Chapter 11

Food Biotechnology

Tahir Zahoor, Muhammad Saeed, Salim-ur-Rehman and Nazia

Khalid*

Abstract
The term food biotechnology is the science to develop living systems that deals
with microorganism’s manipulation in several ways to make raw food materials
into more useful and health caring valued food products. It incorporates various
sciences for enhanced and versatile food productions. Biotechnology can be traced
back from ancient times when processing of food like beverage fermentation, that
is considered as the most important and attractive evolution of 1970s. Food
fermentation is one of the oldest biotechnological processes used for the
preservation of food. The process provides a number of benefits analogous to
preservation, enrichment, increasing digestibility, improving taste and developing
food flavours. It has potential to provide food safety by preventing food from a
large number of pathogens producing various intermediary compounds acting
against bacteria. Principally, fermentation process is categorized into two main
types: solid-state fermentation and submerged fermentation based mainly on the
nature of foods. With respect to foods, another feature of biotechnology is
genetically modified foods. Implication of biotechnology on gene manipulation
also offers genetically modified organisms (GMO) that refers to changes in genetic
material using various genetic engineering techniques. A diversified food can be
achieved with the application of essentially required microbial activity for food
production through GMOs. Fermented food products like dairy, meat, fruit and
vegetables are suitably resourceful for application of GMOs, resulting via lactic
acid fermentation, a main process in valuable dairy products including probiotic

*
Tahir Zahoor˧, Muhammad Saeed, Salim-ur-Rehman and Nazia Khalid
National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan.
˧
Corresponding author’s e-mail: zahoor_t@yahoo.com

Managing editors: Iqrar Ahmad Khan and Muhammad Farooq

Editors: Tahir Zahoor and Masood Sadiq Butt
University of Agriculture, Faisalabad, Pakistan.
282 T. Zahoor, M.Saeed, Salim-ur-Rehman and N. Khalid

foods that are getting additional space in food industry for improving nutrition and
bioavailability through the application of biotechnology.
Keywords: biotechnology, fermentation, food products, value addition, GMOs,
probiotics

11.1. Food Biotechnology

Food biotechnology incorporates several branches including chemistry,
biochemistry, microbiology and chemical engineering etc. for the increased
production of nutritive and biologically available food products. In general, food
biotechnology is the science to develop living systems and organisms to make food
products in useful / modified form. It implies to any technological solicitation that
uses microorganisms (MOs), biological systems, or imitates to make or alter
products for their peculiar utility exhibiting incredible influence on the food
industry and helps to deliver top-grade foods that are more acceptable to the
consumers providing high nutritious, taste and safe food for convenience.

11.1.1. Introduction to Food Biotechnology

Food biotechnology refers to the use of living systems and organisms to extend or
make products, or "any technological application that uses biological systems,
living organisms or derivatives to make or modify products or processes for
specific and intended use.
Broadly speaking, it is the use of living cells, microorganisms, or enzymes for the
manufacture of chemicals, biochemical, drