Thus 80% of
fermentation chymosin is produced as recombinant proteins in microorganisms with
protein produced in Aspergillus and yeast the most widely used.
Expression of yak chymosin in gene-recombinant Pichiapastoris is the method
used for large-scale production of chymosin (Ersoz & Inan, 2019). Some
Lactococcus spp. also help in the production of chymosin at an industrial scale.
Some other common microorganisms that are involved directly or indirectly in the
production of chymosin are E. coli, Kluyveromyces lactis, P. pastoris, Aspergillus
awamori.
Aminopeptidases: Enzymes that hydrolyzes the peptide bond of N-terminal
amino acids in peptides and proteins. Cleavages by the peptidases will change the
flavors of the proteins in a food matrix. Therefore, aminopeptidases are used in the
production of cheese, beverages, flavorings, meats, and milk products (Nandan &
Nampoothiri, 2020). They are specifically meant to enhance and optimize aroma and
flavor. The addition of aminopeptidases can accelerate the maturing of cheese.
Various recombinant aminopeptidases have been produced from Lactobacillus
rhamnosus, Lactococcus lactis, and Aspergillus sojae.
The activity and stability of proteases depend on the amino acid composition and
their respective position. Protein engineering modifies the protease structure in such
a way that enhances the activity, specificity, and stability of protease. Bacillus spp.
are the main producers of alkaline proteases with high thermal and pH stabilities.
The cold activity at 10  C and alkali-resistant properties of a Bacillus alcalophilus
alkaline protease were enhanced through directed evolution using error-prone PCR
for use in cold-temperature food processing (Liu et al., 2014). Similarly, an alkaline
serine protease from mesophilic Bacillus pumilus was engineered to have increased
hydrolytic efficiency at 15  C without compromising thermostability (Zhao & Feng,
2018). Acid protease from a mutant A. oryzae strain was produced using solid-state
fermentation with potato pulp powder, with enhanced glycine releasing activity
(Murthy & Kusumoto, 2015). A truncated neutral protease from A. oryzae had
optimum pH of 8.0 and an optimum temperature of 55  C, and its enzymological
characterization suggested that it was efficient in antihypertensive peptide produc-
tion, debittering, and food oil processing (Ke et al., 2012).
374 S. Muthusamy et al.

Lipases: Lipases are the enzymes that catalyses the hydrolysis of long-chain
triglycerides (fats and oils). These are used for the following applications: dairy,
baking, cocoa butter substitutes, human milk fat substitutes, egg processing, and
edible oil production. In baking and dairy applications, the addition of lipases
enhances and accelerates the development of aromatic notes (Goncalves et al.,
2019). In vegetal oil processing, lipases allow a significant increase in oil yield
and enhance the appearance of end product, i.e., improves their texture and softness.
Also, lipases are used to enhance the emulsification properties of egg yolk lipids.
These enzymes help in developing new functional ingredients and functional foods,
such as cocoa butter equivalents or human milk fat equivalents. Lipases help in the
breakdown of milk fats and provide characteristic flavors to cheeses. The flavor is
due to the release of free fatty acids produced during the hydrolysis of milk fats.
Microbial lipases are produced by fungal, yeast, and bacterial species. Candida sp.,
Aspergillus sp., Rhizomucor sp., Rhizopus sp., Humicola sp., Yarrowia lipolytica,
and Pseudomonas sp., etc. are microbial sources for lipase (Tan et al., 2003).
Lipases are used widely for different applications in the food industry and thus it
is necessary to meet the increasing demand and enzymes capable of tolerating
industrial conditions. Lipase secretion by Rhizopus oryzae was improved by rational
design of the N-glycosylation sites especially for the use in the edible oil and fat
industries (Yu et al., 2017). Genetically modified Candida rugosa by site-directed
mutagenesis produces lipases having high catalytic efficiency for producing fatty
acid methyl esters and diglycerides as food emulsifiers (Chang et al., 2014).

13.4 Regulatory Management of GMO-Derived Enzymes

The Generally Recognized as Safe (GRAS) is well-known process that gives general
supporting materials such as methodology involved and safety of the enzymes used
in food industries from genetically modified organisms. Section 201(s) of the Act
[21 U.S.C. 321(s)] exempts the use(s) of a substance that is GRAS from the
definition “food additive.” In general, the enzyme preparations should confirm the
purity and specifications according to “Enzyme Preparations” of the 6th (2008) or
the current edition of the Food Chemicals Codex (FCC). According to the USA, if an
enzyme is produced to use in meat and poultry products, FDA has to approve it in
consultation with the Food Safety and Inspection Service (FSIS) of the U.-
S. Department of Agriculture. The Food Allergen Labeling and Consumer Protec-
tion Act of 2004 (FALCPA) is used to prevent food allergens contamination from
enzyme. Genetically modified food enzymes, which are produced from genetically
modified organisms fall under the regulation, (Regulation (EC) No 1829/2003)
according to these regulation food enzymes produced from a part or whole of a
genetically modified organism (GMO) but it does not contain GMO. Processing aid
of the enzymes is not included in this regulation because food produced with GMO
are not included in this regulation. Genetically modified microorganisms (GMM)
which are producing food enzymes and are available in commercialized preparations
need authorization according to the regulations (EC) No. 1332/2008 and (EC) No.
13 Enzymes from Genetically Modified Organisms and Their Current. . . 375

1829/2003. The European regulation (EC) No. 1829/2003 is only regulation for the
material which is produced from genetically modified sources is used in enzyme
preparation or food production. Food enzymes fall under two categories either an
ingredient or processing aid. The risk assessment performed by the European Food
Safety Agency (EFSA) for food additives requires European regulation (EC) No
1129/2011.
There are many other European regulations for the safety evaluation for use of
food enzymes in terms of food additives, enzymes, flavorings they are (1) (EC) No.
1332/2008: Food Enzymes (2) (EC) No. 1334/2008: Food Flavourings (3) (EC) No.
1331/2008: Common Authorization Procedure (4) (EC) No. 1333/2008: Food
Additives (The European Parliament and the Council of the European Union,
2008). Some specific food enzymes had already been given authorization for use
according to regulation (EU) No. 231/2012 (The European Parliament and the
Council of the European Union 2008), which allows the specifications according
to regulations (EC) No. 1333/2008 and (EC) No. 1493/1999 lysozyme and invertase
are authorized for use in food additives. Lysozyme, Urease, and β-glucanase are
authorized for use in wine production according to regulation (Deckers et al., 2020).

13.4.1 Safety Concern for GMO-Derived Enzymes

Deriving food enzymes from Genetically Modified organisms have raised concerns
regarding the chance of contaminating the food with bacterial toxins or mycotoxins,
or some uncharacterized extraneous substances that may act as allergens (de Santis
et al., 2018). Since the organisms itself is genetically modified there is also a chance
for modifying their allergenic properties which increases the safety concerns
(Olempska-Beer et al., 2006) There are situations in which industrial workers
exposed to environments using genetically modified enzymes have developed type
I sensitization (Budnik et al., 2017). The regulation of enzymes including those
produced by genetic engineering is a major concern in the food industry. So before
bringing genetically modified food enzymes into the market, it is necessary to obtain
approval from various regulatory bodies such as the US Food and Drug Administra-
tion, the Association of Manufacturers and Formulators of Enzyme Products, the
European Food Safety Authority, and these processes vary in different countries.
Also, ethical and religious concerns are raised for genetically modified enzymes,
such as the raw materials used in the fermentation of microorganisms for the
production of enzymes should be halal (Ermis, 2017).
Genetically modified food enzymes are typically better suited for specific indus-
trial applications than their native counterparts, and research on their enzymological
properties in comparison to wild-type enzymes enables us to better understand how
to optimize structure-function relationships. Currently, novel genetically modified
food enzymes are mostly used for applications in food processing involving
carbohydrates, followed by lipids. Although there are challenges for safe use,
genetic modification strategies for the development of novel food enzymes are
highly promising for the future, and we expect that more genetically modified
376 S. Muthusamy et al.

enzymes will be introduced in various aspects of food processing. The production of

enzymes by the process of genetic engineering emphasizes on economic production,
enhanced enzyme purity, and naturally responsive production processes. Genetically
modified microbes are used to produce a wide variety of products, ranging from
synthetic insulin to vitamin C and enzymes like chymosin involved in food applica-
tion. Appropriate purification, concentration, and formulation of enzymes with
appropriate substances are crucial for further use in food industries (Hanlon &
Sewalt, 2020).

13.4.2 Pros and Cons of GM Food Enzymes

13.4.2.1 Improvement in Food Processing

Increased scarcity of arable lands with increased population forced to use recombi-
nant DNA technology in food industries. Several platforms are working together to
make and deliver GM food enzymes as risk-free safe product (Fig. 13.6). The
achievements in molecular biology in the past several decades have led to a
widespread use of GM microbes in the production of food substances including
enzymes. Currently, these genetically modified enzymes are employed in several
food industries to improve in food processing. Manipulation of regulatory genes,
restrict feedback inhibition, increase the shelf life, overcome the rate-limiting steps,
reduce the process cost and production time, environment friendly are some of the

Fig. 13.6 Improved technologies currently involved in genetically modified enzyme production
13 Enzymes from Genetically Modified Organisms and Their Current. . . 377

major improvements that GM microbes could provide. For example, the use of
enzyme Amino peptidases to modify by debituminize proteins which helps in the
large scale production of food products such as milk, cheese meat, beverages and
flavoring (Molinaro et al., 2005; Raksakulthai & Haard, 2003; Izawa et al., 1997).
Pectinolytic enzymes are also widely used in the food industry for large-scale
production of wine and juice production (Semenova et al., 2006).

13.4.2.2 Health and Ecological Risks Associated with GM Food Enzymes

Smaller recombinant enzymes are easily over-expressed in E.coli, but the larger
proteins and the proteins that require posttranslation modifications cannot be
expressed. Major health-related risks associated with GM foods enzymes are
allergenicity, genetic hazards, and toxicity. These problems are associated with the
change in pleiotropic effects when a new gene is inserted into the organism which
may also cause the deletion of natural genes in the modified organism (Bawa &
Anilakumar, 2013). Ecological risks associated with the food enzymes are resistant
to antibiotics and in this genetic modification, antibiotics are frequently used as
selection markers to distinguish whether the genes got transformed into bacteria.
Thus genetically modify an organism have the risk of transferring the genes of
antibiotics resistance.

13.5 Conclusion and Future Aspects

It has been more than 15 years, since the use of genetically modified enzymes in the
food industry. With the development of genetic engineering and recombinant DNA
technology, the production of enzymes enhances the natural responsive production
and purity of enzymes. It also offers safe handling and cost-effective production of
enzymes in the commercial market. The prime factor to be considered before
commercialization of a food enzyme is the host organism that expresses an enzyme.
A microbe used in enzyme production should be well characterized, which should
not make any adverse effects by synthesizing byproducts like toxins. The safety and
hazardous risks of the enzymes from genetically modified organisms have to be
evaluated similarly to other enzymes those in use.

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Current Applications of Enzymes in GM
(Genetically Modified) Food Development 14
and Food Chain

Nafiseh Sadat Naghavi, Fatemeh Mahmoodsaleh,

and Masoumeh Moslemi

Abstract

New applications are introduced for food enzymes with regard to their impact on
the improvement of organoleptic attributes and quality of foods other than their
cost-effectiveness compared to chemical alternatives. Along with technological
advances, novel and specified enzymes have been widely used in food industries.
The enzymes may be extracted from plants and animals or be produced through
biotechnological processes. In this regard, microbial fermentation is preferred
compared to the other sources to some extent because it is a convenient process,
needs lower cost, and is conducted consistently in the laboratory. Moreover, the
fermentation process can be optimized by the use of genetically modified
(GM) microorganisms or the addition of recombinant enzymes through which
the production yield will be increased significantly. This chapter overviews the
use of GM microorganisms in order to produce food enzymes. Advantages of the
biological systems including recombinant DNA and metabolic engineering for
the production of food enzymes are also discussed. Due to the importance of
changes that occurred in the structure of GM enzymes, their halal status is further
studied. In addition, the main regulations set by countries and the safety
evaluations by risk assessment agencies on GM enzymes are addressed.

N. S. Naghavi
Department of Microbiology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
F. Mahmoodsaleh
Department of Biology, Faculty of Sciences, Shahid Chamran University, Ahvaz, Iran
M. Moslemi (*)
Halal Research Center of IRI., Ministry of Health and Medical Education, Tehran, Iran

# The Author(s), under exclusive license to Springer Nature Singapore Pte 383
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_14
384 N. S. Naghavi et al.

Keywords

GM enzymes · Halal status · Recent developments · Regulation · Safety concern

14.1 Introduction

It was predicted that the market volume of industrial enzymes will have a significant
growth in the early future years (Robinson, 2015). The utilization of enzymes
especially for the production of food and beverages represents a wide industrial
enzyme market. For industrial use, it is possible to extract enzymes from plant and
animal sources or obtained by fermentation using Wild-Type (WT) or Genetically
Modified (GM) strains of bacteria or fungi for which both have been manipulated to
enhance protein production and limit the production of unfavorable secondary
metabolites. In addition to genetic modifications of the desired strain, it is also
possible to modify the enzyme itself for the purpose of obtaining an improved
yield and improve the characteristics of enzymes (Singh et al., 2016). On the other
hand, by genetic modification of producing microorganisms, the enzyme can be
modified to have better performance and improved properties.

14.2 Food Enzymes Production by Genetically Modified

Microorganisms

Foods that contain, or manufactured from genetically modified organisms (GMOs)

are defined as GM foods (Robinson, 2015). The utilization of GM for the industrial
production of food enzymes (FE) is beneficial because it allows suitable production
sources to be combined with the production of the desired enzymes. For example, in
the case of the native enzyme producer strains that are not comfortable for industrial
applications, such as the enzymes producing pathogenic strains, the coding genes
can be extracted and introduced into other organisms that are more appropriate
according to food safety for the production of enzymes. Genetic modifications are
also used to inhibition the production of unwanted secondary metabolites such as
toxins, by deletion of their native genes to prevent their expression. To build an
effective recombinant strain, various points must be taken into account. First, it is
crucial to select a host organism which has the proper properties for the expression
and production of the desired enzyme and has a relatively inexpensive culture. Then,
vital parameters are used to catch the optimal GM for industrial use including the
expression vector, the methods employed for transformation, and the selection
markers to choose the transformed strains must be specified (Liu & Kokare, 2017;
Deckers et al., 2020).
14 Current Applications of Enzymes in GM (Genetically Modified). . . 385

14.2.1 Selection of Host Organism

The reasonable host strain is selected on a case-by-case analysis. The most used host
are discussed below.

14.2.1.1 Bacteria
A simple and inexpensive host organisms are bacteria, with many expression
systems suitable for recombination, leading to an easy combination of modifications.
However, because of protein folding issues, only simple proteins are made in
bacteria. In addition, bacteria are unable to make posttranslational modifications
(Deckers et al., 2020).
Commonly used bacterial host strains are including Escherichia coli, Bacillus
subtilis, B. licheniformis, and B. amyloliquefaciens. For E. coli, many molecular
toolboxes are identified to ease the construction of expression systems available for
high-yield enzyme production. However, proteins are produced in the cytosol,
leading to the requirement for extra purification steps. Nevertheless, various species
of Bacillus can secrete higher concentrations of the enzymes such as amylase and
proteases into the medium (Zhang et al., 2019; Trono, 2019).

14.2.1.2 Fungi
Fungal host strains contain a subcellular organization, which lets proteins be folded
properly and perform posttranslational modifications. Moreover, fungi secrete few
amounts of their own secondary metabolites into the medium, easing the subsequent
purificationsteps. Any other way, filamentous fungi generally produce low numbers
of non-fungal recombinant proteins (Deckers et al., 2020).
Filamentous fungi that commonly used for the production of recombinant
proteins are Aspergillus niger, A. oryzae, and Trichoderma reesei. A. oryzae, for
instance, has been utilized for decades to produce fermented foods, but the produc-
tion of the mycotoxin, aflatoxin is also known. To solve this problem, the mutant
strain BECh2 was built, in which the cluster of the aflatoxin gene and the
cyclopiazonic acid coding gene was removed and the production of Kojic acid
was declined (Fernandes, 2010; Deckers et al., 2020).

14.2.2 Construction of Expression Vectors and Transformation

Processes

Expression vectors are used for introducing modifications indifferent host strains.
The expression vector usually includes at least One Origin of Replication (ORI), a
Multiple Cloning Site (MCS), where the gene is integrated, and a marker for
selecting the modified strain (Fig. 14.1). Besides, the promoter and terminator
regions are available to control the expression of the desired gene and selector
marker. To prevent an increase in metabolic expressions in the host organism and
a decrease in plasmid stability, small size is considered for the vector (Patel et al.,
2017).
386 N. S. Naghavi et al.

Fig. 14.1 Schematic representation of the expression vector. (a) Expression vectors include the
origin of replication (ORI), the multiple cloning site (MCS) in which the desired gene is integrated,
and a Selection Marker (SM). (b) An episomal plasmid and an integrated plasmid, containing a
flanked integration site can be formed. ARS is an independent replication sequence and IS
represents the integration site (Patel et al., 2017)

Bacterial expression vectors can either be used in the form of integration into the
host genome (integrative plasmid) or they can be extra-chromosomal, utilizing
independent replicating plasmids (episomal vector). Yeast and fungal expression
vectors are usually integrative. In addition, shuttle vectors are commonly used for
eukaryotes. These vectors can inter both prokaryotes and eukaryotes, and contain
selective markers that work in both MB groups. To insert the host genome, the
integrative plasmid consists of two Integration Sites (IS) that flanks via the Multiple
Cloning Site (MCS), allowing the cleavage of the vector and then consolidation by
recombination in the host genome containing homologous ordering to the IS. The
integration usually takes place at a unique locus. In such a case, an extension
homologous to a specific desired site needed to be added to the expression cassette
(Patel et al., 2017).
Episomal plasmids contain an origin of replication (ORI) that is homologous via
the ORI of the host type. Autonomous replication in eukaryotes utilizes an Autono-
mous Replication Sequence (ARS), which is the same as the ORI found in
prokaryotes. Different methods of transformation are employed to introduce the
vector DNA into the host organism. For bacterial host strains, this method contains
conjugation that needs cell-to-cell contact, electroporation that is done by using
competent cells, or vector incubation by constructing protoplasts. Protoplast fusion
is mainly utilized for gene transfer into eukaryotes (Deckers et al., 2020).

14.2.3 Induction of Gene Expression

In order to the purpose of catch a high enzyme yield, an effective expression of the
interested gene is required. One of the most common strategies used to upgrade the
expression level is done by mix integrations (Patel et al., 2017). For this purpose,
14 Current Applications of Enzymes in GM (Genetically Modified). . . 387

various procedures are used, such as the use of high quantities of DNA in the
transformation process; high antibiotic concentration pressure that putting to use;
or simultaneous or sequential integration of multiple expression vectors that involve
different markers for the selection of transforms or via applying copies of two
preferred genes in a bidirectional vector (Deckers et al., 2020).
Other important factor affecting gene expression is the promoter strength. The
expression is usually conducted by a group of inducers and suppressors that distin-
guish constitutive promoters from tunable promoters. Constitutive promoters are
independent of the transcription factors that brought about environmental agents and
regulate the transcription from basal genes. Tunable promoters, as opposed to
constitutive promoters, are induced or suppressed by the biotic or abiotic factors in
the environment (Fitz et al., 2018). For example, amyL and amyMare, respectively
used as promoters for B. stearothermophilus maltogenic amylase and
B. licheniformis α-amylase genes (Deckers et al., 2020), and TAKA amylase
promoter is usually utilized in A. oryzae (Vieille & Zeikus, 2001). The last tunable
promoter is activated via the transcription induceramyR (amylolytic gene expres-
sion) and is suppressed via the regulator creA (carbon catabolite suppressor)
(Nedovic et al., 2011). Another promoter that is commonly used by T. reesei or
some other fungi, is the promoter of cellobiohydrolase I gene (cbh1), and activated
by cellulose (Olempska-Beer et al., 2006; Trono, 2019).
A simple solve for tandem repetition of one-directional expression cassettes is to
apply bidirectional promoters (BDPs). These primers lead to the bidirectional gene
transcription from one or more than one gene (Hui et al., 2006). Combination
expression of the desired gene and the selective marker is also available (Rieder
et al., 2019) while in order to use industrially, larger groups of accessed promoters
are required. These promoters control variable expression rates as well as combining
various regulatory profiles that are required in each expression direction (Yan & Wu,
2017).

14.2.4 New Technologies to Introduce Genetic Modifications

Recently, new technologies become involved in genetic engineering. The example

of these technologies is “Clustered Regularly Interspaced Short Palindromic Repeat”
and “CRISPR-associated protein 9” (CRISPR/Cas9), that derived from archaeal and
bacterial cells (Meyer, 2008). The Cas9 endonuclease makes Double-Stranded
Breaks (DSBs) in the desired DNA. By the application of endogenous repair routes,
the genomic DNA can either be conveyed via offering transformation or by the
insertion of a particular donor sequence. This is done through the Non-Homologous
End-Joining (NHEJ) DNA repair processes or by the Homologous Repair
(HR) technique (Song et al., 2019; Deckers et al., 2020). Opposed to eukaryotes,
the NHEJ DNA repair system is not present in bacterial cells. A, these bacteria are
not able to remedy the CRISPR/Cas9-mediated leakages, leading to the demise of
wild-type strains. Therefore, the CRISPR/Cas9 system can request an election of
recombinant types as a substitute for antimicrobial coding marker genes (Deckers
388 N. S. Naghavi et al.

et al., 2020). Using this method, the production of extracellular pullulanase has been
induced in a strain of B. subtilis. Because proteases can debase other enzymes, like
pullulanase, the genes involved in protease generation have been removed (Dijck
et al., 2003). The genes responsible for the production of unfavorable products
during the fermentation such as foam, or involved in spore formation may also be
disrupted to construct a more suitable strain for food enzyme production (Clyne &
Kelly, 1997). CRISPR/Cas can also be requested for the transformation of
eukaryotes. For instance, a strain of Penicillium subrubescens has been constructed
in which the ku70 gene was deleted. This gene has participated in the
non-homologous end-joining (NHEJ) DNA rebuild system. With this deletion, the
homologous repair (HR) system is preferred, and for this reason, the subjunction of a
wanted DNA sequence into the leakage site is performed (Zou et al., 2012).

14.3 Recombinant Food Enzymes

The origin of enzymes usage in industries of food processing dates back to 1874,
when Christian Hansen obtained rennin (chymosin) from calves’ stomach and used
it for cheese production (Petersen et al., 2006). Chymosin is now made up of
microorganisms that involve the bovine prochymosin gene, which is imported by
recombinant deoxyribonucleic acid (rDNA) technology. The bovine chymosin,
expressed in E. coli K-12, is the first recombinant enzyme approved by the Food
and Drug Administration (FDA) for use in the food industry (Flamm, 1991).
Different enzymes that are utilized in food staffs are developed in recombinant
microorganisms. Enzyme producers use new genetic technologies to obtain enzymes
through desired properties. These kinds of enzymes most of the time are produced by
microorganisms that are fastidious in growing on laboratory or industrial scales. By
selecting suspicious host microorganisms, it is possible to produce recombinant
strains and produce enzymes that are significantly free of unwanted enzymes or
other microbial metabolites. Advanced enhancement of food processing resulted in a
request for a wide range of food processing enzymes via properties reasonable for
food processing conditions (Beilen & Li, 2002). For instance, usually applied
sweeteners like glucose or fructose syrups are usually made from corn starch
utilizing hydrolyzing enzymes. In the first stage of starch hydrolysis, the starch is
recognized by using α-amylase for 2–5 min via heating at 105
C and next put at
90–100
C for around 1–2 h. Advances in rDNA technology have made it catchable
to engineer α-amylases by improving the stability of heat and high compatibility
with other parameters of the liquefaction stage. These improvements have been
made by understanding the α-amylase amino acid sequences via the DNA sequence
of the α-amylase genes. Another enzyme recently used in food processing has also
been cached using rDNA technology. These enzymes are well-known commercially
as enzyme ministration. An enzyme preparation usually contains the interested
enzyme and varied additional substances, such as preservatives, diluents, and
stabilizers. These extra substances are usually well-known ingredients that are
accepted for utilization in food staffs (Olempska-Beer et al., 2006).
14 Current Applications of Enzymes in GM (Genetically Modified). . . 389

There are simultaneous techniques that are possible to applied to reproduce

recombinant enzymes, such as rational design, directed evolution, and semi-rational
design, based on the amount of information about the conformational structure and
task of the enzyme, the enzyme sequences, and the alterations to be made to catch the
expected result (Rajamanickam et al., 2017; Yang et al., 2013; Vogl et al., 2018).
The basis of rational design is the replacement, insertion, or removing of a unique
DNA coding the fragment for an amino acid in relation to a specific activity that
needs to be developed. The knowledge about the structural conformation of the
protein and its chemical interactions is vital. Most of the time, site-directed muta-
genesis is beneficial for changes of a specific amino acid by another to gain the
requested activity (Rajamanickam et al., 2017; Vogl et al., 2018; Song et al., 2019).
For instance, a thermostable pullulanase from B. naganoensis was made from a
rational design. This enzyme is applied to hydrolyze amylopectin and make amylose
in starch (Donohoue et al., 2018).
Directed evolution is consisting of two steps. First, is genetic diversity which is
produced by random mutagenesis, and is an imitation of evolution. This can be done
by error-prone PCR, mutagenesis via chemicals, or UV irradiation. In the second
step, a thorough screening and detection process, which is usually performed by
agar-plate method, to select the modified enzymes that have the desired
characteristics. For this case, it is not necessary to have knowledge about the
enzyme’s structure and activities (Rajamanickam et al., 2017; Vogl et al., 2018).
The semi-rational design combines both the above techniques. In order to this
reason, information is catchable for the enzyme’s structure and tasks. Plus, there is
no understanding about the frequency to be made to gain the desired results.
Therefore, in the first step, amino acids undergo mutations by a specific fragment
(in relation to the desired properties). Then a screening and evaluation step is
completed, like the evaluation of directing (Song et al., 2019; Rajamanickam
et al., 2017).

14.4 Application of Metabolic Engineering and Synthetic

Biology in Food Enzyme Production

Metabolic engineering is used to optimize cellular procedures, native to a particular

organism, to make a combination of interest from a desirably inexpensive and simple
substrate. Utilizes different collections of data, a set of compositions and conditions
are applied to maximize the generalization of the desired compound and to prevent
chemicals and conditions that inhibit the update rate and other specific critical
activities available to reach these goals; metabolic fluxes modifications show the
main option. In summary, metabolic engineering is known as the directorial
advancement of cellular properties by modifying specific biochemical reactions or
the introduction new reactions using recombinant DNA technology. Some common
strategies used in metabolic engineering are overexpression of enzyme-encoding
genes that limit the rate of the biosynthetic pathway, block rival metabolic pathways,
390 N. S. Naghavi et al.

heterologous gene expression, and enzyme engineering (Stephanopoulos, 2012;

Kim et al., 2020).
Synthetic biology point to design genetic composition libraries including
promoters, codons, terminal sequences, transcriptional factors, assembling devices,
genetic circuits, and even a perforated organism; in addition to obtain quantitative
information to create models that can demonstrate the behavior of biological systems
(Cameron et al., 2014).
While synthetic biology provides champers and information on various biological
processes, metabolic engineering tries to utilize all of this information to become
optimal for the biological production of the desired compound. It is possible to note
that some instances of synthetic biology can be arranged as examples of genetic
engineering. However, these two areas are depending on the development of real
methods, and tools for DNA transformation. These fields of research seek to achieve
basic needs such as arbitrary changes in DNA sequences, production of specific
mutations, the recombination between a variety of genetic circuits or biosynthetic
pathways, the deletions of genes, and placement of DNA fragments into the organ-
ism genome or in a plasmid. Although PCR and its related methods are one of the
superior equipment for making some necessary changes (specifically applied for
extracting parts of a particular area) they are ineffective for other purposes (Boyle &
Silver, 2012). Some examples of employing metabolic engineering processes to
evolve in the production of food enzymes are discussed here.
By replacement of the bacterial genes with yeast-preferred genes, Wang et al.
(2011) performed mutation and synthesis of tyrosine ammonia lysine (TAL), which
elevated translation and vigorously increased p-coumaric acid and resveratrol bio-
synthesis. They showed that a low-affinity, high-powerful bacterial araE transporter
could increase resveratrol yield, without direct resveratrol transfer. Yeast cells
containing the araE gene generated up to 2.44-fold more resveratrol than control
cells. They offered the transformed yeast cells for industrial applications in fresh
grape juice production.
Microbial Cell Factories (MCFs) have appeared as evolving technology for the
combination of previous single techniques and complicated multistep procedures
into a unit self-replicating microorganism (Dhamankar & Prather, 2011). The usual
way to create an MCF is to create a different or artificial de novo biosynthetic
pathway in a chassis organism to achieve a new composition. The goal product could
be native to a microorganism that is fastidiously cultured/engineered. The product
also can obtain from a complex organism such as a plant that industrial production
by inexpensive, or from a manner non-native to all microorganisms as a compound
from an artificial metabolism. The MCF has also developed a convenient route to
generate new derivatives of the desired compound. For example, phenylpropanoids,
such as resveratrol, are natural secondary metabolites produced by plants that have
been shown to have therapeutic effects and are commercially valuable. Most bio-
available derivatives of resveratrol have also been obtained from an MCF. A new
biosynthetic route for resveratrol constitution in E. coli was designed using different
enzymes obtained from bacteria and plants (Choi et al., 2010), and afterward evolved
by adding a glycosyltransferase (from Bacillus), which led to the generation of
14 Current Applications of Enzymes in GM (Genetically Modified). . . 391

resveratrol glucoside derivatives like resveratrol 3-O-glucoside and res-veratrol

40 -O-glucoside) in an E. coli MCF (Choi et al., 2014). It is with reckless enzymes
that a new combination of enzymes can give rise to new de novo biosynthetic routes
that lead to the acquisition of new chemicals by using MCFs. So long as there are
different published examples containing the yield of isobutanol (Atsumi et al.,
2008a, 2008b), styrene modification (Pugh et al., 2011), production of
3-hydroxybutyric acid (Tseng et al., 2009), or native silk protein (Xia et al., 2010).
Most enzymes in nature are able to react on a spectrum of substrates in order to
maximize evolutionary compatibility and this development may be engineered
(Khersonsky & Tawfik, 2010).
Rational design is the purification of a target DNA sequence that codes a protein
to desired mutations because of improve their catalytic task, consistency, or some
other representative like linking domain specificity. Devises such as Gibson assem-
bly, the BioBricks or Golden Gate can make different genetic systems to shape
genetic circuits, expression cassettes, or equipment (Casini et al., 2015). Finally, the
utilization of Lambda-red, CRISPR-Cas9, and other new combination systems lead
to imbed varied structures into expression vectors or in vital loci of the genome of the
desired organism. It is possible to use some of these techniques to introduce genes of
a partial or complete metabolic route for the synthesis of a special compound, and
they can also be applied to delete genes from an organism that are antagonistic to the
desired synthesis. Moreover, these methods let point mutations be performed that
reduce the function or expression of native proteins to alter the metabolic flux.
However, in order to the generation of all of these changes, it is necessary to have
specific information about the enzymes captive in the pathway that contains these
actions and information about the organism in which the modifications happen.
(Esvelt & Wang, 2013; Liu et al., 2015). Thus, the genome sequence of organisms,
the characteristics of proteins, and the metabolic studies make very beneficial
equipment and information (Fig. 14.2).
Additional techniques and procedures have been crucial to reach other goals in
the progression of both areas. Some instances include BioBricks, Gibson Assembly,
Gap-repair, Lambda-red, MAGE, Recombinase techniques (integrases), and
CRISPR-Cas9 (Stephanopoulos, 2012). Simultaneous sequencing of defined and
non-defined organisms (metagenomics), allows for gaining more completed infor-
mation. For example, the list of enzymes and processes has been developed, and
some biological phenomena (e.g., infections by viruses or phages) have been
reached (Goodwin et al., 2017).

14.5 Halal Status of Recombinant Enzymes

Halal term is a familiar concept in Muslim countries which have been interested in
non-Muslim populations in recent decades. Today, it is globally accepted that a halal
certificate refers to both religiously permitted and safe/qualified commodities. There-
fore, the halal logo facilitates international trade (Ab Talib, 2017). However, it
makes some difficulties for non-Muslim countries’ exporters such as European
392 N. S. Naghavi et al.

Fig. 14.2 Various steps for the progression of synthetic biology and metabolic engineering
procedures

because of their lower knowledge about halal (Tieman, 2017) As a global rule, foods
and their ingredients should be free of compounds that originated/are made from
haram sources or exclude alcohol. On the other hand, the animals should be
slaughtered in a way that is accepted by Islam (named as Dhabihah or Zabiha)
(Alzeer & Abou Hadeed, 2020).
Halal commitment is a critical issue in the whole food chain at the international
level. In 2001 a Japanese company was condemned due to the production of
non-halal monosodium glutamate as a flavor enhancer to achieve better economical
productivity. The process was included using BactoSoytoneas a culture media
prepared by the enzyme derived from porcine that is not acceptable in the halal
concept. This violation was further conceded by the manufacturers that were given
the halal certificate by Indonesian authorities for their products. The event obliged
other enzyme manufacturers to be approved by the halal certification bodies in the
world (Fischer, 2011). There are two types of certificating for halal foods including
1) site registration to evaluate the ability of suppliers in fulfilling the requirements
and 2) certifying the imported products (Hanzaee & Ramezani, 2011). Unfortu-
nately, the halal certifiers grow slowly compared to other scientific associations in
the world due to the disagreements on the halal concept (Halim & Salleh, 2012). On
the one hand, the term Istahala has different understanding and interpretations
among communities. There is not a consensus on the final products produced from
the non-halal origin when they underwent chemical transformation up to
microstructures or molecular level. On the other hand, there is a doubt about the
synthetic genes designed in the laboratory which are modeled from haram sources
such as pork (Riaz & Chaudry, 2019).
Enzymes are food components that are traded worldwide and their halal assur-
ance is a concern among Muslims. Food enzymes are produced from different
14 Current Applications of Enzymes in GM (Genetically Modified). . . 393

sources of plants, animals, and microbes. Several points are considered in their
eligibilities by Islamic traders. The enzymes should be extracted from halal sources
and produced in media containing halal raw materials. Furthermore, fermentation
media should contain the approved organisms other than halal ingredients if a
microbial source is used for enzyme production. It will be important when geneti-
cally modified (GM) microorganisms activate for enzyme synthesis. Based on the
World Health Organization’s definition, “genetically modified organisms can be
defined as organisms (i.e., plants, animals, or microorganisms) in which the genetic
material (DNA) has been altered in a way that does not occur naturally by mating
and/or natural recombination” (WHO, 2014). The source of genes and method of
preparation determines the fate of GM products. For example, accordance of GM
foods with religious remarks is the most important factor for Malaysians in prepara-
tion of their food basket (Oz et al., 2017). It is also a deterministic factor in Islamic
countries such as Iran through which the GM foods and components such as
enzymes first checked whether they are halal followed by safety evaluation. In this
way, the genome of host cells that is manipulated to encode the enzyme should be
originated from a halal source (Ermis, 2017). Some steps are generally monitored in
halal control points of enzymes extraction and production including the source
(animal or microbial), cleanliness of the production utilities, the agents released
into the environment, acceptable processing-aid, substrate or raw materials, and
packing process. However, up to 0.5% of alcohol may be acceptable when an
optimum activity is required (Riaz & Chaudry, 2019).
Use of animal-based enzymes is limited in some cases such as restrictions on the
dietary intake of vegans other than halal uncertainties (Nugraha et al., 2015). In
comparison, the enzymes extracted from conventional plants are not doubtful
(Khattak et al., 2011) and there is no concern about plant-derived genes inserted in
transgenic microbes (Riaz & Chaudry, 2019). However, microbial sources are used
extensively for enzyme production because of their cost-effectiveness and ease of
process control for optimization. Furthermore, it eliminates the concerns of animal
origin and slaughtering methods to meet halal criteria in enzyme production (Riaz &
Chaudry, 2019). Microbial enzymes are commonly produced by submerged fermen-
tation (Khattak et al., 2011). Several recombinant food enzymes have been
engineered to promote resistance of the wild type against harsh conditions of
temperature/pH and better reactivity of the GM enzyme with substrates in the
environment. Pichia pastoris (which was recognized as safe by US Food and
Drug Administration) (Espinoza-Molina et al., 2016) and E. coli are two species
commonly used as a host cells in this regard (Espinoza-Molina et al., 2016; Zhang
et al., 2019). In addition, GM Streptococcus thermophiles, as a common starter
culture of yogurt, capable of antioxidant enzymes’ expression was evaluated by
Carmen et al. in vitro and positive results were observed in the suppression of
oxidative stress in the colon cancer model (del Carmen et al., 2017). The bacteria
was manipulated by insertion of the gene responsible for antioxidant enzyme’
production isolated from Lactobacillus caseiBL23 as an anticancer and anti-
inflammatory probiotic bacterium (del Carmen et al., 2014; Jacouton et al., 2017)
that is considered as a halal source. Moreover, transfer of the encoding gene of
394 N. S. Naghavi et al.

chymosin from camel (Aboulnaga, 2019) and bovine (Ulusu et al., 2016) to E. coli
and from calve to E. coli, Kluyveromyces marxianus and A. niger (Fernandez-
Salguero et al., 2003) in coagulation of milk protein are other examples. At the
end of fermentation, the enzyme will be in accordance with halal merits if the
aforesaid factor is met in the processing. AbdLatip and Nordin in 2018 listed
endemic psychrophilic bacteria of South Pole as a halal source able to produce
enzymes resistant against the cold condition. In their opinion, bioengineering of
these microorganisms toward specific characteristics would be of interest in the
future because their low-temperature resistance limits the side reactions and lowers
the required activation energy (Abd Latip & Hadry Nordin, 2018).
Compared to foods, therapeutic agents may be exempted from halal regulations,
occasionally. For example, Alzeer and Hadeed in 2020 listed the orally administered
capsule of “Nutrizym 22” containing the pancreatic enzymes for pancreatitis treat-
ment as a highly critical pharmaceutical in view of halal because of its porcine origin
(Alzeer & Abou Hadeed, 2020). It is expected that there would be less restrictions on
therapeutic compounds when there are no halal alternatives.
A big deal in halal assurance is that labeling regulations in some countries suffer
from adequate liabilities. In this case, there is no obligation on listing the ingredients
of GM foods that might be originated from non-halal sources (Alhariri, 2020). With
regard to the enzymes, it is due to the fact that they may decompose during the
process, and no residue is left in the final products. Moreover, some regulatory
bodies believe that the labeling (if the product does not include significant changes in
the composition or make an adverse impact on the consumers) may threaten the
products’ sales because of consumers’ misconception about health risks arising from
GM ingredients (Uzogara, 2000).

14.6 Regulation on GM Enzymes

GM foods were firstly released to the market in 1994 in the United States by
introducing a GM tomato containing the gene expressing an enzyme responsible
for modulation of the ripening process (Ujj, 2016). Since then, the countries have
had different strategies in accepting or withdrawal of such products. In general,
European countries have more restrictions on GM foods than America, and no GM
products are approved and marketed without thorough risk assessment or clear
labeling. Although, the products processed by GM enzymes or the animals fed by
GM feeds may be considered lenient (Yuen-Ting Wong & Wai-Kit Chan, 2016). For
example, the bakery products prepared by GM amylase do not require labeling in the
EU (European Commission, 2003a). In 2019, a member of the European Commis-
sion explained the EU regulation on GM foods. Other than the risk assessment,
market surveillance, and labeling, the regulation includes risk communication of the
results of safety evaluation performed by the European Food Safety Authority
(EFSA) which is responsible for risk assessment in the EU. The regulation was
implemented by the authorities for the engineered targeted gene not conventional
breeding with a long history of safety in humans, animals, and the environment.
14 Current Applications of Enzymes in GM (Genetically Modified). . . 395

However, like many other populations, people are not interested in GM foods in
Europe and the most quantities of authorized GM products are used for animal
feeding (Bruetschy, 2019). The list of authorized GM cops by the EU is available on
the European Commission website (European Commission, 2020a) and EU regula-
tion on genetically modified food enzymes is addressed in the directives of EC
No. 1332/2008 and EC No. 1829/2003 (European Commission, 2003b, 2008). As
mentioned, safety evaluations of GM food enzymes in Europe are done by EFSA
and the results are freely published to be available within the EU and for other
countries. The approved enzyme by EFSA can be commercialized under the rule of
the two above directives. The EU directive on GM modified food and feed, which is
in accordance with the Cartagena Protocol on Biosafety, clearly mentioned that the
processing aids are out of the scope of the directive and considered differently
(European Commission, 2003b). Therefore, if the food enzyme is used as a
processing aid so that irreversibly denatures, degrades, or removed before consump-
tion, is not covered by the directive. On the contrary, if the food enzyme is an
ingredient or additive in the food, it will fall under the scope of the directive of GM
food and feed that should be mandatorily listed on the labeling. Importantly, there is
an exception about the enzymes are or possibly contain allergenic or intolerant
compounds that should be mentioned on the labeling every time (European Com-
mission, 2014).
The United States has the biggest share in the production of GM foods in the
world (Oz et al., 2017). It was estimated that the arable land in the United States is
1.4 times more than that Europe but the GM crops are cultured approximately
600 times in the United States (Ujj, 2016).
Three governmental bodies in the US are involved in the safety evaluation of GM
foods from farm to fork that in turn included the Department of Agriculture (USDA),
Environmental Protection Agency (US EPA), and Food and Drug Administration
(US FDA). The first one engages in the protection of GM plants in the farm from
pests and diseases and also protects the environment against possible risks arisen by
GM crops. The second body is responsible for the safety of both environment and
humans faced with the GM traits. The last administration is responsible for the safety
of GM foods and feeds taken by humans and animals (Schiemann et al., 2019). The
US regulatory bodies focus on the GM characteristics rather than the method of
product’s synthesis. The USDA classified the GM seeds to regulated and deregulated
articles which are those that require direct- and exempted from overseeing, respec-
tively. It is based on the known safety level of the seeds. A joint collaboration of
USDA and US EPA is required for the safety evaluation of GM edible crops
containing pesticides. In this way, the producer should examine and present the
amount of pesticides existed in the final food to US EPA. The producer is allowed by
US EPA to conduct a field trial of the edible pest-resistant GM crops on a farm of 0.5
hectares or larger under US EPA consultation. The USDA studies source of the gene
producing the toxin, its characteristics, the environmental impact, and its impact on
nontarget organisms while the US EPA is responsible for toxicological,
allergenicity, and gastrointestinal fate studies on the toxin in the GM corps (Craig
et al., 2012). Other than plant-sourced biotechnological products, the safety
396 N. S. Naghavi et al.

investigation of GM microorganisms and their byproducts such as enzymes and

other chemicals is led by US EPA (Wozniak et al., 2012). After approval, the product
can be commercialized preferably under the consultation of the US FDA which
oversights the safety of GM foods through which the producer is the holder of safety
responsibility (Craig et al., 2012). List of FDA-approved GM crops is freely
available on its website for the readers (US FDA, 2019).
Less restrictions exist in the United States on foods containing GM ingredients
that are overseen by the US FDA. The administration has stated that GM foods are as
safe as or better than their non-bioengineered counterparts to some extent (US FDA,
2020a). The agency supposed that GM food does not impose new/different risks to
the consumer compared to its counterpart. Therefore, no need for pre-market
approval is required unless there would be evidence of safety concerns of the
bioengineered product other than those expected in the wild type. The US FDA
believes that an initial assessment of the food additives was undertaken and further
evaluation on the GM counterparts will not be necessary if the new event and trait
introduce no side-effect in humans (Yuen-Ting Wong & Wai-Kit Chan, 2016) It is
against the public perception about this kind of foods. Scott et al. in 2016 reported
that nearly 64% of Americans did not agree with GM foods and most of them would
not be affected by further informing strategies conducted on risks and benefits (Scott
et al., 2016).
Commercialization of GM foods in the US can be accompanied by FDA consul-
tation but is not compulsory to the industries. The US FDA persists in pre-market
examinations conducted by the producers to find out whether the GM foods
containing the gene extracted from the allergenic source makes an immune response
in humans. It would be well feasible for the known allergenic foods such as Brazil-
nut but will be a big deal for unknown sources (Jacobson, 2000).
Nonetheless, no obligation is assigned for labeling GM foods in the United States
because the US FDA believes that the bioengineered food is not majorly different
from the wild type and labeling may make the additional cost to the consumer
(Marchant & Cardineau, 2013). However, some states in the New England region
enacted local rules on GM labeling. For example, Vermont State in 2016 and Maine
in 2013 (northeast of the US) were obliged listing the GM ingredients when they are
added more than 0.9% to foods or Connecticut State (northeast of the US) in 2013
mandated GM infant formula labeling (Oz et al., 2017).
China is a competitor of genetic modification in the world with a strict rule for
approval by using widespread research. Initiation of GM foods’ investigation in
China was later than the European Union and the United States. However, the
current president of China in 2013 emphasized the both issues of safety and
innovation in bioengineering so that the marketed GM products should be approved
by strict regulations and the country does maintain its superior position in GM
commercialization (Feng & Yang, 2019). China is one of the most consumers of
GM crops especially grains in the world. The Chinese government tried to speed up
the commercialization of GM foods in the country but it did not succeed as expected
because of consumers’ concern (Yuen-Ting Wong & Wai-Kit Chan, 2016). Direct
responsible for agricultural genetic modification in China is the Ministry of
14 Current Applications of Enzymes in GM (Genetically Modified). . . 397

Agriculture and the duties are delegated by the ministry to other bodies. They are
included in Inter-Ministerial Joint Meeting for integration and coordination of GM
issues, National Biosafety Committee for safety evaluation of GM products, the
Standardization Committee to amend the safety standards, testing institutions
responsible for characterization and safety examination of GM products, and other
peer governmental departments responsible for a comprehensive inspection of final
products (Jian-ping et al., 2015). There are several steps of evaluation of GM foods
before their commercialization in China. The first one is biosafety assessment which
covers laboratory to field investigations. The committee in charge of biosafety
evaluation categorized the products into 1 to 4 risk groups based on their threat to
the life cycle (Yuen-Ting Wong & Wai-Kit Chan, 2016). In this regard, no or
negligible risk, low risk, moderate risk, and high risk refer to classes 1 to 4 respec-
tively (European Commission, 2020b). Obviously, a restricted regulation is set for
classes 3 and 4. China makes an obligation on GM foods’ labeling through which the
GM ingredients or those additives degraded under processing (such as an enzyme)
should be named on the label (Yuen-Ting Wong & Wai-Kit Chan, 2016). To the
contrary, Feng and Yang stated that adequate specification has not been made in
Chinese law about labeling GM food (Feng & Yang, 2019). Safety regulation on
GM foods in China undertakes three issues of risk assessment (back to the early
twenty-first century for GM agricultural foods), labeling of GM foods to alarm the
consumers about the content, and risk communication (Sun, 2019). However, the
results of a survey showed that the Chinese population tended to lower its intake of
GM foods in 2013 compared to 2002. It may be due to their increased knowledge
about these types of foods. The authors believed that the oriented advertisement of
Chinese media (that are managed by the government) against GM foods might be an
important reason. However, the increased significant rate of neutral citizens on GM
foods in 10 years along with strict regulations and fast-growing research on GM
sources in China are effective factors on GM food development in the country
(Zheng et al., 2017).

14.7 Safety Concerns of Recombinant Genes and Enzymes

and the Allergenicity Assessments for Development
of GM Foods

Genetic engineering is a polar issue in the world so there are several advocates and
critics for and against the technology, respectively. The advocates are interested in
the promising events evolved in new products by the activity of recombinant genes
compared to native ones. Today, other technologies are served to increase the
functionality of recombinant proteins. For example, recombinant dextransucrase
and dextranase were protected by encapsulation techniques to be more efficient
under intended use. The enzymes were coated with edible polymers of alginate
and pectin (Sharma et al., 2019). Such polymers which are nontoxic, biodegradable,
well-defined film-former, and resistant to higher temperatures and gastrointestinal
conditions (Moslemi et al., 2018; Cheraghali et al., 2018; Sharma et al., 2019),
398 N. S. Naghavi et al.

protected the core in favor of non-digestible oligosaccharides’ higher production

from carbohydrates under harsh condition (Sharma et al., 2019). In comparison, the
critics of genetic engineering mainly worry about unknown risks that may be
observed in humans, animals, and the environment in the future.
Bacteria, yeast, and fungus are utilized as donors or hosts for genetic modifica-
tion. Trono in 2019 listed a large number of bioengineered food enzymes which are
used in different industries of dairy, meat, cereal, beverage, sweetener, and emulsi-
fier (Trono, 2019). Some approved genetically modified food enzymes are listed as
GRAS and are available at GRAS Notice of the US FDA (US FDA, 2020b). It means
that those GRAS enzymes can be used in the market without achieving any safety
approval by regulatory bodies. Safety considerations of GM enzymes in the US are
mainly assessed by the suppliers in collaboration with US FDA.
The new traits improve technical feasibility, such as high-or low-temperature
stability, salt tolerance, specificity, and optimized activity under a developed range
of pH. For example, bioengineered α-amylase produced by B. licheniformis showed
high acid resistance by replacing histidine with aspartic and glutamic acid for starch
saccharification or high affinity of thermostable lipase to short-chain fatty acids in
dairy triglyceride help flavor enhancement. Mesophilic B. pumilus was modified to
produce a thermostable protease with increased hydrolytic activity at 15
C (Zhang
et al., 2019). However, some indications of negative symptoms from genetically
modified foods and enzymes arose. For example, allergenicity symptoms were
observed after consumption of StarLink maize engineered by insertion of pesticide
gene from B. thuringinesis which resulted in an unintended impact on humans.
Moreover, the bioengineered soybean by using the gene extracted from Brazil-nut in
favor of methionine overexpression induced an allergenicity response (Dadgarnejad
et al., 2017). Another example is the observed allergenicity of cooked and uncooked
transgene pea following the insertion of the kidney bean gene (Yavari et al., 2016). It
might be due to the intrastructure changes in the new protein that made it resistant to
high temperature and digestion. Budnik et al. performed IgE diagnostic test in the
blood of 813 workers exposed to genetically modified enzymes used in the produc-
tion of foods, drugs, and chemicals from 2007 to 2013. The results boded that 23%
of the all had high level of IgE, most of them sensitized to α-amylase and the least
frequency was reported for lipase while the highest blood IgE concentration was
diagnosed in those exposed to phytase, xylanase, and glucanase (Budnik et al.,
2017).
Importantly, new organisms may produce some enzymes involved in unintended
biological mechanisms and produce unintended or harmful chemicals. In addition,
the new gene may interfere with the gene integrity and silent or activate the gene
toward unwanted metabolic pathways. They may also lead to acquired resistance of
pests on the farm by time and propagation of the resistant genes to other violent
species. There is a concern regarding the use of antibiotic-resistant genes as a marker
in the examination of transgene products by which they remain active in the presence
of antibiotics if well affected by the bioengineering process. Researchers worry
about the transfer of antibiotic coding genes to pathogenic bacteria and resident
flora of human and animal digestive tract (Dadgarnejad et al., 2017). In comparison,
14 Current Applications of Enzymes in GM (Genetically Modified). . . 399

some explanations are presented against the concern. The fans of genetic engineer-
ing believe that a chain of reactions should be occurred to transfer the target gene
into an unintended organism. They are included in excision of the gene, its resistance
within the digestive tract, its insertion into the second organism, its compatibility and
stable joining to the host, and active expression within the unwanted cell. In addition,
the specialists stated that the normal flora within the digestive tract already has the
gene encoding antibiotics and no additional worries would be expected if the known
genes are used for this purpose (Dadgarnejad et al., 2017).
It is believed that glycoprotein provokes the immune response directed by
immunoglobulin E and glycosylation process is a key factor in vital systems toward
the formation of allergenic proteins. Allergenicity of transgenic protein is evaluated
by several methods. At first, bioinformatics tests that examine homology of the new
protein and the known allergens are conducted (structural information of the
allergens is available at databases). Then, the serological tests consisting of immu-
noglobulin E binding assay are done when more than 35% similarity in a sequence of
80 amino acids in the new protein with the allergens is detected by bioinformatics
assay. Pepsin resistance test together with quantity evaluation which is important in
risk assessment studies is another way of safety evaluation. In this way, a quantified
protein is examined because less allergenic foods may be more harmful than the
others due to their high frequency of consumption. Moreover, the heat stability and
in vivo trials referred to as animal studies are common examinations. Although,
in vivo trials may not be reliable enough because it is species-specific and could not
be strictly extrapolated to humans in some cases (Yavari et al., 2016).
A guidance has been presented by EFSA about allergenicity assessment of new
proteins stimulated a non-IgE mediated response like what is observed in coeliac
patients. It includes finding out the source of protein and the population exposure
followed by amino acid sequencing by which its similarity to the known allergens is
determined. Performing in silico tests and examining the binding possibility of the
protein residues to the receptors overexpressed in coeliac disease (HLA-DQ2- and
HLA-DQ8) to evoke immunogenicity is a further step. Finally, in vitro digestibility
test is conducted (Naegeli et al., 2017).
Food enzymes that are commonly used by the industry are mainly produced by
microorganisms and their full characterization especially on the transgene organisms
is done by EFSA. The first step is collecting the taxonomic information up to strain
level for both bacteria and fungi. The whole-genome sequencing of chromosomes
and plasmid is further required. Third, an antimicrobial resistance test is done on the
producing microorganism to avoid propagation of the resistance gene among other
viable organisms. If the microbe comprises antimicrobial-resistant gene, the enzyme
must be free of genetic materials of the producing organism (Silano et al., 2019).
EFSA developed a Qualified Presumption of Safety (QPS) list containing the
microorganisms subjected to and passed the safety evaluation for food enzyme
production. Filamentous fungi are excluded from the list because of the possible
production of toxic secondary metabolites, occasionally (Koutsoumanis et al., 2020).
The QPS document also includes a safety assessment approach for enzymes produc-
ing microorganisms for additional minor evaluation, if required. Food enzymes
400 N. S. Naghavi et al.

produced by the microorganisms not-listed in the QPS or those genetically modified

in the QPS list should be free of viable cells to make sure of safety. In addition, no
genetic materials of GM microbes and non-GM microbes carrying antibiotic-
resistant genes should be found in the products. Toxicity evaluation of food enzymes
by EFSA is done by in vitro genotoxicity assay and micronucleus test via cell culture
method and in vivo 90-days trials in rodents via the oral route. Allergenicity assay is
done by in vitro gastric simulation test which is a marginal way because it is not
reliable enough and should be used in combination with other methods. Food
enzymes within a group are assessed separately because those with similar function-
ality may have different structures leading to different clinical reactions in humans
(European Food Safety Authority, 2020). The applicants are required to present a
full description of genetic modification including the changed gene, the new trait,
and the altered mechanisms in the GM organism (Silano et al., 2019).
Comprehensive data have been released by EFSA about the enzymes produced
by genetically modified microorganisms. Estimation of oral toxicity in the form of
margin of exposure (MOE) by EFSA is based on the maximum recommended
dosage of the enzyme in the industry and European food consumption database
which may be overestimated in some cases. Acceptable MOE in Europe with limited
variety in the population would be 300 and more compared to the varied geographi-
cal countries which may accept MOE 1000 and higher. Therefore, based on the
reference points or reference doses, deciding about the safety of foods is hold on the
risk managers of each country depending on their local dietary intake (Jamali et al.,
2020; Moslemi et al., 2020). EFSA has evaluated the toxicity of GM food enzymes
by the reference point of NOAEL (no observed adverse effect level). Some examples
of GM food enzymes safety evaluations are as follows. They mainly limited to
carbohydrase that are of the most common enzymes in the food industry.
α-amylase produced by GM Pseudomonas fluorescens (BASF Enzymes LLC
Company) intended for glucose syrup production was free of viable cell and genetic
materials. The enzyme was subjected to toxicity assessment within 90 days of trial in
rats and a reference point (NOAEL) of 887 mg/kg body weight per day were
detected. It was not genotoxic and no similarity to the known allergens was detected.
The EFSA scientists believed that induction of adverse reaction by the enzyme might
not be avoidable although it was considered rare. Notwithstanding, the GM
α-amylase contained antibacterial that will pose the consumers at risk of antibacterial
resistance. Therefore, the GM α-amylase was ignored for human dietary consump-
tion (Silano et al., 2020).
α-amylase produced by GM B. licheniformis (Novozymes Company) intended
for glucose syrup production was introduced as safe by EFSA and no genotoxicity
and allergenicity were detected. Toxicological evaluation was not emphasized for
the suggested industrial use as more than 99% of the enzyme will be removed at the
end of syrup production (Silano et al., 2018g).
α-amylase produced by GM T. reesei (Danisco US Inc) for production of distilled
alcohol and brewing purposes was studied and no genotoxicity was detected. The
enzyme was not similar to the allergenic compounds. Nonetheless, a NOAEL of
230 mg/kg body weight per day was reported which led to MOE of 135 by
14 Current Applications of Enzymes in GM (Genetically Modified). . . 401

calculating the dietary exposure of 1.701 mg/kg body weight per day in Europe. The
low MOE might be due to overestimation of dietary exposure as the scientists
presumed its intake from all brewing products and extrapolated the data of restricted
time to longtime exposure. As a conclusion, they reported that the enzyme can be
used but a low concern would not be avoidable under the defined condition of oral
consumption if no residue remained in the foods (Silano et al., 2019a).
α-amylase from GM A. niger supplied by Novozymes Company was not
genotoxic and MOE for bakeries that contain the GM enzyme accounted for
370 in Europe. Two similar segments in amino acid sequences were observed in
the allergenicity test which was matched to respiratory, not oral allergens and
resulted in a safe status of the GM enzyme for oral intake (Silano et al., 2018).
The enzyme glucan 1,4-α-glucosidase produced by two different GM organisms
of A. niger and T. reesei for conversion of starch to glucose syrup, baking, and
brewing purposes was not genotoxic. NOAEL of 1244 and 1149 mg/kg body weight
per day was detected for the enzyme produced by recombinant A. niger and T. reesei,
respectively. But, one segment of 80 amino acid in the enzyme of both origins
showed more than 35% similarity with a respiratory allergen. Although, the EFSA
panel did deny the low safety concern with regard to the oral intake of the enzyme,
particularly in those susceptible to the known respiratory allergen, but the enzyme
was finally considered safe for oral intake. It is due to the fact that more than 99% of
the enzyme is removed in glucose syrup and variable dietary exposure would be
expected for dough and beer at different countries (Silano et al., 2018a, 2020a).
Same results were observed for glucoamylase produced by GM A. niger for conver-
sion of starch to glucose syrup and brewing purposes. A NOAEL of 1360 mg/kg
body weight per day and MOE of 375 were determined for European countries.
Similarity of the enzyme with the known respiratory allergen was neglected
according to the fact mentioned above for glucan 1,4-α-glucosidase. Therefore,
GM glucoamylase was considered as safe for dietary consumption (Silano et al.,
2018b).
Three domains of GM maltogenic amylase produced by recombinant B. subtilis
(produced by Novozymes A/S) showed more than 35% similarity with the known
respiratory allergens but the GM enzyme was reported as safe by EFSA for bakery,
brewing and starch processing because no evidence was found about their
allergenicity under dietary exposure. No genotoxicity and acceptable MOE for
Europe was also reported (Silano et al., 2018c). In agreement, the same results and
conclusion on genotoxicity (negative), oral toxicity (acceptable risk), and
allergenicity (three similar domains with the known allergens) were further made
by EFSA for maltogenic amylase derived from GM B. licheniformis (Danisco US
Inc) (Silano et al., 2020b).
Xylanase is a commonly used enzyme in the cereal industry for brewing and
starch/gluten production. Its counterpart produced by T. reesei (AB Enzymes
GmbH) showed no genotoxicity, high MOE in Europe (more than 43,000), and no
allergenicity. GM xylanase is removed at the end of grain processing but a trace
amount of the enzyme delivered from brewing products might pose the consumer at
low risk. However, the EFSA considered it safe under the defined oral condition of
402 N. S. Naghavi et al.

use (Silano et al., 2020c). The enzyme endo-1,4-β-xylanase from GM B. subtilis of

two different strains of LMG S-27588 and LMG S-24584 (produced by Puratos N.
V.) for baking purposes showed no genotoxicity, allergenicity, and high MOE (1363
and 2176 for European countries, respectively). In comparison, a recombinant DNA
segment was detected in the food enzyme. Although the last finding was against
EFSA requirements, the EFSA panel considered the GM enzyme safe for commer-
cialization under the defined condition of use. It might be due to the amount of MOE
far from the danger cut-off point and the no antimicrobial reaction found in the purity
test of the GM enzyme (Silano et al., 2018h,d). The enzyme endo-1,4-b-xylanase
produced by T. reesei strain DP-Nzd22 (DuPont company) was also certified as safe
for commercialization by EFSA (Silano et al., 2018e). To the contrary, endo-
1,4-β-xylanase from GM A. niger was not accepted to be released because of no
adequate data achieved by the Ames test and the EFSA panel could not conclude
about its genotoxicity (Silano et al., 2017).
Pullulanase produced by GM B. subtilis (Novozymes Company) and
B. licheniformis (Danisco Company) for glucose syrup production from starch and
brewing purposes showed no oral toxicity within the exposure range of European
and no homology with the known allergens was found. Therefore, the GM enzyme
has been considered safe for human consumption under the defined condition
(Silano et al., 2017a, 2019b).
β-galactosidase synthesized by GM E. coli (supplied by Clasado Ingredients Ltd.)
was withdrawn for commercialization despite passing the toxicological and
allergenicity experiments due to the inclusion of genetic materials in the food
enzyme and no adequate data on their viable cells’ containment (Silano et al.,
2020d).
Xylose isomerase produced by GM Streptomyces rubiginosus supplied by
Danisco Company for immobilized isomerization of the sugar into fructose was
studied and no genotoxicity and allergenicity were detected in vitro. The GM strain
has had antibiotic- resistant gene but no viable cell and DNA fragments were
detected in the GM enzyme. Provided that the enzyme will be removed after the
process or not entered into the product through immobilization, little risk of dietary
exposure and oral toxicity is expected. Thus, the GM enzyme has acquired EFSA
acceptance for commercialization in food (Silano et al., 2020e).
Lysophospholipase expressed by GM A. niger (produced by Novozymes A/S)
usable for starch processing and oil degumming showed no genotoxicity and
NOAEL of 1356 mg/kg body weight per day was determined. Nonetheless, no
similar sequence of amino acids with the allergens was detected. The scientists
reported that there may be a low safety concern regarding the frequent dietary
exposure to the enzyme, but the enzyme removal after processing declines any
health concern in the final product (Silano et al., 2020f).
Triacylglycerol lipase produced by GM Ogataea polymorpha (Danisco US Inc.)
showed no genotoxicity, MOE of more than 1280 for Europe, and no similar amino
acid segment with the known allergens. No viable cells and DNA residue were
observed in the GM enzyme and it was considered safe for use in the food industry
by EFSA (Silano et al., 2020g).
14 Current Applications of Enzymes in GM (Genetically Modified). . . 403

The approved acetolactate decarboxylase from GM B. licheniformis (produced by

Novozymes A/S) for brewing purposes had no genotoxicity, allergenicity, and very
high MOE (more than 300,000) (Silano et al., 2018f).

14.8 Conclusion

Enzymes found in nature have been beneficial for the generation of fermented foods
for thousands of years. The enzymes producing microorganisms that obtained from
natural sources date back to the late-nineteenth century. Furthermore, the progress in
molecular genetics and cell biology over the past four decades have altered enzyme
generation processes. It is possible to choose and extract enzyme-encoding genes
and show them in host microorganisms that are well compatible with large-scale
industrial fermentation. In order to utilize effective promoters and introducing
variant copies of the enzyme coding genes, enzyme performance can be significantly
increased. Also, it became possible to adopt enzyme characteristics to food
processing conditions by using recombinant DNA technology. The evolution of
metabolic engineering in relation to systems biology in recent years and use them for
designation of biological systems promises to produce more sufficient food enzymes
in the early future. Currently, USA, EU, and China are the main players in GM foods
worldwide. The GM enzymes approval is based on the results of widespread risk
assessments. Safety evaluation includes homology assessment of the GM enzymes
to the known allergens, immunology evaluation, pepsin resistance, heat resistance,
and in vivo trials. With regard to labeling of the GM foods, the US FDA is more
lenient than the two others because it believes that the approved GM products do not
represent an additional risk to the consumers compared to the traditional foods.
However, all GM enzymes should be listed on the labels of food packages in China.
Upon the fate of the enzymes under processing, the EU exempts the GM enzymes
added as processing aid from labeling while those additives should be mandatorily
listed on the labels. Besides the all-positive points addressed about the GM enzymes;
the improvements have introduced a new controversial issue in Islamic countries. As
a rule, halal food enzymes should be derived from halal sources, produced in
environments containing halal ingredients, and exhausted from alcohol up to the
acceptable level in the final product. In addition, the gene responsible for encoding
recombinant enzymes should be extracted from halal origin.

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Enzyme Immobilization and Its Application
Strategies in Food Products 15
Nafiseh Sadat Naghavi, Nazanin Sanei, and Martin Koller

Abstract

Enzymes are the dominating class of biocatalysts, which are extensively

employed in food industries. Immobilization of enzymes on inactive and not
soluble supports practically increases their efficiency owning to their high stabil-
ity and multiple reuses, while it can negatively impact enzyme activity. The
characteristics of immobilized enzymes are based on the procedure of immobili-
zation and achieved beneficial properties, such as biocompatibility, chemical and
thermal stability, the impossibility to dissolve (leak) in the reaction liquids,
reconstitution, recyclability, and cost efficiency. Various immobilized enzymatic
systems, like proteases, amino acylase, glucose isomerase, β-galactosidase,
aspartase, lipases, or glucosidase have been shown to be techno-economically
used in food industries on a multi-ton’s scale per year. This chapter provides a
general survey of the benefits and applications of the immobilization enzymes
with a major focus on the food industries.

Keywords
Food enzymes · Immobilization · Reuse · Cost efficiency

N. S. Naghavi
Department of Microbiology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
N. Sanei
Department of Biology, Faculty of Sciences, Shahid Chamran University of Ahvaz, Ahvaz, Iran
M. Koller (*)
Research Management and Service, c/o Institute of Chemistry, NAWI Graz, University of Graz,
Graz, Austria
ARENA - Association for Resource Efficient and Sustainable Technologies, Graz, Austria
e-mail: martin.koller@uni-graz.at

# The Author(s), under exclusive license to Springer Nature Singapore Pte 411
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_15
412 N. S. Naghavi et al.

15.1 Introduction

Immobilization is a process used for transformation of biocatalysts (cells and

enzymes) or bioactive components from a soluble phase to an insoluble state by
using an insoluble carrier or by encapsulating them within a matrix material (Van de
Velde et al., 2002; Chen et al., 2016). As a consequence, the properties of the native
catalysts may change significantly, and in order to be able to explain the observed
reaction behavior the laws of heterogeneous kinetics have to be applied. The
immobilization of enzymes has become a major process in manufacture, medication,
and biotechnology over the past decades. Scientists have developed many processes
based on methods from physical adsorption and covalent binding to encapsulation or
entrapment into polymers (Ispas et al., 2009; Alkan et al., 2009; Koszelewski et al.,
2010; Klein et al., 2011; Defaei et al., 2018). Already in 2010, the market volume of
immobilized enzymes amounted to almost 6
109 US-$ with a currently clear
upwards trend, with food/beverage production and pharmaceutical applications
constituting the major fields of application, each of both occupying about 21% of
the entire market for immobilized enzymes (DiCosimo et al., 2013).
In contrast to free, dissolved enzymes, immobilized enzymes are of higher
stability and more resilient against inhibition by heavy metals and other chemicals,
non-optimal conditions of pH-value, temperature, or salinity in the environment, and
turbulent flow regimes. Importantly, the high variety of reported immobilized
enzyme systems provides a range of beneficial aspects, such as convenient recovery
of enzymes and products, which reduces the number of process steps, recyclability
of enzymes, continuous operation mode of enzymatic reactions, high overall pro-
ductivity, possibility of sudden termination of reaction on demand, contribution to
sophisticated development of enhanced bioreactor designs (“immobilized enzyme
reactors”), and decreased environmental pollution and economic costs (Sheldon,
2007a; Defaei et al., 2018). Drawbacks of using immobilized enzymes encompass
lower activity and reaction rates compared to free enzymes, susceptibility to putre-
faction (“fouling”), additional cost for supports (carriers), or the need for disposing
spent (exhausted) immobilized enzymes (Basso & Serban, 2019).
To date, various types of matrices and supports have been utilized to
immobilize enzymes. Polysaccharides, glass beads, nano-metals, petro-plastics,
and biopolymers are the most frequently used matrices. To immobilize enzymes,
the binding strength between support and enzyme depends on the type of chemical or
physical reaction, which heavily depends on the nature of the support surface, and
the interaction between individual enzyme molecules (Kahraman et al., 2007;
Asgher et al., 2014; Rana et al., 2014; Reshmi et al., 2006; Dey et al., 2002;
Defaei et al., 2018). Especially the immobilization of enzymes on nanosized
supports, such as nanofibers, nanobeads, carbon nanotubes (CNTs), and other
nanoparticles for different reactions driven by biocatalysts, is nowadays emerging
as an innovative research area (Sheldon, 2007a; Klein et al., 2011; Reshmi et al.,
2006; Defaei et al., 2018).
Also enzymes of importance for food technology have been immobilized on
various matrices. Some examples are given in Fig. 15.1. The immobilized food
15 Enzyme Immobilization and Its Application Strategies in Food Products 413

Fig. 15.1 Examples for the applications of different supports for immobilization enzymes used in
food processing

enzyme that was the first to be industrially utilized already in the 1970s was amino
acylase (EC 3.5.1.14), a hydrolase which was implemented for the production of
racemic mixtures of D- and L-amino acids from N-acyl-amino acids as substrates.
Industrially, this reaction took and still takes place in columns carrying the
immobilized enzyme, while the substrate solution is washed thoroughly. Two
other effectually immobilized enzymes were the glycosidase invertase
(EC 3.2.1.26), an approved food additive (E 1103) which is used in fructose-rich
corn syrup production, and lipases (EC 3.1.1.x), a versatile group of amphiphilic that
are applied in the hydrolysis and transesterification of oily products, which, beyond
food industry, is also a novel route toward biodiesel production from waste lipids.
The advantages of immobilized enzymes in food industries are increased productiv-
ity, reduction of product recovery cost, and ultimately increased yields for diverse
food products in the future (DiCosimo et al., 2013; Homaei et al., 2013).

15.2 Methods of Enzyme Immobilization

A number of methods can be used to immobilize a given enzyme; these methods

encompass simple reversible physical adsorption, formation of ionic bonds between
enzymes and supports, and generation of firm covalent linkages (Fig. 15.2). Such
immobilization methods are categorized into reversible (adsorption, ionic binding,
disulfide formation, affinity binding) and irreversible (covalent binding, cross-
linking, physical entrapment [matrix entrapment and encapsulation]) methods.
Apart from this classification, immobilization methods can also be classified
according to the type of chemical reaction involved in binding as support binding
and entrapment methods (Homaei et al., 2013).
414 N. S. Naghavi et al.

E
Adsorption
E Method for immobilization Covalent E
of enzymes binding E
-+
E E E
lonic binding E
E
E Irreversible Crosslinking E
E
-+ Reversible E
E

By disulfide
SS E
bonds Physical E
E
entrapment E E
E Affinity
binding
E

Fig. 15.2 The categories for classification of enzyme immobilization methods

15.2.1 Irreversible Methods

In irreversible enzyme immobilization, the biocatalyst that binds to the matrix cannot
be detached without causing an effect on the biological enzyme activity or the
structural properties of the support. The most usual methods of irreversible enzyme
immobilization encompass covalent binding, cross-linking, and entrapment or
encapsulation.

15.2.1.1 Immobilization via Formation of Covalent Binding

This method is widely utilized for enzyme immobilization. Based on this procedure,
covalent bonds are created between the chemical groups of the enzyme molecule and
the chemical groups present on the support. This method leads to formation of a
stable bond between enzyme and support, resulting in the prevention of detachment
of enzyme from the support when in use. This covalent bond is typically formed
between the side-chain amino acids of biocatalysts, such as aspartic acid, arginine,
histidine, and the functional groups, like imidazole, indolyl, phenolic, and hydroxyl
present on the support. This method is used when the complete absence of enzyme in
the product is desirable. However, it leads to chemical modification of enzymes
and, as a result, the activity of the enzyme can get reduced (Homaei et al., 2013). As
an option to increase activity of enzymes immobilized via covalent linking, linear
spacers are often used to combine enzyme and support, giving the enzyme
enough space for mobility and reaction (De Maio et al., 2003). An important
example in this context is the carboxyl- and amine-reactive linker 1-ethyl-3-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), which can link hydro-
phobic polymeric supports with enzymes (Mohan et al., 2015).

15.2.1.2 Cross-Linking
Crosslinking resorts to covalent linking between the enzyme and active molecules,
and is used to generate biocatalytically active polymeric particles, which can conve-
niently be used for given reactions. This mechanism is also known as “copolymeri-
zation”. Enzymes bind to each other with the aid of bi-functional reagents,
such as glutaraldehyde, glutardialdehyde, glyoxal, diisocyanates, hexamethylene
15 Enzyme Immobilization and Its Application Strategies in Food Products 415

diisocyanate, and toluene diisocyanate, which build bridges between individual

enzyme molecules. The major drawback of this procedure is that the multifunctional
reagents used for cross-linking the enzyme may reconstruct or change the structure
of the enzyme, which again causes the loss of catalytic activity (Albayrak & Yang,
2002).

15.2.1.3 Physical Entrapment

This method involves the physical trapping of biocatalysts into a film, gel, fiber,
coating, or microencapsulation (Costa et al., 2005). In this procedure, the enzyme or
reactive molecule can be mixed with a polymer to attach to it, resulting in creation of
a lattice structure that encapsulates/entraps the enzyme. The advantages of this
immobilization method include generation of a vast biocatalytically active surface
area generated by the substrate and the enzyme, in only a low volume. The most
important disadvantages of this method are the probable inactivation of the enzyme
during microencapsulation, enzyme leakage into the environment, and the need for
typically high concentrations of the enzyme. The most frequently used supports that
are used for encapsulation of enzymes are polymers, such as cellulose, collagen,
polyacrylamide gel, gelatin, alginate, starch, silicone, and rubber (Nisha et al., 2012).

15.2.2 Reversible Methods

15.2.2.1 Adsorption
The adsorption of enzymes on supports, such as activated charcoal, alumina, and ion
exchange resins is among the simplest techniques used to limit enzyme mobility
(Brady & Jordaan, 2009). Depending on the nature of amino acids present on the
surface of enzymes and the chemical nature of the support, the enzyme is fixed by
non-covalent binding trough ionic and hydrophobic interactions, or by formation of
hydrogen bonds. This method can be achieved by mixing an aqueous enzyme
solution with a matrix for a defined time, followed by a washing step to remove
the remaining free enzyme from the immobilization matrix. This method of immo-
bilization is simple and has little effect on enzyme activity and can be repeatedly
applied by adding fresh enzyme solution (“recharging” of the support with biocata-
lyst). However, in this method, shortcomings, like fast enzyme desorption from the
support, or loss of activity by changing pH-value, temperature, solvent, and ionic
strength of the surrounding environment, can hardly be avoided (Costa et al., 2005).

15.2.2.2 Ionic Bond Formation

Immobilization by generation of ionic bonds is primarily based on the formation of
ionic bonds between the enzyme molecules or reactive molecules and a solid matrix
that has a charged ionic surface. The main advantage of ionic binding in comparison
to physical binding is the strength of generated bonds that is stronger in case of ionic
bonds than the power responsible for formation of covalent bonds (Torres et al.,
2002). Such non-covalent immobilization assays can be reversed/deactivated by
416 N. S. Naghavi et al.

alterations in the temperature, solvent polarity, and ionic strength conditions (Nisha
et al., 2012).

15.2.2.3 Immobilization via Disulfide Bridges

This method involves the configuration of disulfide (-S-S-) bridges between the
enzyme and the support. The major benefit of this method is the ability to recover the
bonds created between the activated solid surface and the thiol groups on the
enzyme, because the leakage of bounded protein leads to the release of high amounts
of low-molecular-weight thiol from disulfide bridges. The feasibility of reusing the
polymeric matrix after enzyme deactivation may facilitate the actual large-scale
immobilization of enzymes in industrial procedures, where their use is currently
not economical because of the high cost of support materials (Ovsejevi et al., 2013).

15.2.2.4 Affinity Binding

Affinity immobilization combines the properties of the enzyme to maintain under
varied physiological conditions. This can be done via two routes of support
pre-junction by an affinity ligand for desired enzyme, or enzyme attachment to the
organism that has an affinity toward the support. The benefits of this procedures are
that the enzyme is not exposed to any unusual chemical conditions, minimal
conformational changes occur during immobilization, and the high activity of
immobilized enzyme is maintained (Sardar et al., 2000).

15.3 Classification of Different Support Materials

for Immobilization of Food Enzymes

The reaction occurring between the enzyme and a carrier generates an immobilized
enzyme with particular structural, biochemical, physical, and kinetic characteristics.
Carriers can be divided into different groups based on their appearance or their
chemical components. The support can be a synthetic or biological organic polymer,
or an inorganic solid. The support must display certain features, like extended
surface-to-volume ratio, high permeability (mass transference), acceptable func-
tional groups for enzyme binding under non-denaturing conditions, hydrophilic
moieties, insolubility in water, chemical and thermal stability, mechanical strength,
high recalcitrance, applicable particle shape, resistance to microbial attack,
regenerability, biological safety, and low or acceptable price (Sheldon, 2007b;
Garcia-Galan et al., 2011). Furthermore, multi-enzyme biocatalysis on only one
support, especially processes resorting to multi-enzyme cascades, is an emerging
approach to generate high-value chemicals on an industrial scale (Xu et al., 2020).
15 Enzyme Immobilization and Its Application Strategies in Food Products 417

15.3.1 Classification of Different Support Materials Based

on the Chemical Structure

15.3.1.1 Inorganic Solid Supports

Different inorganic solids, such as zeolites, alumina, silica, and mesoporous silicas,
are possible to use for enzyme immobilization. Silica-based supports are the best
applicable matrices for immobilization of enzymes in the industrial manufacturing of
enzymatically produced products, in addition to research purposes (Vianello et al.,
2006; Pierre, 2004; Hudson et al., 2008). In general, the high surface area provided
via silica gel matrixes is nonpareil. Moreover, silica gel can be facilely treated to
obtain the desired shape, pore size, and microchannels to allow reaction between
substrates and ligands. Furthermore, silica gel is mechanically stable and chemically
inactive; it is therefore environmentally benign for manufacturing and industrial
procedures (Blanco et al., 2004). The simplest and cheapest methods for enzyme
immobilizing, namely, attachment on silica, are realized by simple adsorption. For
instance, this method is utilized for generation of enzyme formulations in detergent
powders that release the enzyme into the washing liquid during the washing process
(Deere et al., 2002).

Polymers
A recent method of enzyme immobilization is based on covalent linkage of such
enzymes to polymers that undergo significant structural conformation changes in
response to even minor environmental changes in terms of pH-value, temperature,
and ionic strength (Klouda & Mikos, 2008). A studied sample is poly (N-isopropyl
acrylamide) (poly NIPAM), a thermo- and bio-compatible polymer (Klis et al.,
2009). Aqueous poly NIPAM has its critical solution temperature (CST) at around
32  C. Above the CST, it becomes dissolvable because of release of water molecules
from the polymer fibers. Thus, the bio-conversion can take place under states that
maintain the enzyme solubility, thereby minimizing diffusional restriction. Subse-
quently, an increase in temperature above the CST leads to detachment of the
immobilized enzyme, thus the enzyme is recovered and can be reused (Virtanen &
Tenhu, 2000; Virtanen et al., 2000; Lozinsky et al., 2003). Polyurethane has recently
been proposed as an entrapping polymer that retains the bioactivity of biocatalysts
for long times. The usage of this polymer resulted in a remaining crude oil degrada-
tion capacity of 44.31% by a microbial consortium after more than 6 months
(Kazemzadeh et al., 2020).
Polymers that possess electrical conductivity have already been successfully
synthesized and utilized in different areas, including biotechnology. Recently, a
new class of polymers has been proposed as novel electro-active conjugated
polymers. This kind of supports exhibit interesting electrical and optical
characteristics previously reported only for inorganic systems. Electronically
directing polymers are different from all the familiar inorganic crystalline
semiconductors, such as silicon. They are molecular in nature and long chains are
absent in them. Immobilization of enzymes and biosensor construction are two
applications of these polymers (Cirpan et al., 2003). Lots of theoretical models
418 N. S. Naghavi et al.

have also been associated with the electrochemical entrapment of enzymes to

evaluate the polymer thickness, enzyme configuration, and the level of enzyme
loading in the biosensor design (Gerard et al., 2002).

Nanomaterials
The headway of nanotechnology in the 1990s was preceded by the quick evolvement
of nanobiotechnology including the construction of nanobiocatalysts. In the early
methods applied in nanobiocatalysis, enzymes were immobilized on various
nanostructured materials utilizing conventional procedures, like simple adsorption
and covalent linkage. This method attracted attention for immobilizing enzymes on a
wide range of nanostructured matrices, like porous nanomaterials, electroconductive
nanofibers, and magnetic nanoparticles (MNPs). This large area provides better
enzyme loading, which in turn improves the enzyme mobility in comparison to
immobilized enzyme systems on conventional matrices. One of the special
advantages of nanostructured materials is that the pore size in nanopores, nanofibers,
or nanotubes can be controlled at nanometer scale (Homaei et al., 2013).
Recently, nanobiocatalytic methods have evolved from simple strategies for
immobilization of enzymes (Homaei et al., 2013). By rapid advancement in nano-
technology, MNPs are presently extremely interesting. The physico-chemical
characteristics of MNPs can widely differ from the properties of the bulk
material from which the nanoparticle is made; this has attracted attention in these
materials also for enzyme immobilization. For instance, magnetizing a particle in a
particular direction by magnetic anisotropy is usually done on the surface of a
particle (Schellenberger et al., 2002). Nanoparticles constructed by ultra-small
superparamagnetic iron oxide (Kooi et al., 2003; Keller et al., 2004), cross-linked
iron oxide (CLIO), and mono-crystalline iron oxide (Krause et al., 2004) all were
fabricated as imaging elements in magnetic resonance imaging (MRI). Magnetic
particles are used for enzyme immobilization for the purpose of increasing the
stability of the biocatalyst, to maintain the stability of the catalyst, and, importantly,
they can conveniently be separated from the interaction environment and recovered
by applying an external magnetic field. (Bilal & Iqbal, 2019). MNPs perform best at
typical sizes ranging between 10 and 20 nm, where superparamagnetism emerges
(Netto et al., 2013). Such magnetic particles have been suggested for biotechnologi-
cal applications (Kluchova et al., 2009; Defaei et al., 2018) or for developing
analytical systems, like biosensors (Bilal & Iqbal, 2019; Kouassi et al., 2005).
Nanostructured metal oxides (NMOs) recently became of interest in the area of
enzyme immobilization because these materials have the best structural revising and
high bioactivity, which leads to elevated sensing properties (Antony et al., 2016). As
a part of NMOs, MNPs have been widely utilized in enzyme immobilization because
of their advantageous properties, such as their size, magnitude, higher safety levels,
better reusability, wide surface, and large capacity of enzyme loading. Because they
have an inactive surface that limits direct binding to enzymes, protective molecules
must be coated on MNPs to supply dynamic functional groups for immobilization of
enzymes (Amirbandeh & Taheri-Kafrani, 2016; Mehnati-Najafabadi et al., 2018).
With all these benefits in mind, MNPs are highly inhibited in acidic and oxidative
15 Enzyme Immobilization and Its Application Strategies in Food Products 419

conditions. Therefore, coating of the outer protective surface is so vital to sustain the
consistency of MNPs (Landarani-Isfahani et al., 2015). In a recent study, Defaei
et al. (2018) immobilized the hydrolase α-amylase (EC 3.2.1.1) onto naringin-
functionalized MNPs by ionic reactions. The MNPs were covered with naringin,
which is a biocompatible flavonoid. The appearance, structure, and features of
functionalized MNPs and the immobilization status of the nanocomposite were
determined by analytical instruments, like thermogravimetry (TGA), vibrating sam-
ple magnetometer (VSM), Fourier transform infrared spectroscopy (FTIR), scanning
electron microscopy with energy dispersive X-ray (SEM-EDX), and transmission
electron microscopy (TEM). In addition, the optimum conditions of temperature,
pH-value, interaction time, and enzyme tendency for better immobilization were
evaluated. The results evaluated the optimum conditions for α-amylase immobiliza-
tion on the synthesized nanocarrier at pH 6.5 and a temperature of 55  C. Reuse
experiments showed high maintenance of immobilized α-amylase activity even after
10 reaction repeats. Furthermore, the storage consistency of enzyme immobilization
was repaired by immobilization compared to that of the free enzyme and it retained
60% of its original activity even after 6 weeks of storage at 4  C. Improving the
catalytic properties of enzymes through immobilization has made this
nanobiocatalyst a feasible tool in bio-industrial systems.
Another field of application of MNPs as supports for immobilized enzymes is the
pharma sector; here, it was shown that the enzyme penicillin G acylase (PGA; EC
3.5.1.11) can be immobilized to functionalized MNPs (epoxy-activated magnetic
cellulose beads) due to the cavity and affinity forces in the matrix of activated
cellulose, and applied for hydrolytic removal of the side chain of penicillin G
molecules, generating 6-aminopenicillanic acid as product. Improved biocatalytic
activity and stability of the enzyme were reported for this process in comparison to
the use of free enzyme (Luo & Zhang, 2010). In recent years, this process was
improved by various research groups globally, such as by Liu and colleagues, who
covalently immobilized PGA on hydroxy- and aldehyde-functionalized magnetic
Fe2O3/Fe3O4 nanoparticles (Liu et al., 2020), or Zhaoyu et al., who used novel
di-functional magnetic “nanoflowers,” equipped with epoxy groups and hydrophilic
catechol as well as with phthaloquinone groups enabling the covalent coupling of
penicillin G acylase (Zhaoyu et al., 2020).

15.3.1.2 Organic Solid Supports

Different biological polymers, especially hardly water-soluble or de facto water-
insoluble polysaccharides, like agarose, cellulose, starch, and carrageenans, have
been heavily utilized as matrices for enzyme immobilization. These polymers create
highly inactive aqueous gels, with the property of high gel formation even at low
concentrations (Van de Velde et al., 2002).
The best described and most widely applied synthetic polymers used to carry the
immobilized enzymes are acrylic resins, like sepabeads or eupergit. These
macroporous polymers are copolymerized by using N,N0 -methylene-bi-
(methacrylamide), glycidyl ether, methacrylamide, allyl methacrylate, and glycidyl
methacrylate. They are very hydrophilic and highly chemically and mechanically
420 N. S. Naghavi et al.

stable in the entire pH-range of 0 to 14 and are not inactivated even at a sudden and
tremendous pH-change. A basic disadvantage of hydrophilic resins are diffusion
limitations that have been shown in kinetically monitored procedures. Immobiliza-
tion by covalent binding to acrylic resins has been successfully utilized for a variety
of enzymes in industrial operations (Katchalski-Katzir & Kraemer, 2000; Boller
et al., 2002).
Enzymes may also be immobilized in biological or artificial hydro- or cryo-gels in
an insoluble environment. For example, poly (vinyl alcohol) (PVA) crucibles made
by melting ice are frequently used for whole-cell immobilization (Tripathi et al.,
2010). Also, because of their minor size, free enzymes can spread from the gel
matrix and get dissolved in an aqueous medium. To trap free enzymes, the enzyme
size should be enlarged by mechanisms, such as cross-linking. Another option to
increase the biocatalytic enzyme size is to design a composite material with a
polyelectrolyte. Because of their ampholytic nature, proteins are released either as
polycations or polyanions, based on the environmental pH-value. Therefore, the
typically can form complexes with polyelectrolytes of opposite charges (Homaei
et al., 2013).

15.4 Multi-Enzyme Immobilization

Multi-enzyme immobilization is a technique that co-establishes more than one

enzyme on appropriate supports/carriers, or joins enzymes using a linking agent
(cross-linker) with no carriers (Ren et al., 2019). Enzymes will be adjacent to each
other, and the total material-transport restriction will be decreased during
co-immobilization, which has been shown to boost the activity of enzymes by matrix
canaling and raising the consistency and ability to reuse them. As support materials
can mainly influence the characteristics of enzymes, selection of support has been
mentioned as a hot subject in the majority of reports on enzyme immobilization
techniques. To date, different matrices, like CNTs, graphene, metal-organic
frameworks (MOFs), DNA nanocomposites, silica, and polymers, have been used
for immobilization of multi-enzyme, which can efficiently shelter enzyme activity
from biological challenges, such as heavy metals or high temperature (Sheldon &
Woodley, 2018; Ren et al., 2019). There are three major types for co-immobilization
of enzymes, such as random and positional co-immobilization, and compartmentali-
zation (Hwang & Lee, 2019).
Biocatalytic transformation by a cascade of enzymes (“multi-enzyme catalysis”)
is an emerging technology to manufacture various industrially valuable compounds;
briefly, it mimics the cascadic catalytic steps of pathways in living cells. In this
context, positional multi-enzyme immobilization has been demonstrated as a viable
tool for effective immobilization of co-enzymes, which can trigger the rates of
coenzyme cascade reactions by fine-tuning the sequence of immobilized enzymes,
and which, first of all, improves channeling of the substrate flow, and avoids the
formation of unwanted side products, thus leading to higher overall yields. More-
over, such positional multi-enzyme immobilization approaches result in high
15 Enzyme Immobilization and Its Application Strategies in Food Products 421

Fig. 15.3 Schematic illustration for multi-enzymes co-immobilization using supports, such as
polymers, graphene, silica, MOFs, DNA, and CNTs

stability and reusability of involved enzymes. Polymers, DNA nanostructures,

graphene, or CNTs are the most frequently used supports for positional multi-
enzyme immobilization grace to their expedient capability to manage the relative
positions of enzymes by usual reactions (Xu et al., 2020).
As an imitation of the organization of natural enzymes in cellular environments,
compartmentalization can divide enzymes by varied features and ratios spatially,
which can inhibit enzymes proteolysis, biological decomposition, or exposure to
toxic agents (Marguet et al., 2013). As an example, a compartmentalized co-enzyme
operation was created on the base of inorganic nanocrystal–protein complexes via a
simple precipitation technique that showed increased overall catalytic performance
in comparison to the use of free enzymes. For this purpose, the versatile enzyme
horseradish peroxidase (HRP; EC 1.11.1.7), a well-known oxidoreductase, was
combined with CuSO4 in aqueous environment in order to form HRP-incorporated
complexes; subsequently, glucose oxidase (GOx; EC 1.1.3.4) was attached on the
surface of the complexes via the cooperative reaction between Cu2+ and protein
amino acids (Li et al., 2014). Different organic and inorganic bases can be applied
for immobilization of multi-enzymes as described in Fig. 15.3.
DNA nanotechnology has turned out to be a feasible method to construct
complex bio-molecular nanocomposites because of the ability of programmed
DNA hybridization. For immobilization of multi-enzymes, it is important to
decrease the mass transfer consistency by managing the relative location and
directions of a variety of enzymes in a limited space. Therefore, DNA nanotechnol-
ogy has been used as an effective instrument for the multi-enzyme immobilization in
422 N. S. Naghavi et al.

which notably addressable DNA nanocomposites can ease the suitable self-
aggregation of varied enzymes and optimize the substrate penetration (Xu et al.,
2020).

15.5 Kinetics of Enzyme Immobilization

Compared to measurements of the kinetics of enzymes in absolute solution, the

interactions of immobilized enzymes have only scarcely been shown under spatially
sole substrate and uniform conditions, such as pH-value. In addition, immobilization
can change the innate kinetics of the enzyme by altering the structure of the enzyme
and surrounding microenvironment. To diminish the effect of immobilization on the
enzyme catalytic activity, the molecular configuration of the enzyme should be
considered (Gonzalez-Saiz & Pizarro, 2001).
Immobilization usually enhances enzyme maintenance at the expense of lower
catalytic function (Sheldon, 2007a; Garcia-Galan et al., 2011). Nevertheless, the
appropriate choice of enzyme immobilization techniques may reduce or increase the
enzyme activity (Rodrigues et al., 2013, 2011; Mateo et al., 2007). For example,
enzyme immobilization on highly active supports can improve the multipoint cova-
lent linkage and prevent the enzyme from being inactive after immobilization
(Pedroche et al., 2007; Mohan et al., 2015). Similarly, the selection of enzyme
loading and immobilization matrix may prefer the partition of H+ or OH ions,
and change the local pH-value in the support. The changed pH-value may cause the
immobilized enzyme to work under conditions close to the pH optimum (Pedroche
et al., 2007). The conformation of the enzyme molecule across the immobilization
process may also affect its function in the presence of detergents. MCA immobiliza-
tion of lipases can enhance their activity because the enzyme is immobilized in an
active combination (Fernández-Lorente et al., 2006).
Omitting standard metrics for enzymatic activity assessment, such as Km, Vmax,
and kcat, or extracting separated function metrics from data sets other than interaction
progress curves can be prevented using the available literature to lead to rational
choose of immobilization schemes (Herzog et al., 2005; Koh & Pishko, 2005). To
enhance an acceptable comparison between the studies about kinetics, minimal
reporting standards (STRENDA) have been reported that contain features of
model election and error diagnosis (Tipton et al., 2014; Gardossi et al., 2010).

15.6 The Usage of Enzyme Immobilization for Production

of Different Food Products

Unlike pharmaceutical industries and some chemical industries, the food industry
requires the production of vast quantities of commercial products. For this purpose,
the cost of the biocatalyst should be lowered, thus, using immobilized enzymes show
acceptable operational consistency that allows lots of repetitive production cycles to
be carried out. In the food sector, continuous fermentation processes are preferred to
15 Enzyme Immobilization and Its Application Strategies in Food Products 423

batch processes, especially when large quantities of material are manufactured.

Examples of broad-scale usages of immobilized enzymes for production of food
products are explained in the subsequent paragraphs of the chapter at hand.

15.6.1 D-Glucose/Xylose Isomerase for Production of High Fructose

Corn Syrup

The use of immobilized D-glucose/xylose isomerase (EC 5.3.1.5) in the preparation

of highly fructose-rich corn syrup (HFCS) shows a largely applied commercial
procedure encompassing an enzyme immobilization step, with a high quantity of
enzyme that is used, and expedient product yield (Crabb & Shetty, 1999). Generally,
the enzyme, which belongs to the top three industrially used enzymes (others being
proteases and amylases), catalyzes the interconversion of D-glucose to D-fructose and
D-xylose to D-xylulose, respectively (Gaikwad et al., 1992). HFCS is predominantly
used in fructose production that is applied as a sweetener for beverages and foods, or
utilized directly as a food and beverage ingredient. While D-xylose is the native
substrate for D-glucose/xylose isomerase function, the enzyme has a wide substrate
spectrum, and in its industrial application it produces D-fructose from D-glucose in an
efficient manner (Bhosale et al., 1996).
Today, HFCS production processes are performed in fixed bed reactors that are
ordered in parallel and operated continuously (Nedwin et al., 2014). D-glucose syrup
that is stemming from corn is converted into a mixture that is generally named as
HFCS-42. This syrup contains approximately 42%, 50%, 6%, and 2% D-fructose, D-
glucose, maltose, and maltotriose, respectively, as well as low quantities of other
sugars. Higher concentrations of fructose that are obtained by chromatographic
enrichment of the 42% syrup to a final 90% D-fructose (HFCS-90) content is used
in soft drinks. The two commercially available immobilized D-glucose/xylose isom-
erase preparations, most commonly used for these processes, are on the basis of
inexpensive inorganic supports, like bentonite clay and diatomaceous earth with
cross-linking the enzyme by glutaraldehyde. The resulting composite is dewatered
and then dried in a fluidized bed dryer. The obtained immobilized D-glucose/xylose
isomerase preparations are very consistent, with a half-life of more than 1 year and
are used in a packed bed reactor at 60  C (Basso & Serban, 2019).

15.6.2 Epimerase for Production of Allulose

D-allulose (D-psicose or “pseudofructose”) is a low-calorie monosaccharide sweet-

ener recommended due to its sweeting similarity to dextrose, and about 70% of the
sweetness of sucrose. D-allulose is the C3-epimer of D-fructose. The difference
between allulose and fructose is that allulose is hardly metabolized in the human
body and has almost zero calories (only 0.007 kcal/g)). Globally leading food and
beverage manufacturers are the main target markets for allulose that replace dex-
trose, fructose or HFCS in their products, which is important to decrease calories in
424 N. S. Naghavi et al.

parallel with maintenance of the properties obtained by the sugar ingredient, such as
browning, bulking, texture, and sweetness. Allulose is also supposed to display
potential antihyperglycemic effects, and was shown to prevent postprandial hyper-
glycemia in humans. More than 0.2 g per day human allulose intake is estimated
when consuming naturally found materials, such as processed cane and beet molas-
ses, coffee treated via steam, wheat products, and HFCS. In 2012, allulose was
labeled as generally recognized as safe (GRAS) by the FDA and approved as
sweetener in food products (not yet approved in, e.g., the European Union!), so it
is commercially utilized as food additive in some parts of the world. Moreover,
ketose-3-epimerase (EC 5.1.3.31), an isomerase which is found in various
microorganisms, can interconvert fructose to allulose and vice versa (Basso &
Serban, 2019).

15.6.3 b-Galactosidase for Production of Tagatose

Another emerging sweetener is the ketohexose tagatose, which can be isolated from
animal origins. Tagatose is a kind of sweetener with 92% fructose sweetness, but
contains only 38% of fructose calories. Its catabolic route is different from sucrose;
therefore, it has insignificant effect on insulin and blood glucose levels. Moreover,
tagatose is considered a “tooth-friendly” compound for dental care products.
Tagatose can be gained from lactose with only 16% of the sweetening of the later.
The disaccharide lactose (β-D-galactopyranosyl-(1 ! 4)-D-glucose) is a natural sugar
found in milk and normally makes up 2–8% of its total mass. The process of tagatose
production utilizing the immobilized glucosidase β-galactosidase (3.2.1.23) has been
reported in 2014. This process suggests preparation and valorization of lactose
present in whey at a concentration of 18 wt.-%. It is possible to obtain tagatose by
lactose hydrolysis using immobilized β-galactosidase to produce the monomers
glucose and galactose. Then glucose is removed from the mixture by
deglycosylation using baker’s yeast; now, tagatose is obtained by epimerization of
galactose with aerated Ca(OH)2 (Basso & Serban, 2019). There have also been
reports on the utilization of immobilized L-arabinose isomerase (EC 5.3.1.4) to
obtain tagatose in stirred tank reactors or continuous flow systems (Lim et al.,
2008; Oh, 2007).

15.6.4 Other Sweeteners Produced by Immobilized Enzymes

Another sweetener for food and beverage industry, also accessible by means of
biocatalysis, is the canonic amino acid L-aspartate (2-aminobutanedioic acid). Aspar-
tate production is performed by amination of fumaric acid; this reaction is catalyzed
by the lyase enzyme aspartase (aspartate ammonia-lyase, EC 4.3.1.1). Already in
1973, Tosa et al. reported the application of aspartase from Escherichia coli for
aspartate production from ammonium fumarate; these authors immobilized the
enzyme by different methods: ionic binding on cellulose derivatives or sephadex,
15 Enzyme Immobilization and Its Application Strategies in Food Products 425

physical adsorption to silica gel or calcium phosphate gel, covalent binding to

cellulose azide or diazonium derivatives of cellulose, or entrapping in polyacryl-
amide gel lattice. The authors obtained the most active immobilized aspartase by the
entrapping process; regarding optimum pH-value, temperature, and ion concentra-
tion, no differences were observed between the immobilized and free enzyme in
terms of kinetic constants and heat stability. Excellent conversion yields were
reported for this process when operated in continuous mode using columns packed
with the immobilized aspartase (Tosa et al., 1973). Later, the same group of authors
immobilized E. coli entrapped in a polyacrylamide gel lattice as whole-cell biocata-
lyst for continuous aspartate production in a packed column reactor (Tosa et al.,
1974). Currently, aspartate is produced on an annual scale of 104 tons by cross-
linked whole cells or by the isolated enzyme immobilized by different methods
(reviewed by DiCosimo et al., 2013).
Moreover, aspartame is another sweetener produced as a dipeptide of L-aspartate
and L-phenylalanine. For this purpose, in 1981, Oyama et al. immobilized the
hydrolase enzyme thermolysin (EC 3.4.24.4) by different methods: physical adsorp-
tion to Amberlite XAD-7 and XAD-8, ionic binding to the ionic ion exchange resin
Amberlite IRA-94, adsorption on glass beads, and covalent linking to a hydrophilic
ethylenediamine-derivatized polymer gel by glutaraldehyde. The immobilized
enzyme assays prepared via these different ways were tested for formation of
aspartame by reaction of the precursors N-(benzyloxycarbonyl)-L-aspartic acid
with L-phenylalanine methyl ester by incubating the mixture of the substrates in
the solvent ethyl acetate, which normally would denature the enzyme. Substrates
moved from the organic phase to the aqueous phase in the support, where the
reaction took place, and the product (aspartame) diffused back to the organic
phase, from which it could be recovered. Among the different tested immobilization
strategies, physical adsorption to Amberlite XAD-7 and XAD-8 resulted in the best
yields (Oyama et al., 1981). Later, this immobilization technique for thermolysin
was successfully used by Miyanaga et al. for continuous production of the aspartame
precursor N-(benzyloxycarbony1)-L-aspartyl-L-phenylalanine methyl ester from
N-(benzyloxycarbony1)-L-aspartic acid and L-phenylalanine methyl ester in a
mixed organic solvent system consisting of tert-amyl alcohol and ethyl acetate in a
column reactor. Here, excellent conversion yields of 99% were achieved under
optimized conditions (Miyanaga et al., 1995).

15.6.5 Enzyme Immobilization for Debittering of Citrus Fruit Juices

The polyphenol naringin, a flavonoid, is responsible for the bitter taste of citrus
fruits. There is increased interest in fruit juice industry by using highly efficient
immobilized enzymes for debittering of citrus fruit juices (Puri et al., 2008). Already
in 1979, Olson and co-workers reported the immobilization of commercially avail-
able naringinase (mixture of the hydrolases α-rhamnosidase, EC 3.2.1.40, and
β-glucosidase, EC 3.2.1.21; hydrolyzes naringin to naringenin, glucose, and rham-
nose) in a reactor system consisting of polysulfone hollow fibers; immobilization
426 N. S. Naghavi et al.

took place by ultrafiltration of the enzymes into the sponge region of the hollow
fibers. After 210 min of continuous operation, 50% of naringin contained in grape-
fruit juice was hydrolyzed at 25  C and a flow rate of 300 mL/min (Olson et al.,
1979). Other approaches to immobilize naringinase encompass entrapping in algi-
nate, which resulted in 60% debittering of kinnow juice after 3 h when using a total
enzyme activity of 30 U (Puri et al., 1996), immobilization on electrospun cellulose
acetate nanofibers (Huang et al., 2017), on chitin (Tsen, 1984) or chitosan
microspheres (Bodakowska-Boczniewicz & Garncarek, 2019) by linking with glu-
taraldehyde, or adsorption of the enzyme on mesoporous molecular sieves via
glutaraldehyde for naringin hydrolysis in white grapefruit juice (Lei et al., 2011).
A rather bizarre protocol for naringinase immobilization was developed by Puri and
colleagues, who attached the enzyme on chicken egg white beads obtained by cross-
linking the protein with glutaraldehyde; debittering of Kinnow juice achieved an
efficiency of 68% (Puri et al., 2001). In addition, Busto et al. immobilized thermo-
philic Aspergillus niger naringinase by entrapping it into a PVA hydrogel matrix,
which was cryostructured in liquid nitrogen, to generate beads biocatalytically active
for naringin hydrolysis. Authors reported high stability of the beads; after storage at
4  C for 2 months, they retained 75% of initial activity (Busto et al., 2007).

15.6.6 Lipases for Production of Vitamin C Esters and Cocoa Butter

Analogs

One of the main water-soluble natural antioxidants is L-ascorbic acid (vitamin C). L-
ascorbic acid and its derivatives act as free radical scavengers, reacting with oxygen,
and destroying it. Moreover, hydrophobic long-chain fatty acid ester derivatives of
L-ascorbic acid are used as antioxidants in fat-rich food because of their higher
ability to dissolve in fats in comparison to the typical hydrophilic compound
vitamin C, which is insoluble in oils (Burham et al., 2009). In this context, ascorbic
palmitate and stearate are currently prepared by reaction between ascorbic acid with
sulfuric acid, followed by re-esterification with the corresponding fatty acid; finally,
a purification step by re-crystallization is carried out (Ferreira-Dias et al., 2013). In a
biocatalytic approach, immobilization of Candida antarctica B lipase (CalB) was
utilized for generation of ascorbyl esters. The biocatalytic conversion can reach a
yield of approximately 95%, depending on process temperature, the level of removal
of the side product water, and fatty acid chain length. In spite of the fact that
enzymatic synthesis suggests some benefits to the current chemical procedures,
such as interaction in the lower temperatures than chemical reactions temperatures,
higher material purity, and decreased downstream processing expenditure, many of
the manufactures of ascorbyl esters still carry out this synthesis by chemical pro-
cesses, because of the long interaction time needed by the enzymatic procedure and
the high price of the immobilized enzymes in contrast to the chemical catalysts
(Villeneuve, 2007).
The source of vegetable oils, such as palm, rapeseed, canola, and sunflower,
specifies the physical characteristics of fats and oils present in food products,
15 Enzyme Immobilization and Its Application Strategies in Food Products 427

because each oil has a different arrangement and type of saturated mono- and
polyunsaturated fatty acids in the 1, 2, and 3 locations of triacylglycerides. To obtain
the suitable melting properties of fats and oils, especially in the generation of
margarine and baking fat, chemical hydrogenation, fractioning, and esterification
have been applied. The enzymatic transesterification of food oils and fats is one of
the benefits because of the option to better monitor the product composition com-
pared to chemically transesterified products due to the removal of the hydrogenated
trans fats that have important health challenges (Marangoni & Rousseau, 1998; Asif,
2011).
Enzymatic transesterification was first investigated to produce an equivalent of
cocoa that used the sn-1,3 specificity of different fungal lipases. Cocoa butter
homologs are semisolid oils that commonly have a melting temperature of 37  C.
They are obtained from more cost-effective origins than cocoa, like palm, sunflower,
or rapeseed oil. A variety of commercial processes have been developed to produce
the equivalent of cocoa butter with elevated amount of the demanded triglycerides,
1(3)-palmitoyl-3(1)-stearoyl-2-monooleine, and 1,3-distearoyl-2-monooleine,
required for chocolate production. Most systems are made by using fungal lipases
immobilized by surface adsorption or encapsulation in liposomes (Basso & Serban,
2019).

15.6.7 b-Galactosidase for Lactose Hydrolysis

Bovine milk contains 4.3–4.5 wt.-% lactose that exposes 38–40% of the whole milk
solids. Lactose in milk and milk products is not hydrolyzed in the stomach or in the
initial part of the small intestine; it enters to other parts of the intestine and gets
hydrolyzed into the monosaccharides D-galactose and D-glucose by the glycosidase
β-galactosidase (lactase, EC 3.2.1.23) excreted by the intestinal microflora. About
65% of the entire human population (up to 90% in some Asian countries) are unable
to secrete sufficient quantities of β-galactosidase, causing many health disorders.
Elimination of lactose from milk and milk products makes them suitable for con-
sumption by people with lactose intolerance (hypolactasia), so the dairy industry has
demonstrated great interest to develop advanced lactose hydrolysis processes based
on β-galactosidase. Because the sweetening potential of lactose, glucose, and galac-
tose is 20, 70, and 58%, respectively, of sucrose, lactose-hydrolyzed milk is sweeter
than pristine milk (Panesar et al., 2010).
The simplest but most expensive solution for this problem is to add free
β-galactosidase to whole milk. Enzyme activity is stopped after complete substrate
hydrolysis, typically combined with pasteurization. Another procedure is the usage
of immobilized β-galactosidase for processing of skimmed milk; after completion of
hydrolysis, the fat fraction is added again to the hydrolyzed milk to reassemble its
nutritious components. This technique, of course, displays the benefits of recycling
and reusing the immobilized enzyme in contrast to adding free enzyme, and the final
product is free from additional ingredients, like enzymes or components or the
enzyme formulation that can constitute putative allergens.
428 N. S. Naghavi et al.

The techno-economic evaluation of lactose removal by immobilized

β-galactosidase from the fungus Aspergillus niger dates back as far as to 1990;
that time, Axelsson and Zacchi calculated the cost for a tank reactor operated in
batch mode, free and immobilized β-galactosidase, a continuously operated stirred
tank reactor (CSTR), and for a plug-flow tubular reactor (PFTR). For all cases, the
mass transfer behavior and enzyme deactivation were considered. As outcome, the
authors concluded that enzyme immobilization indeed is economically more feasible
when compared with application of free enzymes, although the high cost for enzyme
immobilization itself still constitute an obstacle. The lowest cost of 0.48 Swedish
crowns (SEK) per kg lactose to be hydrolyzed, calculated for a half-life time of
80 days, were calculated for immobilized enzyme in the PFTR; however, calculated
costs for using immobilized β-galactosidase in a batch reactor were only
insignificantly higher with 0.66 SEK/kg lactose. In any case, these two modes of
using immobilized enzyme were considerably lower in costs than for the case of free
enzyme in a batch reactor (2.10 SEK/kg lactose) (Axelsson & Zacchi, 1990).
In 2003, Roy and Gupta used the commercially available β-galactosidase prepa-
ration Lactozym™ (Novozymes, Denmark) from the yeast Kluyveromyces fragilis.
This preparation is GRAS for hydrolysis of whey to produce lactose-poor milk. The
authors immobilized Lactozym™ on cellulose beads via covalent epichlorohydrin
coupling. In a column serving as fluidized bed reactor, whey lactose was hydrolyzed
by >90% within 5 h, while the same enzyme, when applied in continuous batch
mode, took 48 h for the same hydrolysis outcome. It was possible to reuse the
immobilized enzyme three times without decrease in the biocatalytic performance of
the fluidized bed reactor column. In the same fluidized bed reactor, also lactose in
whole milk was converted to glucose and galactose up to 60% within 5 h (Roy &
Gupta, 2003).
A more recent example for the use of an immobilized β-galactosidase is the
enzyme isolated from the yeast Saccharomyces lactis. This enzyme was
immobilized by entrapment in cellulose triacetate fibers. The entrapped enzyme
was reused for 50 times with the reduction of enzyme activity less than 9% in a
rotary horizontal column reactor; 10 tons milk were processed per day via this
process on industrial scale (reviewed by Basso & Serban, 2019). Moreover, Asper-
gillus oryzae β-galactosidase was immobilized by covalently binding the enzyme to
an ion exchange resin based on polyphenolic formaldehyde (Hirohara et al., 1981;
reviewed by Basso & Serban, 2019).

15.7 The Usage of Immobilized Enzymes in Transforming Food

Waste

A notable amount (approximately 40%) of all types of food are disposed as waste
(Godfray et al., 2010), and these losses not only lead to environmental pollution but
also affect the entire food chain. This amount varies between different geographic
regions, and one should differentiate between food waste sensu stricto and agricul-
tural waste. These waste streams are responsible for a major global challenge both in
15 Enzyme Immobilization and Its Application Strategies in Food Products 429

causing environmental pollution and ethical concern considering the huge number of
people starving worldwide. With the global population expected to increase to 9.8
billion until 2050, suitable technological solutions should be developed to solve this
problem. Some technical proposals are represented at the food processing level.
Liquid food processing waste contains numerous organic carbonaceous compounds;
therefore, it has high biological oxygen demand (BOD) that causes problems for
direct disposal of them to wastewater removal plants. Here, the disposal of about one
million liters of lactose-rich whey per day only on the Northern Italian region
constitutes a prime example (Koller et al., 2016). Hence, the lipid, carbohydrate,
and protein contents of food and agricultural waste liquids are leading to high BOD;
however, at the same time, they have the potential to be converted to valuable
products, thus upgrading waste liquids into potential recoverable sources. Examples
of such conversions include oxidation, hydrolysis, acylation, and phosphorylation of
carbohydrates as well as glycosylation and deamination of amino acids, and esterifi-
cation and hydrogenation of lipids. In particular, esterification processes are widely
used for production of different value-added food and agricultural products. Waste
oils and animal waste lipids from the slaughtering and rendering industry can be
transesterified with alcohol to generate biofuels (Koller et al., 2018). Esterified
sugars can be applied as surfactants, and esterified starch may be used as biodegrad-
able plastics and adhesives. Esterification of flavonoids was reported to increase their
life time, health, and acceptance characteristics (Walle, 2009). Traditional processes
to these transformations require significant amounts of chemical catalysts and energy
resources that have limited reactivity, and lead to formation of by-products, espe-
cially when done in complex matrices, like food waste liquids (Alissandratos &
Halling, 2012; Fang et al., 2002).

15.7.1 Carbohydrate Wastes

Food processing waste streams which are carbohydrate-rich can easily be converted
by the enzymatic valorization catalyzed by hydrolases and isomerases into more
valuable products, like sweeteners and prebiotics. In fact, some of the best accepted,
well-known procedures in food and agricultural systems begin with the use of
carbohydrate substrates. In this context, immobilized thermophilic enzymes
(“thermozymes”), which have been studied for the production of high fructose
corn syrup, could be progressed and used for valorization of food waste liquids
that are carbohydrate rich (Andler & Goddard, 2018). Emtiazi et al. (2001) used
immobilized cellulase enzymes from Aspergillus terreus to decrease chemical oxy-
gen demand (COD) by cellulose removal (40–80%) from pulp manufacturing waste.

15.7.2 Lipid Wastes

According to the significant impact of environmental and economic waste stream

valorization, waste oil can be converted to value-added products, such as biodiesel,
430 N. S. Naghavi et al.

surfactants, and lubricants, by the use of enzymes. Lipases, like most other enzymes,
can be mined from different microbial sources with different performance properties.
For instance, lipases produced by Thermomyces lanuginosus and Candida
antarctica were used for lipid hydrolysis and esterification, respectively, yielding
valuable products, such as biofuel, from waste cooking oil. More than 90% conver-
sion was obtained after 10 h of hydrolysis and 10 h of esterification reactions.
Noteworthy, after 5 catalysis repeats, the lipase from C. antarctica retained its
activity, while the lipase from T. lanuginosus lost some of its activity after each
use (Vescovi et al., 2016). In another study, Rhizomucor miehei lipase and
C. antarctica lipases were immobilized on silica particles that were epoxy-
functionalized and used to enhance the performance of biofuel generation from
waste cooking oil. A 91.5% conversion rate was achieved during 10 h (Babaki
et al., 2017).
Other examples for immobilization of C. antarctica lipase for conversion of lipid
substrates to value-added products encompass the use of support materials as diverse
as core–shell MNPs for conversion of waste cooking oil to biodiesel (Mehrasbi et al.,
2017), immobilization by adsorption on poly (styrene) nanoparticles (Miletić et al.,
2010), covalent attachment on chitosan-based hydrogels (Silva et al., 2012), or
adsorption to green coconut fibers (Brígida et al., 2007).

15.7.3 Proteinaceous Wastes

Proteinaceous food waste may stem from different origins like dairy products (whey
retentate), grains (Zhi et al., 2017), oilseeds (Doshi et al., 2014), soybeans (Laskar
et al., 2018), eggs (Hong et al., 2019), or even poultry feathers (Pernicova et al.,
2019). Proteases are used for hydrolyzing proteins from waste streams and
converting them to biological peptides or useful chemicals, such as the monomers
that build up polymers. Here, enzymatic pathways are more favorable than chemical
reactions, which are not easily controllable, as observed for the degradation of
tryptophan via acid hydrolysis (Kumar et al., 2015). Immobilized trypsin has been
used for hydrolysis of dairy waste, such as whey protein (retentate fraction
remaining after ultrafiltration of full whey), as an alternative to well-established
acidic hydrolysis (Koller et al., 2019). Immobilization of bovine pancreas trypsin on
porous polymethacrylate with a pore size of 2.1 μm has led to 9.68% hydrolysis. The
hydrolysis degree had reached ~6% under the same conditions when using free
trypsin, which indicates the need for optimization of the immobilization process.
Most significantly, the peptide analysis differed between the immobilized and free
trypsin that showed the effect of immobilization methods on enzyme selectivity for
hydrolysis of amino acid sequences (Mao et al., 2017). Trypsin has also been
immobilized on reusable matrices containing spent grain and lignocellulose for
hydrolysis of whey protein (Tavano, 2013; Bassan et al., 2016).
15 Enzyme Immobilization and Its Application Strategies in Food Products 431

15.8 Commercialization of Immobilized Enzymes for Usage

in Food Industries

For successful commercialization of procedures based on immobilized enzymes, the

overall process cost is the decisive factor. Some of the major factors determining the
cost of immobilization, such as the used matrix (support), the enzyme itself, the
chemicals used in the immobilization procedure, and the special equipment that may
be needed should be considered. Current requests for immobilized enzymes make up
only a small portion of the whole enzyme market (DiCosimo et al., 2013). Different
uses of immobilized enzymes may be developed in food manufacturing, medical
applications, and food research studies (Homaei et al., 2013). Other examples for
commercial usages of immobilized enzymes are synthesis of organic materials on
laboratory scale as well as analytical and pharmaceutical usage. Moreover, apart
from the food sector, the application of immobilized enzymes for removal of
eco-pollutants from aqueous environment might be an important future field of
research and development, as it was suggested for the removal of endocrine-
disrupting compounds from wastewater by covalently immobilizing HRP on filtra-
tion membranes (Mohan et al., 2015; Rathner et al., 2017). For each commercial
application, the appropriate selection of a particular immobilized enzyme or strategy
of immobilization should be based on the advantages and disadvantages of the
performance of free and immobilized enzymes, respectively. This shows that the
establishment and use of immobilized enzymes needs profound understanding of
affecting functional and economic factors, in-depth kinetic studies, plus the knowl-
edge about the current market requirements and trends (DiCosimo et al., 2013).

15.9 Conclusions

Enzymes usage in soluble form for food processing is well established. Although a
high variety of enzymes have already been immobilized and used in different food
manufacturing industries, only few procedures have become practical and economi-
cal, and succeeded in getting established on the long term. Numerous recent
concepts have been attempted or are being used in this field. The future of such
applications and operations will depend mainly on their cost and also political
decisions. Despite the fact that not all established transformations involved in food
processing and food production can to date be replaced by biocatalytic techniques
resorting to immobilized enzymes, and although many immobilization processes
holding promise in lab-scale experiments are not yet scalable to industrial
dimensions, the outlook for immobilized enzymes in food industry is indeed
promising considering current food industry trends to become more efficient and
sustainable, combined with the rapid progression of immobilized enzyme
techniques. In any case, due to the growing human population on Earth and a
remarkable decrease in limited natural food resources, the future use of immobilized
enzymes may elevate significantly in order to produce higher amounts of food
products, and even to unlock alternative food sources for enhanced global food
security.
432 N. S. Naghavi et al.

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Prospects and Challenges in Food-Grade
Enzymes Industrial Production 16
Musliu Olushola Sunmonu and Mayowa Saheed Sanusi

Abstract

Food-grade enzymes are biocatalyst in industrial production of food and they

have numerous advantages over conventional chemical processes due to their
ability to achieve efficient and sustainable processes and products. It has found
extensive application in bakery, beverages, meat, cheese, alcoholic and juice
industries. Commercial food-grade enzymes have been preferred to be sourced
from microorganisms (bacteria, fungi, yeast, mold) rather than from plants and
animals because of their high production yield and controlled conditions. The
relevance, applications and challenges of some novel technologies; enzymes
screening, enzymes immobilization, solid-state fermentation, recombinant
DNA, protein engineering and bioinformatics as techniques for producing food-
grade enzymes were discussed. The sources and the industrial applications of
some selected food-grade enzymes, such as amylase, proteases, lipase, rennet,
cellulase, lactase, invertase, glucose oxidase, glucose isomerase and catalase in
the food industry, and food-grade enzymes safety issues, were also discussed.

Keywords
Industrial application · Food-grade enzymes · Food industry · Novel
technologies · Safety

M. O. Sunmonu (*) · M. S. Sanusi

Department of Food Engineering, Faculty of Technology and Engineering, University of Ilorin,
Ilorin, Nigeria

# The Author(s), under exclusive license to Springer Nature Singapore Pte 439
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_16
440 M. O. Sunmonu and M. S. Sanusi

16.1 Introduction

The emergence of the food-grade enzyme for industrial production can be tagged as
green revolution approach due to its catalytic biological nature in transforming food
into various value-added products in different food industries, such as in beverages
and juices, baking, development of functional foods, meat, wine, dairy, fats and oils.
The food-grade enzyme has been projected as a potential replacement for chemical
additives or harsh physical conditions (temperature, pressure, pH) used in industrial
production, therefore guarantees sustainable production for safe and nutritious food
supply. Also, with an increase in the awareness on the need to consume a healthier
diet which has increased consumers’ preference for new and convenient food
products manufactured from natural food additives has further increased the use of
food-grade enzymes. Presently, food-grade enzymes are being used in the food
industry to increase diversity, quality and varieties of food produced. The food-
grade enzymes can be generated from the plants, animals and microbial sources.
However, out of the entire sources used for enzymes generation, microbial is the
most preferred choice in the food industry. Enzymes from microbial sources can be
produced in control conditions at a faster rate with minimal harmful by-products and
in larger quantities. Large-scale Industrial production has explored the use of
Saccharomyces cerevisiae, Aspergillus oryzae, Fungi Aspergillus niger and Bacillus
subtilis bacteria as sources for microbial enzymes production. Some of the commer-
cially important food-grade enzymes produced from this sources are proteases,
rennet, pectinases, invertases, cellulases, lipases, amylases, catalase, lactase,
raffinase, pullulanase, glucose oxidase, etc. Most of naturally occurring enzymes
from plants, animals or microbial sources are not often suitable for economical
production processes and sustainability. Therefore, there is a need to adopt novel
technological approaches that can be used to improve the industrial production of
food-grade enzymes. Uzuner and Cekmecelioglu (2019) reported that industrial
enzymes market is competitive with minimal profit margin and the enzymes are
currently manufactured by few companies in the United States, Denmark and
Germany with approximately twenty (20) enzymes being commercially produced
at present. Hydrolase represents 75% of food-grade enzymes in the market. Carbo-
hydrase (i.e. tannase, phytases, amylases, pectinases, cellulases, β-galactosidases),
lipase and proteases are family of hydrolase usually employed as food-grade
additives or processing aids in the food industry. This chapter provides an overview
of the prospect and challenges of a novel application of techniques for improving the
industrial production of food-grade enzymes through screening, enzyme immobili-
zation, solid-state fermentation, recombinant DNA technology, protein engineering,
and use of bioinformatics tool in enzyme engineering. The potential application of
some selected food-grade enzymes used for industrial production and the safety of
food-grade enzyme were discussed.
16 Prospects and Challenges in Food-Grade Enzymes Industrial Production 441

16.2 Food-Grade Enzymes Screening

Screening technique is used for isolating enzymes by judiciously selecting plant,

animal and the species and strains of microorganism or cells. Maximum volumetric
productivity is used as an objective function for selecting strains or original cultures
with the capacity to produce the preferred enzyme of high productivity rate (Ramos
et al., 2011). Also, screening is used in finding alternative strains with improved
possibility of cloning proposed enzyme. The process of isolating enzymes from
extremophile source using screening techniques is useful in stabilizing, showing
high activity and turnover numbers, and specificity of enzymes when operated under
severe environmental conditions for industrial application (Synowiecki et al., 2006).
For instance, there is a rising interest in the sourcing of enzymes from isolations of
bacteria from the marine environment, such as proteases from several genera esterase
from Vibrio fischeri, inulinase from Cryptococcus aureus, amylolytic enzymes,
glucosidases and invertase/inulinase from Thermotoga neapolitana DSM 4359
(Dipasquale et al., 2009; Rastogi & Bhatia, 2019). In the meat and dairy industry,
cryophiles are needed potential microbes for screening. Since the industry operates
under room-temperature lactase, protease and pectinase can be obtained for the
production of lactose-free milk, milk coagulation and cheese maturing, fruit juice
or oligosaccharide production, respectively (Nakagawa et al., 2004, 2006; Wang
et al., 2008; Mao et al., 2010). In the screening technique, the major challenge is to
cultivate the screened and identified microbes in laboratory conditions most espe-
cially the extremophile. However, if the extremophiles are expressed in mesophiles
using genetic engineering, they can be easily cultured in the laboratory with easier
protein extraction and purification (Rastogi & Bhatia, 2019).

16.3 Food-Grade Enzyme Immobilization

Food-grade enzyme immobilization simply means confining enzymes in a given

portion of space with the use of microencapsulation or by the use of solid matrix,
such as cross-linking, ion exchange, adsorption and covalent binding, so that
the enzyme can be physically separated from the reaction medium. The prospect
of the industrial application of food-grade enzyme immobilization is that it permits
the reuse of the enzymes and also extends its usefulness to be incorporated in the
food products or food processing to improve productivity. Among the food-grade
supports used of immobilization are alginate, carrageenan, agar, glass beads, poly-
urethane foam, polyacrylamide, metal surfaces and vermiculite (Ramos et al., 2011).
Ramos et al. (2011) reported that for protease enzymes, vermiculite which is less-
expensive support using adsorption had been used for immobilization. It is a must to
consider food-grade support for any food-grade enzymes immobilization. According
to Sumantha et al. (2006), immobilization of microorganism during the production
of the enzyme has been reported to produce higher enzyme yields than
non-immobilized cells of bacteria or mycelia. Some of the immobilized enzymes
that had been applied in the food industry are immobilized glucose isomerase for
442 M. O. Sunmonu and M. S. Sanusi

manufacturing high-fructose corn syrup, immobilized aminoacylase for

manufacturing amino acids, immobilized lactase for producing tagatose,
immobilized invertase for producing inverted sugar, immobilized lipases for pro-
ducing trans-free oils (cocoa butter equivalents, and for modification of
triacylglycerols), immobilized β-fructofuranosidase for producing
fructooligosaccharides and immobilized isomaltulose synthase for producing
isomaltulose, respectively (Rastogi & Bhatia, 2019). From an economic perspective,
it is advisable to immobilize expensive enzymes to have a less-expensive process
(Fernandes, 2010). The challenges associated with immobilization are that if the
procedure of immobilization is not properly done, it might lead to the loss of enzyme
activity which might be as a result of mechanical and chemical stress (Rastogi &
Bhatia, 2019).

16.4 Solid-State Fermentation

Fermentation is a technique used for the synthesis of food-grade enzymes. The

conventional fermentation method of synthesizing enzymes is through submerged
fermentation. The type of substrate used in the submerge fermentation is a determi-
nant in obtaining either high or low enzyme activities. For instance, complex
substrate suspended or dissolved in an aqueous medium is expected to give enzymes
with high activities as compared with a simple substrate in submerged fermentation.
Aside from the type of substrate, the continuous operation has been reported to
favour enzyme yield as compared with the batch operation process which is simple
to handle and also versatile (Ramos et al., 2011). Optimization of submerge fermen-
tation parameters through the use of advance experimental design, such as response
surface methodology, artificial neural network and radial basis function, had been
reported as tools that can be used to obtain optimum parameters combination aimed
at given maximum volumetric productivity of the enzymes during fermentation
(Sumantha et al., 2006). However, solid-state fermentation is gaining greater interest
in recent years than the conventional submerged fermentation. The reason is that in
solid-state fermentation, cultures are grown of moist solid rather than in aqueous
liquid as in submerge fermentation. Also, the culture media are not usually complex;
the substrate media for solid-state fermentation can be enriched with nutrient. The
enzyme of interest in solid-state fermentation is not diluted, thus paves the way for
easy purification after synthesis. There is also low effluent generation, catabolic
repression hardly affects the synthesis of enzymes and higher inoculum and rela-
tively low humidity content used in solid-state fermentation reduces extraneous
microbial contamination (Ramos et al., 2011). The wide use of agricultural waste
as a substrate in solid-state fermentation is another advantage of this technique over
submerge fermentation as it aids in reducing the cost associated with enzyme
synthesis (Aguilar et al., 2008). Sumantha et al. (2006) reported that the use of
wheat bran as a substrate for solid-state fermentation gives a higher yield of
proteases than in submerge fermentation using Aspergillus spp. and Mucor pusillus.
16 Prospects and Challenges in Food-Grade Enzymes Industrial Production 443

The use of two or more substrates has been reported as a way of increasing the yield
of the enzymes in solid-state fermentation (Aguilar et al., 2008).

16.5 Recombinant DNA/Genetic Engineering

According to the Food Drug Administration (FDA) in the USA, Escherichia coli
K-12 also known as bovine chymosin is the first recombinant enzyme. The recom-
binant deoxyribonucleic acid (rDNA) techniques or genetic engineering has now
been used to produce chymosin by transforming microorganisms with bovine
prochymosin gene. At least there are thirty-six (36) enzymes which are genetically
modified microorganisms (GMOs) among the one hundred and sixty (160) enzymes
produced for use in the food industry based on Association of Manufacturers and
Formulators of Enzyme Products (AMFEP) enzymes lists. To achieve the synthesis
of efficient enzymes that is free of undesirable enzymes or metabolites of microbes
using rDNA, appropriate selection of host microorganisms as recombinant strains
with potential DNA sequences that can code for the defined enzyme amino acid
sequence is needed. For instance, engineering microbial host stains were applied to
some fungi strain to manipulate or eliminate their capacity to release metabolites that
are toxic to the formulation of the synthesis enzyme (Olempska-Beer et al., 2006).
The application of rDNA has immensely increased the availability of food-grade
enzymes with distinct properties to meet the food process and food matrices require-
ment. Also, it has assisted in synthesizing enzymes that have desirable sensitivity
and specificity at a low cost of production.
Rastogi and Bhatia (2019) stated that the application of rDNA can be
implemented in batch or continuous operations using cheaper raw material, therefore
lessens the criteria for enzyme extraction and purification. The use of food-grade
enzymes from genetic engineering has minimized the cost connected with upstream
product processing and also increases productivity (Panesar, 2010; Rastogi &
Bhatia, 2019). ɑ-amylases, proteases, lipases and pullulanases used in food
applications, such as in hydrolysis of starch, are produced by using improve strains
(Ramos et al., 2011; Rastogi & Bhatia, 2019). Despite this entire prospect, the main
challenge of genetically improved strain is that is not yet certified safe for consump-
tion by the FDA and European Union due to the rise in public consciousness
regarding the non-consumption of genetic modified processed foods. Also, the
lack of standard in its production and purification procedures has caused a restriction
in its application food technology.

16.6 Food-Grade Enzymes by Protein Engineering

The improvement of the food-grade enzyme in terms of stability and tolerance to

severe temperature, pH and organic solvents can be achieved through protein
engineering where the catalytic rate is improved and amino acids are modified for
enhancing substrate specificity. Rational protein design and direct evolution are two
444 M. O. Sunmonu and M. S. Sanusi

approaches used by protein engineering either exclusively or alongside each other

(Jackel et al., 2008; Tiwari et al., 2012; Rastogi & Bhatia, 2019). According to
Singhania et al. (2010), previous knowledge of the function and structure of the
protein to introduce the desirable traits using sited mutagenesis is required for
rational protein design approach, while for direct evolution, information of the
protein is not compulsory and random mutagenesis is performed by error-prone
PCR or chemical mediation. In order to forecast the overall structure of enzyme
using rational protein design, multiple sequence alignment technique, homology
modelling and protein treading methods are used (Gulati & Poluri, 2016). Rational
design is easier to improve properties during screening due to an infinite number of
protein variants generated (Singhania et al., 2010). Quax et al. (1991) used rational
design to generate mutant glucose isomerase from Actinoplanes missouriensis with
enzyme activity that is thermal and pH stable as against the parental enzyme. Double
mutant isomerase (G138P, G247D) generated using rational design showed 2.5-fold
increases in half-life and 45% specificity increment as compared with the wild type
(Zhu et al., 1999). A similar approach was also reported by Lin et al. (2008) for
generating amylase mutants from the strain of TS-23 Bacillus sp.to enhance the
thermal stability of the enzyme. However, some of the biocatalysts generated by
direct evolution are glucoamylases, cellulases, ɑ-amylases, lipases and proteinases
(Singhania et al., 2010).

16.7 Application of Bioinformatics Technique in Enzyme

Engineering

In order to analyse the information in enzymes data within a limited time either
online or off the network, the novel application of bioinformatics technique in
enzyme engineering is the recent trend useful in performing sophisticated
computations analysis (Alderson et al., 2012). The enormous of structural data and
sequencing coming out of nuclear magnetic resonance spectroscopy, X-ray crystal-
lography, mass spectrometry and NGS next-generation sequencing (NGS) for
enzyme informatics are now better understood by the application of bioinformatics
technique (Rastogi & Bhatia, 2019).

16.8 Application of Food-Grade Enzymes for Industrial

Production

The use of food-grade enzymes for industrial production has widened their applica-
tion in the development of different food products. The application of some selected
food-grade enzymes for industrial production of food are as follows.
16 Prospects and Challenges in Food-Grade Enzymes Industrial Production 445

16.8.1 Application of Food-Grade Amylase Enzymes

ɑ-amylase and β-amylase have been used over years for the industrial production of
maltose and glucose by applying the enzyme on starch. The application of ɑ-amylase
has paved way for the production of glucose syrup and has replaced the chemical
hydrolysis of starch in most starch manufacturing companies (Reddy et al., 2003).
The sources used for commercial production of ɑ-amylase are some selected strains
of bacteria (Bacillus spp.: B. amyloliquefaciens, B. licheniformis, B. subtilis,
B. cereus, B. stearothermophilus, B. licheniformis; halophilic bacteria:
Chromohalobacter sp., Halobacillus sp., Haloarcula hispanica, Halomonas
meridiana; fungi: Aspergillus species and few species of Penicillium:
P. brunneum, P. fellutanum) (Konsoula & Liakopoulou-Kyriakides, 2007; Erdal &
Taskin, 2010). ɑ-amylase had been used in the baking industry to play an important
role in enhancing the quantity, aroma and taste of baked products. Jegannathan and
Nielsen (2013) reported that the addition of amylase and lipase enzymes in bread
helps in extending its shelf life and reduces crystallization. Antistaling in bread and
improved softness can also be achieved by the addition of ɑ-amylase (Gupta et al.,
2008). However, more research still needs to be done on the application of
ɑ-amylase in the baking industry as a small overdose of ɑ-amylase can result in
sticky bread (Singh et al., 2019). For the fermentation of raw starch into ethanol, a
combination of ɑ-amylase produced from Streptococcus bovis and glucoamylase
from Rhizopus oryzae has been reported to improve the ethanol production (Singh et
al., 2019).

16.8.2 Application of Food-Grade Protease Enzymes

Protease enzymes can be the source from plant, animal, humans and microorganisms
(bacteria, fungi and virus) and are known to hydrolyse peptide bones of proteins.
Protease enzymes produced from microorganisms’ source are the most preferred for
industrial production due to ease of genetic manipulation and extensive diversity,
respectively. They are also known to favour low manufacturing cost, extensive
chemical and physical characteristics, lack of seasonal variation, large production
in industrial fermentators and quick culture development (Singh et al., 2019).
Protease enzymes of a wide variety have been exploited for industrial production
in food and pharmaceutical industries because of their specificity in action (Kalpana
et al., 2008). Almost 60% of the total food-grade enzymes in the market fall under
the category of protease enzymes (Mala et al., 1998). Alkaline and neutral proteases
produced for commercial purpose are usually generated from Bacillus genus. Pow-
dered bacteria proteases derived from Bacillus can be stable and remain 95% active
for a year. In the food industry, proteases can be applied in cheese production, baked
products making, soy hydrolysates production and meat tenderization. Proteases
from A. oryzae can improve bread colour and texture when mixed with flour dough.
More so it also aids in the reduction of mixing time. Heredia-Sandoval et al. (2016)
stated that the gluten content of bakery products can be decreased and degraded
446 M. O. Sunmonu and M. S. Sanusi

using proteases. In the dairy industry during cheese manufacturing, vegetable rennet,
animal rennet, genetically engineered chymosin and microbial milk coagulants are
milk coagulating enzymes used. Paracasein and micropeptides are generated from
the hydrolysis of peptide bone using protease enzymes in cheese making. However,
chymosin is good for generating casein. In beer production, application of protease
and amylase enzymes have been used as an efficient alternative way of achieving
malting process, thus saves energy and agricultural land that would have been used if
the conventional malting that involves drying was to be used (Jegannathan &
Nielsen, 2013). Microbial proteases have been reported to hydrolyse proteinaceous
in beer that causes haze formation, while production of wort can be achieved by the
use of B. subtilis proteases to solubilize proteins from barley adjuncts (Singh et al.,
2019). For commercial purpose, improved tenderness of meat which is the organo-
leptic quality of meat as desired by the consumers and for easy marketability can be
achieved by using thermophile protease derived from Bacillus strain, caldolysin
from Thermus strain, collagenase from Clostridium histolyticum, and aspartic prote-
ase from A. oryzae (Bekhit et al., 2014). For industrial production of aspartame
which is known as nutrasweet (zero-calorie sweetener), enzymatic synthesis of
aspartame has been preferred over chemical method due to their ability to maintain
stereospecificity. Proteases have been reported to be used to synthesize aspartame by
catalysing the reverse reaction in two amino acids, thus maintain stereospecificity
and reduce the cost of production (Singh et al., 2019).

16.8.3 Application of Food-Grade Lipase Enzymes

Lipases are important food-grade enzymes due to their biocatalysts activities in

breaking down fats and oils, esterification, hydrolysis, interesterification, acidolysis,
alcoholysis and aminolysis. Lipases can be sourced from the plant (barley, corn and
cotton), animal (adipose tissues, blood, gastric juices, intestinal juices and pancreatic
secretion), yeast (Candida and Torulopsis), filamentous fungi (Geotrichum,
Humicola and Rhizopus), bacterial species (B. subtilis, P. fragi, B. megaterium,
S. aureus, Burkholderia cepacia, P. aeruginosa, P. pseudoalcaligenes) and fungi
species (Helvinal anuginosa, Rhizopus delemar, Eurotium herbariorum, A. niger,
M. circinelloides and Penicillium citrinum) (Hasan et al., 2006; Sachan & Singh,
2015; Singh et al., 2019). However, the microbial source is mostly used for
commercial production of lipase due to their ability to reduce the cost of production,
greater widespread ability in animal and plant lipases, and greater stability (Singh et
al., 2019). Hasan et al. (2006) and Treichel et al. (2009) noted that lipase enzymes
have found application in food processing, pharmaceuticals, agrochemical,
detergents, paper and cosmetic industries. The first commercial lipase from recom-
binant technique was isolated from Thermomyces lanuginosus which is a fungus and
since then many other recombinant lipases have been produced for commercial
purpose (Singh et al., 2019).
Lipases have been applied extensively in the dairy industries for hydrolysis of
milk fat, lipolysis of cream, producing cheese products and in enhancing the flavour
16 Prospects and Challenges in Food-Grade Enzymes Industrial Production 447

of cheeses in order to accelerate cheese ripening (Aravindan et al., 2007). In the

bakery industry, lipolytic enzymes have been used to produce emulsifying liquids in
situ by degrading wheat lipids instead of using traditional emulsifiers. Also, the
application of lipolytic enzymes helped in the release of short-chain fatty acids
through esterification, thereby aids in improving the flavour of baked products
flavour. Apple wine has been produced by the Japanese company Tanabe Seiyaku
with improved alcoholic content and aroma by isolating lipase enzyme from
R. delemar or a Candida species during fermentation (Singh et al., 2019). Lipases
have also been used to improve the quality and texture of mayonnaise, whipping and
dressing products. In the meat and fish processing, lipases are used to enhance and
remove fat. Ramarethinam et al. (2002) reported that volatile flavours and the aroma
of black tea were increased using lipase secreted by Rhizomucor miehei. Lastly, in
the fat and oil industry, cheap oils have been converted into nutritional-rich oils,
oleic acids oils, low calorie triacylglycerols and polyunsaturated fatty acid using
microbial lipases (Gupta et al., 2003). Besides commercial production in fats and oils
modification, low energy consumption and high level of specificity can be achieved
using lipase enzyme modification when compared with chemical modification.

16.8.4 Application of Food-Grade Rennet Enzymes

Rennet is enzyme made up of chymosin, lipase and pepsin and it is usually referred
to as complex enzymes. Rennet can be generated from the plant, animal and
microbial sources. Rennet has been extensively applied in the commercial produc-
tion of different types of cheese. However, rennet from the microbial source is
mostly used for cheese production worldwide. For instance, R. miehei, Irpex lactis,
A. oryzae and R. pusillus are the good source of microorganism that can be used to
generate rennet for cheese production (Neelakantan et al., 1999; Singh et al., 2019).
Also, microbial rennet has served as a replacement for mammalian rennet used in
cheese production due to consumers’ demand of non-animal derivative. Unlimited
availability, low cost of production, bulk production and no risk of disease transmis-
sion are some of the advantages of microbial rennet over animal rennet (Singh et al.,
2019). The application of rennet enzyme is one of the most important uses of
enzymes in the food industry.

16.8.5 Application of Food-Grade Catalase Enzymes

Catalase enzymes also known as hydroperoxidases and is popularly known com-

mercially for converting hydrogen peroxidase to water and oxygen. According to
Singh et al. (2019) one molecule of catalase can transform millions of hydrogen
peroxide to water and oxygen per second. Catalase can be found in all living
organism but mostly source from microorganisms and bovine liver (Chelikani
et al., 2004; Tukel & Alptekin, 2004). Plant leaves with high antioxidant properties
are M. sylvestris L., black gram (Vigna mungo) seeds, cotton, sunflower and
448 M. O. Sunmonu and M. S. Sanusi

pumpkin (Eising et al., 1990; Kandukuri et al., 2012). Catalase found industrial
application in the removal of hydrogen peroxide used in sterilizing, oxidizing and
bleaching agents (Ertaş et al., 2000). The combination of catalase and glucose
oxidase has been used for the preservation of some selected foods. The enzyme
can also be used in egg processing but has limited usage in cheese production.

16.8.6 Application of Food-Grade Lactase Enzymes

This enzyme is very important in the dairy industry, most especially in the produc-
tion of low-lactose or lactose-free food, such as flavoured milk drinks, ice cream,
yoghurt and frozen dessert. Lactase can be the source and isolate from animal
organs, plants, yeasts, bacteria or molds. For industrial production of lactase, it is
usually source from A. niger, A. oryzae and Kluyveromyces lactis due to their safety
of use history and the numerous safety tests they undergo before use. The source
used for generating the lactase and the method of preparation determines its
properties, such as the temperature and pH. For optimal performance of immobilized
lactases, the procedure of immobilization and the type of carrier are important.
Acidic pH of 2.5 to 4.5 is favourable for fungi lactase production, while neural
region pH is favourable for bacterial and yeast lactase with ranges of 6.5–7.5 and
6–7, respectively. The prospects in the utilization of lactase, hydrolase and
β-galactosidase have been extensive research due to the discovery of enzymes
immobilization techniques (Mehaia & Cheryan, 1987). The application of lactase
in milk has assisted in preventing diarrhea and severe tissue dehydration which affect
lactose intolerance people. It has also been reported to increase digestibility, sweet-
ness and scoop and creaminess of ice cream, yoghurt and frozen dessert. Increase in
sweetness and sugar addition level reduction has also been ascribed as another
advantage of lactase-treated milk in the production of flavoured milk drinks.

16.8.7 Application of Food-Grade Cellulase Enzymes

Cellulases are enzymes used for extracting constituents from soy protein, essential
oils, aromatic products, green tea and sweet potato in the food industry. Dillon et al.
(2004) described cellulase enzymes as enzymes that break the glucosidic bond of
cellulose microfibrils and release oligosaccharides, cellobiose and glucose.
Cellulases can be produced from filamentous fungi (Aspergillus, Chaetomium,
Fusarium, Penicillium, Phoma and Trichoderma), anaerobic bacteria (Acetovibrio,
Bacteroides, Butyrivibrio, Caldocellum, Clostridium, Eubacterium, Erwinia,
Pseudonocardia, Ruminococcus and Thermoanaerobacter) and aerobic bacteria
(Acidothermus, Bacillus, Cellvibrio, Pseudomonas, Streptomyces, Staphylococcus,
and Xanthomonas) (Soares et al., 2012; Singh et al., 2019). Among all the sources of
producing cellulolytic enzymes, Aspergillus has been reported to be the most
outstanding (Singh et al., 2019). Combination of cellulases, hemicellulase and
16 Prospects and Challenges in Food-Grade Enzymes Industrial Production 449

pectinase is currently being used in the food industry to extract and clarify fruits
juice. Cellulase can also be used to degrade solid phase, thus enhances liquefaction.

16.8.8 Application of Food-Grade Invertase Enzymes

This enzyme has a great commercial significance in the production of inverted sugar
and is known for its sweetness effect. Invertase can catalyse sucrose to glucose and
fructose through hydrolysis. Temperature and pH within the range of 40–60  C and
3–5, respectively, are more favourable for more active invertase. At the industrial
level, invertase is mostly isolated from yeast, such as S. cerevisiae, and there is
ongoing research for the use of high yielding filamentous fungi for production of
invertase. Examples of the filamentous fungi are Rhizopus sp. And Aspergillus
casiellus. The filamentous fungi Rhizopus sp. was cultivated in the medium of
wheat bran to obtain invertase (Soares et al., 2012); Aspergillus casiellus was
inoculated in a medium of soybean meal and the invertase was isolated after 72 h,
while in yeast, invertase (S-b D-fructofuranosidase) is isolated from S. cerevisiae
(Singh et al., 2019).

16.8.9 Application of Food-Grade Glucose Isomerase Enzymes

In the food industry glucose isomerase has found extensive application in the
production of fructose-rich corn syrup. The enzyme is also known as d-xylose
ketol isomerase which aids in catalysing reversible isomerase from d-glucose and
d-xylose into d-xylulose and d-fructose, respectively. Protein engineering technique
is being explored to generate thermostable glucose isomerase enzymes (Hartley
et al., 2000). Glucose isomerase has also been used for bioconversion of hemicellu-
lose into ethanol (Singh et al., 2019). For commercialization of glucose isomerase,
the application of biotechnology in isolating mutants of promising prospect is
important.

16.8.10 Application of Food-Grade Glucose Oxidase Enzymes

Glucose oxidase is majorly used in the food industry to eliminate harmful oxygen in
food product containing probiotic microorganisms. The enzyme allows catalysing
oxidation–reductions reaction through the prosthetic group flavin adenine dinucleo-
tide (FAD). High level of glucose oxidase can be isolated from filamentous fungi,
such as Aspergillus versicolor and Rhizopus stolonifera (Guimarães et al., 2006).
Singh et al. (2019) suggested that the use of glucose oxidase through biotechnology
can be used to improve the stability of probiotic bacteria in yoghurt without adding
chemical additives.
450 M. O. Sunmonu and M. S. Sanusi

16.9 Safety of Food-Grade Enzyme

Industrial food-grade enzymes are generally considered safe and are known as food
additives, while some considered them as food processing aids. The safety regulation
on the use of food-grade industrial enzymes differs among countries, premarket
approval of the enzymes and the information provided by the manufacturer. The
prominent occupational food safety risks that have been associated with the use of
industrial food enzymes are mainly allergic reaction (in the respiratory tract), irrita-
tion risks, residual microbiological activity, chemical toxicity and oral toxicity
among consumers (Spök, 2006; Ramos et al., 2011; Singh et al., 2019). Ramos
et al. (2011) stated that enzymes are sensitizers and can cause allergic reaction in the
respiratory tract (asthma) upon inhalation and also irritation upon contact with the
skin. However, aside from the occupational risk, the final consumers of food
products containing food-grade enzymes rarely report allergic reaction because
low levels of the enzymes are usually used in food formulation which would have
eventually be deactivated upon processing and before reaching the consumers. The
future challenges that might be associated with the safety concern of food-grade
enzymes are toxic substance arising from the by-product of contaminants present in
the preparation of the enzyme. Adequate attention must be paid towards fermenta-
tion broth contaminate or environmental conditions of some phylogenetical strains
used in enzyme production. Although with the advent of genetic engineering
technique, toxicity problems can easily be eliminated. Food industry and national
or European regulatory bodies must develop strategies to harmonize and enforce
legislation on the available food enzymes that are categorized as a novel or those
with unusual properties and their food safety assessment practice to eradicate
occupational risks that may arise from their use. One of the current issues related
to food enzymes legislation is that in the USA, Japan and Canada food-grade
enzymes are considered as food additives, while Australia regards food enzymes
as food processing aids. WHO/FAO, joint committee of food additives, does not
differentiate food-grade enzymes either as a food processing aids or food additives
(Ramos et al., 2011). However, EU food legislation considered more than 160 food-
grade enzymes as food processing aids, while invertase and lysozyme enzymes are
the only two food-grade enzymes considered as food additives. National legislation
of France, Hungary, Denmark and Poland do subject food-grade enzymes that are
referred to as food processing aids to authorization before use, while in the UK the
authorization is voluntary (Ramos et al., 2011). Therefore, it is imperative for
legislation of each country worldwide to define the ranges of the permitted enzymes
and their applications.

16.10 Conclusions

For the future purpose, there is need to explore the benefits of novel approaches, such
as recombinant DNA technology, screening, protein engineering, enzyme immobi-
lization, solid-state fermentation and use of bioinformatics tool in enzyme
16 Prospects and Challenges in Food-Grade Enzymes Industrial Production 451

engineering in generating food-grade enzymes, and to adapt the enzymes for sus-
tainable and economical industrial production processes. Understanding the novel
approaches can aid the industrial production of food-grade enzymes which can be
useful in improving food product quality and consistency, preventing potential
harmful by-products in the food, reducing raw material dependence during
processing, replacing chemical additives, reducing process time, increasing shelf
life and creating more opportunities for enhancing the aromatic, structural and
textural properties of food. In addition, application of food-grade enzymes for
industrial processes should be able to lead to the development of new or improve
healthy food products and food properties within the food processing industry;
bakery, dairy, beverages, juice, alcoholic, meat, fats and oils, vegetables and other
industries. Proper selection of enzymes source, enzymes isolating method and
purification technique that is safe and non-toxic must be carefully selected as they
influence the cost of production of food-grade enzymes and yield. Solid-state
fermentation produces higher enzymes than submerged fermentation at a lower
cost. National legislation of countries should make provision for permitting and
regulating products from food-grade enzymes and their applications. More research
should be channel towards the application of enzymes technologies in food
processing to broaden our knowledge and future use because consumers are now
demanding for food products or additives that are free of chemicals.

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Nanotechnology and Food Grade Enzymes
17
Zahra Beig Mohammadi, Khadijeh Khoshtinat, Sanaz Ghasemi,
and Zahra Ahmadi

Abstract

Public concerns about food safety and its health, on the one hand, and the rapid
advancement in emerging technologies and the green ones, on the other hand,
have led to less interest in chemical interactions occurring in the food industry.
Enzymes are proteins and consequently sensitive to environmental effects such as
pH and temperature. Additionally, they are less likely to be reused and the cost of
their recovery is exorbitant. Therefore, their application as natural catalysts has
been limited. Having tackled the problems associated with free enzymes, enzyme
immobilization made it possible to reuse and store enzymes. Then, nanotechnol-
ogy implemented this technique to end in nanobiocatalysis which merges nano-
technology and biotechnology and stabilizes enzymes accordingly. Nano carriers
improve enzymes’ activity and efficiency, stability, and storage ability as they
possess unique features such as nanoscale size, high surface/volume ratio, and
variety in composition and structure. Nanobiosensors are another breakthrough in
bio and nanotechnologies which can substitute chemical and electrochemical

Z. B. Mohammadi (*)
Department of Food Science and Technology, North Tehran Branch, Islamic Azad University,
Tehran, Iran
e-mail: z.beigmohammadi@iau-tnb.ac.ir
K. Khoshtinat
Institute of Nutrition and Food Technology Research Institute, Nutrition and Food Technology
Faculty, Shahid Beheshti Medical Science of University, Tehran, Iran
S. Ghasemi
Faculty of Food Science and Technology, Isfahan University of Technology, Isfahan, Iran
Z. Ahmadi
Food Hydrocolloids Research Center, Department of Food Science and Technology, Ferdowsi
University of Mashhad (FUM), Mashhad, Iran

# The Author(s), under exclusive license to Springer Nature Singapore Pte 455
Ltd. 2022
A. Dutt Tripathi et al. (eds.), Novel Food Grade Enzymes,
https://doi.org/10.1007/978-981-19-1288-7_17
456 Z. B. Mohammadi et al.

sensors for analysis, quality control, and food safety measurement. Recently,
nanomaterials-based biosensors have acted as the focal point in food contamina-
tion detection, namely pesticides, antibiotics, and heavy metals, due to their
variety, highly specialized activity, and high precision. In this chapter, we
investigate nanotechnology and food enzymes, probable risks, and legal aspects
of nanomaterial application in food bioprocess.

Keywords
Nanotechnology · Enzyme · Nanobiocatalysis · Nanobiosensor · Food analysis ·
Food safety

17.1 Introduction

The prefix “nano” comes from the Greek word for dwarf which means “one-
billionth” and the nanometer is equal to one-billionth of a meter that is extremely
small to see even by a conventional light microscope (Ravichandran, 2010;
Sundarraj, 2019). The late Norio Taniguchi initially applied the prefix “nanotech-
nology” in 1974 and Richard Feynman first gave its concepts in 1959 (Handford
et al., 2014; He & Hwang, 2016).
Nanomaterials are tiny particles at least ranging from 1 to 100 nm in size in one
dimension, bio-persistent or insoluble in nature, synthesized through numerous
ways. Nanotechnology is a branch of science that manipulates the materials at
nanoscale which are synthesized in various routes, and are used in different fields
including electronics, medicine, agriculture, and food industries (He & Hwang,
2016; Ghasemi et al., 2017).
This reduction in size results in highly impressive features and functions.
Nanomaterials are able to be applied in different forms such as nanotubes, nano
rods, nanoparticles, nano sheets, and nanofibers in various technologies and
industries of food, electronics, catalysts, energy, and agriculture (Jafarizadeh-
Malmiri et al., 2019; Handford et al., 2014). Comparing nanomaterials and materials
in bulk form, it can be concluded that changing the particle sizes of materials into
nanoscale increases the ratio of surface area to volume, leading to unique physical,
mechanical, chemical, optical, and biological properties such as solubility, absorp-
tion of the cells, delivery properties, and the residence time in the cells of body. On
the other hand, it affects the bioavailability, safety, nutritional value, and efficiency
properties (McClements & Xiao, 2017). This factor plays a critical role in creating
unique features, so engineered nanomaterials have raised more attention in the
industries of agro-food, medicine, sewage water treatments, and others (Haroon &
Ghazanfar, 2016).
Recently, the industries of food and agricultural have had significant investments
in nanotechnology which enhance the quality of product and reduce total prices
(Yu et al., 2018a). Firstly, Pasteur introduced nanotechnology by using it in pasteur-
ization process in food industries to omit the bacteria of spoilage (1000 nm) which
17 Nanotechnology and Food Grade Enzymes 457

made the first step to transform the food processing and improve food quality.
Watson and Crick first built a model for the structure of DNA at almost 2.5 nm
which enabled the applications in biomedicine, biotechnology, production pro-
cesses, and agriculture (Hansen et al., 2013). Nanotechnology has immense potential
in the postharvest food processing. The main applications of nanotechnology in the
food and agriculture structures are food processing and security improvements,
production, packaging of food which enhances its functionality, food bioavailability,
nutrition and flavor, pathogen detection, protection of the environment, nutrients
absorption of plants, and the cost-effectiveness of storage and distribution. It also
modifies the particle size, size distribution, surface charge, possible cluster forma-
tion, and delivery methods (Rashidi & Khosravi-Darani, 2011; Ghasemi et al.,
2018). Study in nanotechnology has high potential to advantage humanity in food-
related industries (Fig. 17.1).
Generally the potential of applying nanotechnology in food industries has been in
nanocarriers (such as nanoparticles, nanoemulsion, nanocomposites,
nanobiocomposites, and nanolaminates), nanosensors and nanobiosensors (for the
control of safety and quality of the food), processing (such as nanofiltration, nano-
scale enzymatic reactor, heat and mass transfer, nanofabrication and nanocapsules
for modification of absorption), delivery, packaging, formulation, DNA recombinant
technology, evaluation, etc. (Ghasemi et al., 2018; Jafarizadeh-Malmiri et al., 2019),
but the main nanotechnology applications in food industries are food nanostructured
components and food nano sensing to increase the safety and quality of food.
Improvements in the production of inorganic nanoparticle have permitted the prepa-
ration of effective nanosensors to identify pathogens or pesticides rapidly in food
(Neethirajan & Jayas, 2011; Nile et al., 2020).
Our main goal in this chapter was to provide a brief overview of the use of
nanotechnology and nanobiotechnology, namely enzymes, in/on nanocarriers on the
food industry improvements especially food packaging. Also, the potential
applications and future perspectives of nanobiotechnology on food safety were
discussed. Also this chapter overviews the information usually accessible about
nanomaterials’ risk assessments, marketing concerns, safety and toxicity aspects,
and regulatory aspects.

17.2 Overview of Nanotechnology and Nanobiotechnology

Applications in Food Industry

Request for extra innovative improvements and technologies in valuable tools and
materials for the life study are supported by the technologies of nanotechnology and
biotechnology which are among the promising technologies in the twenty-first
century (McClements & Xiao, 2017). Nanotechnology is a technology that uses
materials and devices possessing at least one dimension sized in nanoscale, which
plays an essential role in the food and agriculture parts, contributes to improve the
crop, progresses the food quality and safety, and supports the health of humans
through innovative and novel approaches (Jafarizadeh-Malmiri et al., 2019).
 458

NanoTechnology

Primary Production Food Processing Food Packaging

Animals Plants Nutrition & Feed Processing Equipment Smart Packaging Active Packaging

Fortification Smart Sensors Nutraceutical Insulation Detection Antimicrobials

Mineral & Vitamin

Diagnostic Detection Sanitization Sensors
fortification

Nano-Formulated Novel Products (improved taste,

Agrichemicals texture, Color)

Nutrient delivery

accessible about nanomaterials’ risk assessments, marketing concerns, safety and toxicity aspects, and
regulatory aspects.

Fig. 17.1 The nanotechnology applications in food areas

Z. B. Mohammadi et al.
17 Nanotechnology and Food Grade Enzymes 459

Biotechnology

The process of food The improvement of food taste, The genetically

(Fermentation) nutrition and yield and shelf life modification of food

Fig. 17.2 The applications of biotechnology in food industries

Generally, biotechnology contains physiological processes of biological agents

such as microorganisms, enzymes, and living cells. Actually, the techniques and
knowledge of biology in biotechnology have been used to manage the cellular,
genetic, and molecular processes for developing services and products in different
fields such as food and agriculture and medicine (Faramarzi et al., 2020; Jafarizadeh-
Malmiri et al., 2019). Biotechnology is helpful in the processing of foods (the
processes involved enzymes and fermentation) and in removing malnutrition, hun-
ger, and illnesses from all the countries (Haroon & Ghazanfar, 2016). The most
important biotechnology applications in industries of food are categorized into three
groups which are shown in Fig. 17.2.
In food industries biotechnology is applied to food processing as shown in
Fig. 17.1 by using microorganisms and their enzymes to improve characteristics
including the aroma and taste, texture, nutritional values, and shelf life in foods
which is known as fermentation (Haroon & Ghazanfar, 2016; Ruane & Sonnino,
2011). In other words, in the process of fermentation the microorganisms break and
catabolize the organic compounds under aerobic and anaerobic circumstances for
manufacturing the ultimate products. Also biotechnological tools can be applied for
modifying food genetically, which means that genetically engineered
microorganisms are applied in order to produce organic acids, vitamins, sweeteners,
amino acids, nutritional supplements, and edible oils. This process can be developed
from the insertion of functional gene on DNA into a host bacteria (such as lactic acid
bacteria) (Haroon & Ghazanfar, 2016; Jafarizadeh-Malmiri et al., 2019).
Nanobiotechnology has obtained more attention nowadays because of its varied
applications in the industries of food. Progress in nanobiotechnology leads to a better
viewpoint in enzyme technology and has more importance toward commercializa-
tion of enzymes, as well as a further development in catalytic performance
(Jafarizadeh-Malmiri et al., 2019).
Nanobiotechnology is the integration of nano-engineering and molecular biology
which combines the design of novel materials in nanoscale and devices with the
wonderful characteristics for cells, biological molecules, and enzymes to find the
solution for the most important difficulties in biology (Ghouri et al., 2020;
Jafarizadeh-Malmiri et al., 2019).
The investigation in the field of nanobiotechnology in the food industries mainly
contains adding biosensors, antimicrobial, antioxidants, and further nanomaterials in
the form of nanoparticles, edible nanocomposite films, and nanobiosensor at
460 Z. B. Mohammadi et al.

packaging. In recent years, Nanobiotechnology has been a center of attention in food

packaging. Innovative food packaging has significant results on preserving safety
and quality of foods, during storage and transportation to customers. Actually,
packaging can be able to extend the shelf life of foods by controlling disagreeable
conditions and factors such as microorganisms, moisture, enzyme activities, and
chemical contaminants. Many studies have been done on the application of
nanobiotechnology in packaging, which are described in Table 17.1.
As shown in Table 17.1, the merging of the materials for food packaging and
active ingredients is a novel way for surface microbial contamination control in
foods. Several nanomaterials such as silver exhibit antimicrobial influences. The
potential perspectives of nanocomposites for the applications of packaging for foods
based on nano and bio ingredients, including biodegradable and edible
nanocomposite films, have achieved consideration because the coatings on food
products have significant effects in preservation, distribution process, and selling
various foods (Morais et al., 2014).
Nanosensors can identify particular indicators of the metabolism of pathogens or
can inform the customers of the light, temperature of product, or changes of humidity
in the environment during storing. This can omit the need for expiration date in
various occurrences and causes the consumers know the precise state of spoilage in
food and develop the products’ shelf life (Jafarizadeh-Malmiri et al., 2019). The
sensor which is applied for the biological organisms is known as biosensor.
Biosensors are used for the detection of biomolecule such as several microorganisms
and pathogens, glucose, and urea and as a complementary tool to classical analytical
methods such as high-performance liquid chromatography which is required for
food analyses. It is clear that the applications of nanomaterial in biosensors prepare
chances to produce a novel aspect for biosensor technologies for applying them in
food analysis (Jafarizadeh-Malmiri et al., 2019; Sundarraj, 2019).
Nanobiosensors gained their specificity from the reaction of biological binding,
which includes enzyme/substrate/cofactor, antigen/antibody, nucleic acid
hybridization, chemical interactions, and ligand/receptor in combination with differ-
ent transducers. Nowadays enzymes are applied as nanobiosensors much more than
the others (Thakur & Ragavan, 2013).
Today, enzymes can improve texture, nutritional value, and appearance and may
produce desirable aromas and flavors in a wide range of applications such as
processing of the cheese and starch, bakery, and producing the various fruit juices
and further drinks. Food enzymes sometimes are originated from plants or animals
(such as amylase, an enzyme for starch digestion), but most of them come from a
range of useful microorganisms (Fathi et al., 2018).
Enzymes are biological catalysts which can lower activation energy due to
accelerating the chemical reactions (Jafarizadeh-Malmiri et al., 2019). Due to their
excellent properties such as specificity, selectivity, and high activity, enzymes have
various applications in food productions such as yogurt, bread, cheese, and
fermented beverages (Sheldon, 2007; Jafarizadeh-Malmiri et al., 2019). But use of
native enzymes in food have following limitations which are effective keys in
enzymatic production and their commercialization (James et al., 1996):
 17

Table 17.1 Nanobiotechnology applications in food industries

Method of
nanobiotechnology Type of nanomaterial Matrix Application Reference
Nanoparticles Silver Fresh-cut melon, poultry Delays the growth of aerobic An et al. (2008), Fernández et al.
meat, asparagus, orange psychrotrophics, yeasts, and molds; (2009), Fernández et al. (2010),
juice, beef meat exudates effective as an antimicrobial Fernandez et al. (2010),
material in killing Staphylococcus Emamifar et al. (2010)
aureus and Escherichia coli
Titanium oxide Chinese Strawberry, jujube Decreasing browning, slow- Li et al. (2009)
senescence, down ripening, and
decay
Zinc oxide Liquid egg albumen, orange Decreased Lactobacillus Emamifar et al. (2010), Emamifar
juice plantarum, yeast, Salmonella, and et al. (2012), Jin and Gurtler
mold counts with no changes in the (2011)
parameters of quality
Nanotechnology and Food Grade Enzymes

Silver oxide Apple slice Delays microbial spoilage Zhou et al. (2011)
Edible Silica in situ improved Cherries Significant reduction in the Yu et al. (2018b)
nanocomposite the biodegradable films permeability of moisture and
film of PVA/chitosan (CS) oxygen by 10.2% and 25.6%,
respectively, and extending the
preservation time of cherries
Edible film with chitosan Ready-to-eat (RTE) roast Chitosan exhibited antimicrobial Beverlya et al. (2008)
beef activity against Listeria
monocytogenes
Nanosensors Cadmium telluride 2,4-D (herbicide) The pesticide of Vinayaka et al. (2009)
quantum dot nanoparticle 2,4dichlorophenoxyacetic acid was
(CdTe QD) detected
Hetero-structured silicon/ Suspension of salmonella Au/Si nanorods based biosensor Park et al. (2007)
gold nanorod bacterial in 1% BSA could detect Salmonella
solution
461
462 Z. B. Mohammadi et al.

• High prices
• The need of various enzymes
• Low stability of the enzyme
• High residual enzyme remained in the product and difficulties for their recovery
and reuse
• Incompatibility of the continuous process
• Low activity
• Higher labor costs

Therefore, immobilization appeared as a new and powerful method to overcome

these limitations and achieve enzyme separation, recovery, lifetime cycle rate, and
good stability. By enzyme immobilization, the enzyme mobility artificially becomes
restricted that leads to change of its properties, structure, and activity. On the other
hand, this process is based on absorption of enzymes onto nanocarriers known as
nanobiocatalysis (Jafarizadeh-Malmiri et al., 2019). The methods and conditions for
immobilizing the enzymes are explained in Chap. 14 completely. Application of this
nanotechnology as a novel method can improve the material properties such as
remote addressability, multifunctionality, multi-compartmentalization, and stability
(Cushen et al., 2012). So use of carriers for enzymes in nanoscale can improve the
enzyme characteristics such as maximum surface area per unit mass, diffusional
limitation, the loading of enzyme, the activity of enzyme, the possibility of magnetic
separation, resistance to proteolytic digestion, and better rate of electron transfer
which causes more applications for nanobiocatalyst in food industries (Faramarzi
et al., 2020).
The nanoencapsulated enzymes, antigens, antibodies, glycol-proteins, and
polysaccharides can be recommended as effective nanobiosensors in food packag-
ing, which can detect numerous chemical or microbial spoilage of foods and
eliminate pathogenic microorganisms, thus reducing diseases in people as a conse-
quence of food consumption (Bajpai et al., 2018; He & Hwang, 2016). Immobiliza-
tion of enzymes in/on the nanocarriers is a critical and essential step for the design of
nanobiosensors. Nanobiosensors were advanced for being sensitive, small, portable,
very exact, quantifiable, reproducible, stable, and trustworthy (Kumar & Neelam,
2016). So in this chapter the use of enzymes as nanobiosensors was discussed
completely.

17.3 Application of Nanotechnology with Food Enzymes

Recent advances in biotechnology have shown increasing attention to the use of

modern technologies for the development and growth of green and sustainable
biotechnology using enzymes. Immobilization of suitable enzymes along with the
use of biodegradable has gained currency as reduced chemical consumption has
received much attention for many attractive applications, including biocatalysts and
biosensors. The main challenge of using bioprocesses with enzyme catalysts is high
operating costs due to low stability and inability to reuse enzymes on large-scale
17 Nanotechnology and Food Grade Enzymes 463

industrial processes. Enzyme immobilization has been growing rapidly in recent

years. Immobilization of enzymes means that they are physically restricted to a
specific area by contact with the support, while trying to keep their catalytic activity
at a maximum level. This technology is usually able to protect enzymes against
adverse chemical and environmental conditions such as changes in temperature and
pH level. Importantly, immobilized enzymes can be recovered and reused on a large-
scale continuous industrial scale. However, immobilization may lead to a series of
structural changes that may lead to serious changes in the properties, activity, and
performance of enzymes relative to their natural state. To solve these problems, the
process of immobilization of enzymes in various methods and various carriers is
optimized to consider the best option with the highest efficiency for the intended
purpose (Jafari, 2019; Netto et al., 2013).
Nanotechnology as a new and advanced technology offers more efficient methods
than previous conventional methods to overcome technological barriers in many
areas, especially for food production (Jafari et al., 2015). Many researches have been
conducted on the use of nanotechnology in food for the production, preservation,
packaging, and delivery of biologically sensitive food and pharmaceutical
compounds. Nanoscale food, compared to micro type, leads to increased solubility,
more bioavailability and more accurate and controllable release. Nanomaterials
(NMs) have been highlighted in technological advances due to their adjustable
physical, chemical, and biological properties with better performance than their
bulky types. NMs are classified based on their origin, composition, size, and
shape. The ability to predict the unique characteristics of NMs increases the value
of each classification for their industrial applications. In the following section, more
attention will be paid to the different nanostructures and their applications for food
enzyme immobilization (Shahiri Tabarestani & Jafari, 2019).

17.3.1 Enzyme Immobilization in/on Nanocarriers

The protein and sensitive nature of enzymes justifies the need to select appropriate
immobilization methods to maintain their structure and activity. Significant
advances in the recognition of enzymes and their immobilization have led to the
use of NMs in various structures and compounds as a support to immobilize
enzymes. Enzyme carriers, in general, should have a large surface area, functional
groups, as well as biodegradability. NMs have a much higher surface-to-volume
ratio than conventional carriers and can provide more and better enzymatic immobi-
lization (Jia et al., 2014; Saallah et al., 2016).
Enzyme immobilization methods are generally divided into three main
categories: entrapment or encapsulation, binding to a carrier or matrix, and cross-
linking. Immobilization may be reversible or irreversible, involving direct adsorp-
tion to the surface. Different bonds on the surface of enzymes lead to various
interactions, such as hydrophobic, electrostatic, and van der Waals forces and
hydrogen bonds. Types of irreversible immobilization include immobilizing the
enzyme by covalent bonding to the carrier surface, entrapment in the matrix, or
464 Z. B. Mohammadi et al.

encapsulating the enzyme. In this case, it prevents the enzyme from being washed
during the reaction, but these supports cannot be reused after finishing the work and
reducing the activity of the enzyme and should be discarded. In contrast, reversible
immobilization allows recovery of enzymes after inactivation of the enzyme, but
some of the enzyme may be lost during washing. Cross-linking agents (such as
glutaraldehyde) are commonly used in cross-linking method. These agents have very
low molecular weight relative to the enzyme, so in this method the enzyme remains
almost unchanged and is also called a carrier-free biocatalyst with 100% activity. It
is undeniable that in order to achieve optimal immobilization, a proper understand-
ing of support and enzyme properties is required. On the one hand, changes in the
three-dimensional structure of enzymes and the amphiphilic nature of proteins
(hydrophobic inner part and hydrophilic outer part), the functional groups of
supports, and the type of bonds they can form should be carefully considered
(Netto et al., 2013).
Advanced developments in nanotechnology have created different modes for
enzyme immobility in different nanomaterials (Husain, 2010). The combination of
enzymes with nanomaterials as nanobiocatalysts (NBs) provides a better option than
a variety of chemical catalysts that can be referred to green technology. Enzyme
immobilization has been used by various nanostructures such as nanofibers,
nanotubes, and nanoparticles. These supports are preferred because of their large
specific surface areas (great surface area-to-volume ratios) which can provide
extreme immobilization enzymes (Agustian et al., 2016; Beig Mohammadi et al.,
2016). However, some nanostructured materials also have their own disadvantages.
For example, mesoporous silica typically binds enzyme molecules to its inner
surface, which restricts the diffusion of the substrate to the enzyme and reduces
activity. Nanoparticles and nanotubes also dramatically reduce mass transfer
limitations, while their dispersing and recycling is more challenging. Conversely,
electrospun nanofibers have a high ability to solve these problems and can be
favorable supports for enzyme immobilization (Beig Mohammadi et al., 2016).

17.3.1.1 Nanofibers
Nanofiber membranes with special properties such as continuous, porous, and nano-
open structure allow easy access of the substrate to the active sites of enzyme
molecules. Therefore, compared to other nanostructures, substrate can more easily
diffuse in this continuous porous space and perform the necessary reactions. In
general, nanocarriers for enzyme immobilization can be solid or liquid, spherical or
non-spherical, fibers, tubes, and network characteristics (Wen et al., 2017). During
the last few years, materials based on one-dimensional nanostructures (nanofibers
and nanotubes) have created a subject of substantial interest due to their unique
properties. Nanoelectrospun fibers, for instance, have achieved more attention com-
pared to other thicker fibers made of the equal material, and their applications in
different industries are relatively new. Immobilization of enzymes and encapsulation
of nutraceutical compounds or probiotics in/on nanofibers for maintaining the
quality and effectivity of foods have gained due to the specific properties of
nanofibers. In addition, nanofibers produced by electrospinning method have many
17 Nanotechnology and Food Grade Enzymes 465

advantages over their counterparts by traditional methods, such as drawing, template

synthesis, self-assembly, phase separation, and melt-blowing techniques (Neo et al.,
2018).
Nanofibers are continuous filaments with diameters ranging from a few
nanometers to 1 μm. In general, nanofibers are produced by electrospinning process
with different apparatus programs. Due to its unique properties (high surface-to-
volume or surface-to-mass ratio, high porosity, and easy to use for fabricating
various structures), nanofibers are potentially useful for different fields in food
industries such as enzyme immobilization, nutrient delivery, food packaging, food
indicator, and biosensing (Ghosal et al., 2018).
Electrospun nanofibers are formed by various materials such as synthetic and
natural polymers alone or in combination with repulsion of electrostatic charges and
elongation of the solution. Electrospun technique began in 1897 and it was
revitalized during the 1960s by Taylor. Finally, in the 1990s, with the advent of
nanotechnology, much attention was received. In recent years, this technology has
been developed due to valuable features such as high efficiency, good mechanical
properties, low-diameter distributions, and usability for many natural or synthetic
polymers or their combinations (Neo et al., 2018).
The electrospinning mechanism is considered as the combined effects of electro-
static repulsions by the charges collected on the exterior layer of the polymer
solution and the Coulombic forces exerted by an external electric field. A remarkable
degree of sufficient molecular interaction and cohesion to resist electrical tension
leads to the formation of a fiber, and this process is referred to as electrospinning.
Generally electrospinning does not alter the structure of systems, but reduction of
material to the nano size may lead to change some of their properties (Ghorani et al.,
2017; Alehosseini et al., 2017).
The desired polymer is prepared to produce nanofibers in a suitable solvent.
Solvents are evaporated using high-pressure evaporative electric fields. Finally,
ultrathin polymer structures as continuous fiber structures are collected dry by the
collector plate. This mechanism is visible in Fig. 17.3.

• All ingredients must be nontoxic.

• Their preparation is cost-effective and affordable.
• They must be resistant during production (heating, freezing, drying, and mechan-
ical stresses), storage, and transportation.
• No adverse effects on organoleptic properties of food products must be observed
(appearance, texture, aroma, and taste of the mouthfeeling).
• This not only must not reduce the bioavailability of enzymes and nutrients but
also it must have the ability to increase them (McClements et al., 2009).

Enzymes are actually biocatalysts that have many applications in the commercial
and industrial regions of food processing, bioreactors, and biosensors. Commercial
and recyclable use of biocatalysts depends on the degree of efficiency of the
immobilization technique and the residual activity of the enzymes. Methods of
enzyme immobilization are divided into different categories: carrier-free.
466 Z. B. Mohammadi et al.

Syringe Solution Needle Taylor Conee

High Voltage Power Supply

ly

Fig. 17.3 Schematic of the electrospinning process and nanofibers production

Enzyme immobilization on these nanofibers can enhance and retain their activity
and binding of enzymes to the nanofibers as well as enzyme separation for recycling.
Enzyme encapsulation in nanofibers can also be used as an efficient method to
preserve the third structure and activity of the enzyme. The main limitation for
immobilized enzymes on nanoelectrospun fibers is the nature of a liquid system for
dissolving enzymes and water-insoluble polymers that can remain resistant in a
variety of environments and are easily separated. Fiber materials are generally the
most desirable supports due to their inherent properties such as good mechanical
strength, high specific surface area, and inter-fiber porosity (Gabrielczyk et al., 2018;
Rojas-Mercado et al., 2018).
Enzymes can be immobilized after electrospinning by covalent or noncovalent
bonding on the fibers, or encapsulated coelectrospinning methods within polymer
fibers. Encapsulation of ficin and papain enzymes in nanofiber polyvinyl alcohol
(PVA) electrospun and glutaraldehyde (GA) as cross-linking agents were studied.
The results showed phycin and papain remained stable 92% (after nine consecutive
use periods) and 40% (after four consecutive use periods) of their initial activity,
respectively (Rojas-Mercado et al., 2018).
Phospholipase A1 was successfully immobilized on modified surface
(MS) chitosan:polyethylene oxide (CS:PEO; 90:10) nanofibrous mat (NFM) with
atmospheric plasma (AP) at different times (2, 6 and 10 min). Scanning electron
microscopy (SEM) images showed that the membrane structures maintained the
nanofiber porous structures and uniformity before and after AP treatments. PLA1
was successfully immobilized by covalent bonding with functional groups of
chitosan NFM. The results showed 80% of initial activity of immobilized PLA1
on APSM CS: PEO NFM was still maintained after ten consecutive use periods
(Beig Mohammadi et al., 2016).
17 Nanotechnology and Food Grade Enzymes 467

17.3.1.2 Nanoparticles
Nanoparticles are generally unique and discrete particles with three dimensions with
a size of 100 nm or less and have very different properties from bulk materials.
Magnetic nanoparticles are composed of various elements, especially iron, nickel,
cobalt, and their various chemical compounds. In the food industry, due to the
existing safety limitations, nontoxicity of good biocompatibility, nanoparticles com-
monly used include iron oxides such as Fe3O4 magnetic nanoparticles.
Like other nanoscale materials, they perform better than bulk magnetic materials
in sizes of 10–20 nm. Magnetic nanoparticles are very efficient as they can be easily
separated and recovered using a magnetic field during the process. Since surface
energies play an important role, surface interactions between magnetic particles and
proteins must be carefully considered. Bates noted that the adsorption capacity of
proteins is highly correlated with the activation of chemical bonds on the particle and
the protein surface. The absorption capacity of proteins is highly related to the
activation of chemical bonds on the particle and the surface of the protein.
Nanoporous materials have a lower enzyme load as support but no restriction on
substrate diffusion. In contrast, porous materials have a lower restriction of substrate
diffusion while showing a high enzymatic load (Netto et al., 2013). Immobilization
of α-amylase on carboxylated magnetic nanoparticles, lipase on magnetic Fe3O4
chitosan to synthesize ascorbyl palmitate, invertase on silica-modified magnetic
nanoparticles in order to hydrolyze the potato starch to glucose and fructose are
successful examples of the application of nanoparticle-immobilized enzymes in the
food industry (Cao et al., 2016; Wang et al., 2015; Bayramoglu et al., 2017).

17.3.1.3 Nanosheets
The carbon family is one of the most widely used materials to immobilize enzymes.
For this reason, different methods of producing carbon-derived nanomaterials have
been studied. Among these materials, graphene is used in the immobilization of
enzymes due to its remarkable optical, thermal, electronic, and mechanical
properties. It is composed of carbon atoms with a dense two-dimensional structure
similar to a honeycomb network. Graphene, in addition to successful drug and gene
delivery, has been able to immobilize enzymes due to maintaining the natural protein
structure and activity of enzymes (Singh et al., 2014).
Immobilization of Laccase obtained from Aspergillus oryzae (for ethanol produc-
tion, clarification of wine and tea) and α-galactosidase (for hydrolysis of raffinose
oligosaccharides (RFOs) cause of flatulence in soybeans) on graphene nanosheets
are significant examples of the application of nanosheets in food industries (Singh
et al., 2014; Skoronski et al., 2017).

17.3.1.4 Nanotubes
In recent years, carbon nanotubes (CNTs) have been considered for enzyme immo-
bilization due to their special surface properties along with their desirable structural
and mechanical properties as well as their very specific surface. The main types of
CNT, including single-walled (SWCNTs) and multiwalled (MWCNTs), are used to
immobilize enzymes which provide higher enzyme loading and lower mass transfer
468 Z. B. Mohammadi et al.

resistance. MWCNTs are generally composed of multiple layers of graphite that

surrounded a central pipe, which are less expensive and more stable in harsh thermal
and mechanical conditions. Both covalent and noncovalent methods could be carried
out for binding enzymes to nanotubes (Homaei & Samari, 2017).
Mukhopadhyay et al. (2015) immobilized pectate lyase by entrapment in
SWCNTs. The results showed enzymatic behavior and its stability boosted at
different temperatures, namely 4  C and 80  C and repeated freeze-thaw cycles. In
other study enzyme activity of Laccase immobilized on MWCNTs maintained was
more stable than the free one after nine cycles and storage time (Tavares et al., 2015).

17.3.2 Application of Nanobiocatalyst in Food Industry

17.3.2.1 Food Products

Different immobilized enzymes have been used by different nanostructured supports
in different food industry processes, some of which are discussed in this section
(Table 17.2).
Mosafa et al. (2013) immobilized papain onto silica-coated magnetic
nanoparticles for applying in pomegranate juice clarification. The immobilized
papain, compared with the free papain, improved storage stability and exhibited
enhanced enzyme activity, good reusability, and well tolerance to different pH and
temperature of the medium. Both the immobilized and free enzymes were influenced
for the pomegranate juice clarification (Mosafa et al., 2013).
Sojitra et al. (2016) prepared nanobiocatalyst of magnetic tri-enzyme by
co-immobilizing three enzymes simultaneously, pectinase, cellulase, and
α-amylase, onto nanoparticle of amino-functionalized magnetic for clarification of
juice. The results showed that up to 75% of residual activity of the immobilized
enzymes was retained even after reusing eight cycles consecutively. Finally, the
clarification of grapes, pineapple, and apple juices using magnetic tri-enzyme was
obtained, with 46, 53, and 41% reduction in turbidity, respectively, during 150 min
treatment (Sojitra et al., 2016).
Shahrestani et al. (2016) successfully immobilized xylanase onto 1,3,5-
triazinefunctionalized silica encapsulated magnetic nanoparticles (MNPs) for
clarification of fruit juice. The results showed that the immobilized xylanase on
functionalized MNPs (Xy-MNP) had excellent catalytic activity at 65  C and pH 6.5.
Also, Xy-MNPs had a significant influence on juice clarity and showed quite
impressive stability so that up to 55% of the initial activity of the immobilized
enzymes were retained even after 10 reaction cycles consecutively in the enrichment
of the juices (Shahrestani et al., 2016).
Patel et al., (2018) immobilized Smt3-D-psicose 3-epimerase onto nanoparticles
of the functionalized iron oxide magnetic for the clarification of wash from the
pomace of kinnow and apple fruits. The results showed that the immobilized enzyme
had excellent storage stability by missing only 20% activity during 60 days of
storage at 4  C. Up to 90% of initial activity for the immobilized enzyme retained
even after reacting 10 cycles of catalyzing D-fructose epimerization consecutively.
17 Nanotechnology and Food Grade Enzymes 469

Table 17.2 Application of nanobiocatalyst in food industry

Type of
Product Types of nanocarriers enzyme Results References
Soybean oil Superparamagnetic Lipase Immobilizing lipase Xie and Ma
Fe3O4 nanoparticles improved storage (2010)
stability, exhibited
enhanced enzyme
activity until 70%
initial activity, 90%
soybean oil
transesterification
Pomegranate Silica-coated Papain Immobilizing papain Mosafa et al.
juice magnetic improved storage (2013)
nanoparticles stability and
exhibited enhanced
enzyme activity,
good reusability, and
well tolerance to
different pH and
temperature of the
medium.
Immobilized
enzymes could be
influenced for the
pomegranate juice
clarification
α-Amylase Magnetic Up to 70% of the Baskar et al.
nanoparticles initial activity of the (2015)
immobilized
enzymes was
remained. Simply
recovered
Fruit juice 1,3,5- Xylanase Up to 55% of the Shahrestani
triazinefunctionalized initial activity of the et al. (2016)
silica encapsulated immobilized
enzymes were
retained even after
10 reaction cycles
consecutively
Grapes, Nanoparticle of Pectinase, Up to 75% of Sojitra et al.
pineapple, amino-functionalized cellulase residual activity of (2016)
and apple magnetic and the immobilized
juices α-amylase enzymes were
retained even after
reusing eight cycles
Wash from Functionalized iron Smt3-D- Excellent storage Patel et al.
the pomace oxide magnetic psicose stability by missing (2018)
of kinnow nanoparticles 3 epimerase only 20% activity
and apple during 60 days up to
fruits 90% of initial
activity for the
immobilized enzyme
(continued)
470 Z. B. Mohammadi et al.

Table 17.2 (continued)

Type of
Product Types of nanocarriers enzyme Results References
retained even after
reacting 10 cycles of
catalyzing
D-fructose
epimerization
consecutively
Soybean oil Atmospheric plasma PLA1 78.50% Beig
surface modified Immobilization Mohammadi
chitosan:polyethylene efficiency retained et al. (2020)
oxide (90:10) 80% of its initial
nanofibers activity after 10 time
of recycling
decreased
phospholipids to
8.6 mg/kg after 6 h
Pineapple Polyethylene glycol Pectinase Up to 59% turbidity Kharazmi
juice grafted magnetic reduction occurred et al. (2020)
nanoparticles in treated pineapple
juice
Up to 55% of the
initial activity of the
immobilized
enzymes was
retained after
10 repeated uses

Immobilizing made significant improvements in storage stability of the enzyme, its

recycling efficiency, and thermal tolerance (Patel et al., 2018).
Kharazmi et al. (2020) immobilized pectinase covalently onto nanoparticles of
polyethylene glycol grafted magnetic via trichlorotriazine for fruit juice clarification.
The immobilized pectinase exhibited improved operational stability, enhanced cata-
lytic activity, and simple reusability. Up to 59% turbidity reduction occurred in
treated pineapple juice with immobilized pectinase. In addition, it exhibited high
operational stability of immobilized pectinase and the enhanced reusability, and up
to 55 and 94% of the initial activity of the immobilized enzymes was retained after
10 repeated uses for clarification of the pineapple juice and 125 days storage at
25  C, respectively. It can be suggested for juice and food processing industries
(Kharazmi et al., 2020).
Lipase was immobilized onto superparamagnetic Fe3O4 nanoparticles by cova-
lent bonding for soybean oil transesterification. The results demonstrated the resid-
ual activity to be almost 70% of its initial activity after four cycles (Xie & Ma, 2010).
The immobilization of PLA1 on APSM CS:PEO NM by Beig Mohammadi et al.
(2020) for soybean oil degumming was carried out. The result showed the
immobilized PLA1 can be reused 10 times without a significant loss in the enzyme
activity. In addition, high enzyme loading, effective catalytic activity, and easy
17 Nanotechnology and Food Grade Enzymes 471

recovery reusability were observed. The residual phosphorus content by

immobilized PLA1 decreased to less than 10 mg/kg after 6 h, which is acceptable
according to the standard for the physical refining of soybean oil (Beig Mohammadi
et al., 2020).
The magnetic nanoparticles were used as a carrier for immobilized fungal
α-amylase. This nanobiocatalyst is used for the production of glucose from starch.
The results showed the potato starch had more affinity toward immobilized
α-amylase than free type. In addition, the α-amylase simply recovered after each
progression (Baskar et al., 2015).

17.3.2.2 Application of Nanotechnology with Food Enzymes in Food

Packaging
Materials used in food packaging should improve mechanical, barrier, and antimi-
crobial properties, as well as imbedded nanosensors to monitor packaged food
during transportation and storage. Nanofibers amplify mechanical property to protect
foods with brittle texture; adding green tea and oregano extracts to edible films
prepare antioxidant activities for packaging materials. Monitoring of changes of
food quality during storage could be possible by smart packaging, such as applica-
tion of multiple pH dyes in fibers used as packaging materials. Nanofibers, as a
product of nanotechnology, as well as antimicrobial nanoparticles with controlled
release properties, and bioactive components could have significant role in food
industries. The structured polymeric films containing nanofibers make great surface
area and mass transfer rates, they could be used to detect pathogens in food.
There have been many efforts to innovate nanostructure functional food system,
in order to control food safety by nanosensors and nanobiosensors; make tools for
objective transfer of nutrients for their nanotherapy; extend controlled release system
of nanoencapsulated materials; and form enzymatic reactor in nano size for exten-
sion fortification of food with valuable ingredients such as essential fatty acids.
Bionanocomposites, such as polymer nanocomposites, with hybrid nanostructure
can improve gas barrier and thermal and mechanical features. Biodegradable types
could be environmentally friendly, because of reducing the need to use plastics.
These materials need not only inorganic particles to create biopolymer structure but
also some additives such as surfactants.
Nanotechnology-derived food products are significantly expected to be reachable
for people, because of development of some new innovations such as electrospun
nanofibers as a food packaging material or to encapsulate valuable components
(Dwivedi et al., 2018).
Nanoparticles, nanoemulsions, nanocomposites, and nanostructured materials are
used for food processing, delivery, formulation, and packaging, which are very
important for food safety and biosecurity. Application of nanosensors in food
packaging has many advantages, and there are some examples including:

• Palladium, platinum, and gold based nanosensors to detect any sort of color
changes in food, any gases produced by spoilage, any changes in light, heat,
472 Z. B. Mohammadi et al.

humidity, and chemicals into electrical signals, and toxins such as aflatoxin
in milk.
• Single-walled carbon nanotubes and DNA to monitor soil condition needed to
crop growth and detect pesticides on fruit and vegetable surface.
• Carbon black and polyaniline to detect carcinogens and pathogens in food.
• Array biosensors, electronic noses, nano-test strips, and nanocantilevers to moni-
tor color changes resulting from food spoilage.
• Nano-smart dust in order to detect any type of environmental pollution.
• Nanobarcodes to detect agricultural products quality.
• Nanobiosensors in order to detect viruses and bacteria.
• Biomimetic sensors (protein and biomimetic membranes) and smart biosensors to
determine mycotoxins and some toxins, act as pseudo cell surfaces to assist
pathogens detection and removal.
• Surface plasmon-coupled emission biosensors (containing gold) to detect
pathogens.
• Cerium oxide immunosensors and chitosan-based nanocomposites to detect some
toxins like ochratoxin A.
• Carbon nanotubes and silicon nanowire transistors to detect staphylococcal
enterotoxin B and cholera toxin.
• iSTrip of time-temperature indicator/integrator to detect the spoilage of food
based on the history of temperature.
• Abuse indicators to detect desired temperature has been achieved or not.
• Partial temperature history indicator, when the temperature exceeds a certain
pre-determined value.
• Full-temperature history indicator to register continuous temperature changes
considering time and detect frozen food temperature changes.
• Reflective interferometry in order to detect E. coli contamination in packaged
foods (Pradhan et al., 2015).

To keep food quality and safety during storage and transport, food packaging
plays a key role, by extending the shelf life. Therefore, the type of food packaging
materials is very important, as well as being biodegradable and using natural
polymer instead of petroleum-based plastics. Some biodegradable polymers used
in food packaging are:

• Polysaccharide-based polymers, such as alginate, cellulose, chitin, chitosan,

gellan, pectin, starch, and xanthan.
• Protein-based polymers, such as collagen, gelatin, whey protein, soy protein,
and zein.
• Aliphatic polymers, such as polylactic acid and polyhydroxybutyrate.

Today, in order to control microbial contamination on surface of foods, active

components are to be combined with edible films. The application of biopolymer
needs using some additives to make better their mechanical and thermal properties as
well as retarding oxidation, controlling of respiration rate, migration of moisture, and
17 Nanotechnology and Food Grade Enzymes 473

microbial growth. In active packaging, shelf life of foods would be prolonged by

using antioxidant and antimicrobial agents in formulation of packaging materials.
Organic acids, bacteriocins, and enzymes are some examples of active components
used for the formulation of packaging with antimicrobial property.
Because of some specific features of bionanomaterials, like improvement of
sustainability and also biological activity, they might be used to encapsulate and
deliver some micronutrients. Chitosan, a biopolymer having particular properties,
which has been extracted from shells of shrimp and crab, is nontoxic and biodegrad-
able and possesses film forming and antimicrobial properties. Nanoparticles of
chitosan have more surface area-to-volume ratio and more effectiveness compared
to bulk ones, which make them appropriate for formulation of food packaging
materials.
Metal nanomaterials, such as silver and gold, have excellent antimicrobial activ-
ity and can show inactivation of microorganisms. As silver nanoparticles could
absorb and break down ethylene, they are able to prolong the senescence of fruits
and vegetables in packages with films containing them. Also, it is shown that they
have good effectiveness for strains such as E. coli, which are resistant to silver
(Jafarizadeh-Malmiri et al., 2019).
In order to detect E. coli O157:H7AQ19 -the pathogen separates by
immunomagnetic method- after hydrolyzing of p-nitrophenyl phosphate by alkaline
phosphatase the absorbance of p-nitrophenol will be red. Another example of
enzyme application for meat freshness monitoring is using yeast sensor
(Trichosporon cutaneum), in which signals of flow-injection analysis will show
polyamines and amino acids producing by meat, and increasing of bacteria count
during its storage.
Most sensors for quality control of meat packaging, which have been introduced
by researchers, are not commercialized and should be developed for releasing to
market. The application of antimicrobial ingredients in coating in smart packaging
by nanomaterials can be a promising impact on food packaging. Of course, fast
monitoring methods are needed to check a lot of food packages during storage and
shipping for a long period of time (Fatima Mustafa & Andreescu, 2018).
Biocatalysts can have many advantages as an aid in bioprocessing and bioactive
packaging, because these components or any reaction adjusted by enzymes help
bioactive materials to bring a substrate to react. In fact, an enzyme is a specific
catalyst that can increase product quality by producing useful ingredients or
decomposing unpleasant ones. Immobilized enzymes not only can be simply recov-
ered, and used again, but also have a low cost for environment, used materials, if the
suitable method for immobilizing of enzyme be used. By using nanomaterials for
applying immobilized enzymes in food packaging, increasing the ratio of surface
area to volume, production scaling up, and mechanical resistance will happen
(Wong, 2016).
The intelligent packaging using nanosensor will have a lot of advantages in food
industries. Very small chips will be put in the food packages, which act as an
electronic bar code, in which food would be monitored in all steps including process,
474 Z. B. Mohammadi et al.

storage, and even consumption. Developing of wireless nanosensor networks is

necessary to cover limitations of nanosensors.
In future, intelligent food packaging using nanotechnology could be a necessity
for food safety and quality control by precise and fast methods. These new packag-
ing tools need innovation and advancement of nanosensors as well as lowering the
cost (Fuertes et al., 2016).
Time temperature integrators (TTIs) are one of the newest commercialized
methods for monitoring fresh foods, which need cold chain during distribution and
handling. TTIs functions are based on some reactions, such as enzymatic, microbial,
and photochemical, which are dependent on temperature, and reaction speed
increases by temperature enhancement.
In a kind of TTIs, lipase shows color changes which are dependent on pH
changes. As the spoilage of meat usually is made by microbial growth and enzymatic
autolysis, TTIs using enzymatic biological reaction are very effective tool as a
freshness indicator and fresh and frozen meat spoilage trend. TTIs can simply be
applied into packaging material formulation for frozen fish, fruits and vegetables,
and dairy products. Available commercialized TTIs demonstrate changes of color
which are simultaneously dependent on temperature and time.
As in meat spoilage, most bacteria use glucose; predicting of meat shelf life can
be possible by glucose measuring. The color of some indicators which work on
chemical reaction will change.
In rigor mortis, glucose changes to lactic acid and protons in muscles, by
anaerobic fermentation. This happens in cold packages of meat as well as freeze-
thawed meat products. Lactic acid will simply be detected by pH indicator dyes. In
order to control the quality and freshness of food products, in commercialized
glucose sensors, glucose oxidases is immobilized on the electrodes surface and
ferrocene acts as a mediator.
Electrochemical biosensors are used to monitor food quality in smart packaging.
Two main electrochemical biosensors are biocatalytic devices and affinity sensors.
The first one redox enzymes, whole cells, or tissue slices would produce electrical
signal, and in the second type, antibodies, antibody fragments, or aptamers are
targets. In another kind of electrochemical biosensors, enzymes are used to recog-
nize analytes. These are very specific, not expensive, and could simply be suited to
materials of packaging (Park et al., 2015).
In active packaging, silver nanoparticles are investigated by many researchers due
to their antimicrobial property against foodborne pathogens, and inhibition of fungi
and some viruses. Because of silver bactericide effect, binding to bacteria DNA,
proteins, and enzymes will happen. Nanoparticles of silver can destroy outer and
cytoplasmic membranes of bacteria by inhibition of their respiratory chain enzymes
and be effective against E. coli, when used in agar film formulation.
The antibacterial activity of nano titanium dioxide particles (nano-TiO2) is also
known, although it be limited by exploring UV irradiation. DNA damage through
hydroxyl radicals and attack to outer and inner membrane of bacteria are two
proposed mechanisms for their bactericide effects. It is shown that plastic films
17 Nanotechnology and Food Grade Enzymes 475

coated by TiO2 nanoparticles could stop Penicillium expansum spoilage in apple,

tomato, and lemons, because of their photocatalytic activities when exposed to light.
Also, it is observed the effectiveness of lactase or cholesterol reductase to
increase food quality, when are enclosed by packaging materials, as well as adsorp-
tion into nanoclays incorporation by polymers (Sharma et al., 2017).
Some sensors utilize enzymes like laccase or tyrosinase for biocatalytic conver-
sion of phenolic antioxidants to their quinones, which will be measured by electro-
chemical reduction at a low applied potential.
Antimicrobial packaging by inhibition of microbial growth will extend shelf life
of food products. In this packaging, active agent, such as organic acids, enzymes will
be coated on inner layer or enclosed by packaging material. Silver nanoparticles
have been used in biodegradable film, and their antimicrobial activity against E. coli
and Staphylococcus aureus and increasing cherry tomatoes packages shelf life was
shown. It is suggested to study on the toxicity of AgNPs, and their potential
migration from packaging into food, before commercialization (Mustafa &
Andreescu, 2020).

17.3.3 Application of Immobilized Enzyme on/in Nanocarriers

as Biosensors in Food Analysis and Safety Aspects

Biosensing is a way for detection, in which combination of biological components

and physicochemical methods are used. By increasing of food adulteration, the
application of nanobiosensors in safety analysis has got an important potential as
high sensitive and selective detection methods. Better identify of target, fast recog-
nition by enhancement of signal production, boost of selectivity and sensitivity, and
reduction of time analysis are some of the advantages of biosensors. Various
nanomaterials have had effectiveness as biosensors, which can be classified into
metallic nanoparticles, metal compound nanomaterials, carbon materials,
non-metallic nanomaterials, nanostructures, and composite nanomaterials. Tradi-
tional and time-consuming methods to detect microorganisms, as well as increasing
demands for quick tests, without culture, led to find the other selective and sensitive
techniques. Initial screening of food bacteria could be one of the nanobiosensing
advantages. Trace quantity of pathogens’ DNA could be detected by nanomaterials
and amplification methods such as polymerase chain reaction (PCR), or recognize by
antibodies and or aptamer (antibodies as a part of a sensing). Inappropriate storage
might make contamination of animal feed and agricultural products with mycotoxins
or bacteria toxins; therefore, the detection of toxins is very important in point of food
safety. Detection by nanomaterials in biosensing technique could be particularly way
as well as fast, susceptible, compared with ordinary methods. For example, aflatoxin
B1 has been detected by nanoparticles based biosensors, using antibody-
functionalized mesoporous carbon (MSC). Also, fluorescent nitrogen-doped carbon
dots on gold nanoparticles (AuNPs) have been used to detect ochratoxin A, an
aptamer-based assay method, which is highly selective for other toxins, such as
deoxynivalenol, fumonisin B1, and zearalenone. Gold nanoparticles doped
476 Z. B. Mohammadi et al.

ferrosoferric oxide nanoparticles, cadmium telluride quantum dots-glucose oxidase,

and silver nanoclusters are some of metal compound nanomaterials used for toxins
detections. Application of nano-extraction using mass spectrometry to extract
aflatoxins from liquid samples by an absorbant (polydopamine-coated on nanoparti-
cle) has been reported, as well as to quantify, that was coupled with HPLC-MS/MS.
Pesticides are another harmful components, if accumulate in humans and animals.
Therefore, to control the Maximum Residue Levels, using suitable determination
methods are necessary. Inhibition of acetylcholine esterase by pesticide is a fast and
widely used method, in which organophosphorus compounds and carbamates spe-
cifically are inhibited by the enzyme. By nanobiosensing assay, thiocholine genera-
tion by acetylcholine esterase catalysis would cause the accumulation of gold
nanoparticles, and better their recovery. Another method is direct detection by
organophosphorus hydrolase, in which ordered mesoporous carbons are used to
immobilize cell surface-displayed enzyme, and could detect paraoxon, parathion,
and methyl parathion. Some pesticides would be detected by nanobiosensors based
on their electrochemical activities, such as malathion and lindane. The most preva-
lent method to detect pesticides in food is an immune test by nanobiosensors.
Prevention of photochemical property of nanomaterial by pesticides would be the
principle of antibodies reorganization, for example, blocking of gold nanoclusters/
grapheme hybrid’s electrochemiluminescence by pentachlorophenol.
The uncontrolled usage of some antibiotics in animal feed to raise the growth
would result in the increase of antibiotic residues in their meat and milk. Therefore,
some recognizing assays based on aptamer, antibody, and liposome are used to
detect antibiotic residues in food, by using some nanomaterials like glucose oxidase
and nanoparticles of gold.
Detection of heavy metals, as hazardous elements, can be done by recognizing
some biomolecules such as nucleotides (with colorimetric assay for Hg2+ using gold
nanoparticles), DNAzyme (for lead ions with DNA-stabilized AgNCs), amino acid
(cadmium and leads ions with graphene-enhanced electrochemical signals), and
antibodies (for cadmium biosensing based on coreshell of bimetallic nanoparticles
of gold and silver enhanced Raman scattering).
Existence of some components could be recognized by biosensors, like mela-
mine, as a food adulteration (to increase protein content milk), by aptamer of triplex
molecular beacon and fluorescent nanocluster of silver or nitrite, as a hazardous
additive, by electrochemistry of myoglobin on a reduced glucose oxidase-
multiwalled carbon nanotubes-platinum nanoparticles.
Biosensor is an apparatus or tool which contains a biological sensing material
composed of a chemical or physical transducer, to convert chemical or biological
signals into electrical ones (Fig. 17.4).
The potential of a biosensor will be increased by application of nanomaterials
such as carbon nanotubes, nanowires, magnetic nanoparticle, and nanorod. These
materials increase sensitivity and decrease limits of detection.
However, formal methods for detecting foodborne microorganisms, like enzyme-
linked immunosorbent assay, polymerase chain reaction, and the application of fast,
reliable, specified, and low-cost analysis assays, such as electrochemical biosensors,
17 Nanotechnology and Food Grade Enzymes 477

Analytes Bioceptor Transducer Detector

Measurable signals

Electrical signals

Fig. 17.4 A schematic biosensor

Biosensors

Bioreceptor Transducer

Antibody Mass (Piezoelectric)

Enzyme

Nucleic Acid Optical (Surface Plasma Resonance)

Bacteriophage
(DNA)

Electrochemical, Potentiometric,
Molecular Imprinted Polymer Conductometric, Impedimetric

Fig. 17.5 Biosensor classifications and methods

have their own importance. E. coli and Salmonella are examples which have been
detected by amperometric and electrochemical DNA biosensors. Classifications and
methods of biosensors can be seen in Fig. 17.5.
Transducers, the main part of biosensors, cause physical responses, which may
include heat absorption (calorimetric biosensor); variation of charge distribution
(potentiometric biosensor); motion of electrons made by a redox reaction (ampero-
metric biosensor); or total effects of reactants (piezo-electric biosensor).
478 Z. B. Mohammadi et al.

Three creations of biosensors are included: ordinary one, an electrical reply of

converter resulted by product reaction; the next one set by particular mediator among
convertor and reaction for better reply; in the newest one reaction makes reply
without involving product or mediator, straightly.
The main features of a biosensor are having high particularly for the reason of
analyses, stability in ordinary storage and a lots of experiments; precise and repro-
ducible reactions with no affected by physical variables such as temperature; no
toxic affect; low price, small size, and user-friendly application.
Raghu et al. (2014) immobilized acetylcholinesterase on carbon paste electrode
by silica sol–gel method, which worked as an electrode to monitor Malathion and
Acephate in foods. The detection and quantification of these two pesticides were
done directly, without extraction and pre-concentration as well as less quantity of
enzyme. They demonstrated that this immobilized enzyme can work as a biosensor
with high sensitivity and low cost.
Biosensors are made of three parts: biological recognition segment, transducer,
and a signal processing part. In biosensors, in which enzymes are immobilized on the
surface of transducers, enzyme work as a bridge with analyte. These biosensors have
many usages in different applications, such as monitoring of food safety. Good
sensitivity and specificity and the least preparation of samples are some of their
advantages.
One of the biosensor groups which is the greatest one and are produced commer-
cially is electrochemical enzyme-based ones. The use of nanocomponents in
biosensors would improve their sensitivity, stability, and detection limit.
Electrochemical enzyme-based biosensors have had successfully and commer-
cially application as a bioanalytic system with a good selectivity and stability, much
better than previous ones. These biosensors in nanoscale have more efficiency in
being sensitive, selective, and stable as well as detecting limit and response time,
comparing to macroscale ones, because nanomaterials increase sensitive surface of
the transducer and effectiveness of enzyme immobilization by increasing the ratio of
surface to volume. Nanomaterials have good electrical conductivity, magnetic
properties, and catalytic activity, which could also improve biosensor properties.
These materials have been classified into two groups: organic and inorganic; metals,
quantum dots, and zeolites are the examples of the former group, and carbon
nanotubes, graphene and graphene oxide, and calixarenes for the latter one.
Inorganic nanomaterials accelerate transfer of electron and catalyze electrochem-
ical reactions, while the organic ones amplify electrochemical signals to increase the
level of biocompatibility. These biosensors could have application in food
industries, because of their good biocompatibility, which would make them stronger
with more specific usage.
Biosensors can be used to recognize a variety of biomolecular complexes, such as
antibody, antigen, living cells, and enzyme-substrate interactions. Their nanoscale
can detect molecules like glucose because of being small, transportable, having
linearity of response, as well as more selectivity, sensitivity, and stability.
In fact, a biosensor is composed of three important parts: a bioreceptor molecules
(to classify target analyte), a biotransducer component (to convert a chemical or
17 Nanotechnology and Food Grade Enzymes 479

biochemical signal into an electrical signal), and finally an electronic system includ-
ing amplifier, processor, or recorders. The biosensors could be classified into
electrochemical, potentiometric, calorimetric, amperometric, resonant,
ion-sensitive, and optical. Nanobiosensors are included to optical, bio, chemical,
physical, and sol-gel types. Nanosensors matrix would protect food from effects of
enzyme or dye toxicity. Of course, their usage in food industries depends on the
detection type. For example, chemical or bio detection for optical nanosensors, in
which their color changes by reaction with chemical or biological reagents. In
biological nanosensors, antibody/antigen; DNA; enzyme interactions would happen.
While, chemical composition or molecular concentration may occur in chemical
type, or pressure, force, mass, and displacement in physical ones.
Knowing the mechanism of nanobiosensors is very important, for example, by
spraying a nano bioluminescent on a food, visual glow could indicate microbial
contamination, or released nano particles, such as nano-silver used in active packag-
ing will react with microorganisms. In amperometric nanosensors, organophospho-
rus pesticides could be detected by mechanism of dual enzyme electrode system.
Technology of biosensors effects on many sections, including pharmacology,
medical care, and food industry, in which safety has an important role, as well as
easy, quick, and precise control of nutrients and pollutants.
In fact, enzymatic biosensors are electrochemical ones for food analysis, in which
substrates would be detected either from their transformed products obtaining by
enzymatic catalysis or by the detection of enzyme inhibitors.
To detect xanthine in fish, immobilized xanthine oxidase (MWCNTs) on
multiwalled carbon nanotubes and a copolymer to act as a biosensor were used. It
was shown that MWCNTs could have increased sensitivity more than biosensor
when was applied to immobilize alone. By immobilization of glutamate dehydroge-
nase in MWCNTs, detection limit of monosodium glutamate was improved.
Nanotechnology has an important role in the agri-food industries for very dis-
tinctive properties of nanomaterials. Recently, they are used to detect food
adulterants, pathogens, and toxins. Enzymatic nanobiosensor is made of an enzyme
immobilized on the surface of a transducer.
Enzymes in nanobiosensor adjust the sensitivity and stability of them; therefore,
more substrate specificity makes more enzyme turnover number and thermostability,
the main necessity for a biosensor. Bioconjugation is a biosensing method with cost-
effectiveness, based on the nature of interaction between nanomaterials and the
enzyme surface functional groups. In nanobioconjugation technique, physical
(adsorption, entrapment), covalent (covalent binding, cross-linking), and bioaffinity
types are used to plan a strong nanobiosensor. Weak bioconjugation could have a
little activity of biosensor, following the change of the enzyme active site conforma-
tion. Therefore, by more understanding functionality of a biomolecule at interface,
better choice to plan and improve a bioconjugation would happen.
Nanobiosensors have been used to detect the pathogens in foods, and the optical
assays like fluorescence and surface plasmon resonance are the most usable
techniques. It was demonstrated that by using covalently co-immobilized enzymes
choline oxidase and peroxidase on zinc oxide nanorod films, detection limit would
480 Z. B. Mohammadi et al.

be improved, as well as xanthine, when xanthine oxidase immobilized on

nanocomposite.
Another example of developing nanobiosensor is detection of glucose in fruit
juice by immobilizing pyranose oxidase on gold nanoparticles, which was consid-
ered as a quick and sensitive method. The other technique to detect this analyte is
immobilization of glucose oxidase on nanoclusters. Fructose has been detected in
honey by fructose dehydrogenase which was immobilized on carbon nanotubes. To
detect Escherichia coli, immobilization of b-galactosidase on carbon nanotubes has
an effect on the lowering boundary of detection. It is supposed that enzymatic
nanobiosensors get a perfect and important part of food quality control.

17.4 Risk Assessment of Nanomaterials in Food Industries

17.4.1 Legal Aspects

It is true that universities, companies, and governments have become intensely

interested in the application of nanotechnology and nanomaterials in the food
industry. However deceiving this area might seem, it is of utmost importance to
consider its regulatory issues if this is supposed to be successful. In other words,
population acceptance is highly dependent on regulations that assure nanomaterials’
safety. According to the latest guidance of EFSA (European Food Safety Authority)
any new food which contains nanomaterials is considered a novel system that must
be fully tested and authorized to be consumed by society. EFSA is also responsible
for verifying the latest approaches which are useful for measuring their safety. It
must not be forgotten that for all kinds of nanoengineered foods, it is still vital to
follow all regulations regarding conventional, non-nanotechnological food
components.
Additionally, it has been proven that size reduction process carried out in
nanotechnology can induce specific changes in features and biokinetics of food
matrices which consequently leads to toxicological effects compared with their
conventional counterparts. So, their safety must not be assumed identical to their
conventional non-nanomaterial peers, which means for a specific nanomaterial,
specific considerations and data must be provided individually.
Detailed characterization, impurity recognition, and physicochemical knowledge
also provide pivotal insight into safety assessment. Material composition and purity,
empirical formula of a complete matrix, particle size, particle shape, structure,
surface chemical composition, surface area, surface charge, appearance, melting
point, boiling point, density, porosity, dustiness, pH, and the final form of the
material (if it is powder, dispersion, etc.) are among physicochemical properties
that must be taken into account while testing the nanomaterials’ safety.
It is also probable that changes in production process may add new impurities or
modify physicochemical and morphological properties of the food matrix, and hence
alter its safety.
17 Nanotechnology and Food Grade Enzymes 481

Direct and indirect exposure to nanomaterials is another influencing factor when

analyzing a nanomaterial safety. Feed additives, inhalation, pesticides, dermal expo-
sure, and migration or transfer of nanomaterials from the packaging agent into food
are some areas which must be closely examined to assure a safe component being
accepted in food chain.

17.4.2 Toxicity

As a result of the extremely wide application of nanomaterials in food industry, their

safety has become a complicated matter to deal with as there has always been a gap
in data provided for emerging nanoparticles’ safety. These days, nanomaterials are
being used from the farm up to the moment that the food is consumed by people at
homes. Nanoscale pesticides, fertilizers, functional ingredients namely food
additives, flavor enhancers, or packaging enzymes, and nano sensors are part of
this hugely implemented technology. Therefore, precise investigation of its
properties, potential threats, and daily safe intake dose is mandatory.
Silver, silica, iron, and titanium dioxide are considered as most consumed
nanoparticles in food industry, especially in beverages. For instance, Ag NPs are
used as an antimicrobial agent in food products. However, consumption of Ag NPs
in edible products has caused some concerns as various experiments declared Ag
NPs toxicity for cells as they can change the normal functionality of mitochondria,
produce reactive oxygen particles, and boost membrane permeability.
Silicon dioxide (SiO2) nanoparticles, which are widely used in powdered
products as an anticaking agent to improve their flow properties, have been proven
to be risky when accumulated in liver to a certain extent. Silica nanoparticles which
are used as anticaking agents can be cytotoxic in human lung cells when subjected to
exposure (Athinarayanan et al., 2014). On the contrary, Yun et al. reported no
toxicity of these nanoparticles when consumed orally by rats during 13 weeks.
Therefore, more studies are needed to avoid any ambiguity concerning the
nanoparticles safety.
Titanium dioxide NPs are widely consumed in cosmetics and medicine; in food
products titanium dioxide nanoparticles act as white coloring agent. Higashisaka
et al. (2015) claimed that titanium dioxide shows no toxicity in human and animal
cells. Iron oxide nanoparticles, which are among interdisciplinary substances,
showed to be noncytotoxic when used at concentrations below 100 mg/mL. How-
ever, massive consumption may end in accumulation in a targeted organ, possibly
leading to iron storage, which can be hazardous toxic. Hence, their consumption is
safe provided that they are used in a limited amount below 100 mg/mL
(Buyukhatipoglu & Clyne, 2011).
482 Z. B. Mohammadi et al.

17.5 Future Trends

Enzyme immobilization is a matter of concern in both academia and industrial

sections. As a consequence, combination of nanotechnology and biotechnology
boosts actions which result in immobilized enzymes’ success. In order to increase
the use of nanobiocatalysts, the following factors must be taken into account: (1) the
ability to be separated and recycled easily and reusing high activity enzymes,
(2) minimizing the structural changes of enzymes during stabilization and decreasing
the inactive sites of nanobiocatalysts to reduce costs, (3) increasing the biocompati-
bility and stability of nanobiocatalysts in alimentation and pharmaceutical
applications in various temperatures and pHs, and (4) simultaneous use of multiple
enzymes with high compatibility in sequential reactions. Rapid advancement of
nanotechnology can create opportunities to produce nanobiocatalysts to be used in
food, medicine, pharmaceutical, and biological applications.

17.6 Conclusion

The latest attentions to nanomaterials and their wide applications have made it
possible to immobilize enzymes, maintain their enzymatic activity, reuse, and finally
recycle them. Various nanostructures such as nanofibers, nanoparticles, nanotubes,
nanosheets, and others have been proven to act as efficient media for enzyme
stabilization. If produced under good manufacturing practice and according to health
regulations, these particles are preferred in comparison with their chemical
counterparts. Enzymatic biocatalysts have become the center of attention as sensors
in order to ensure food quality and safety. They are used as indicators of pesticides’
residues, pathogenic microorganism, and their toxic metabolites. Nanotechnology
and stabilization on nanostructures have been widely used in the production process
of these biosensors.

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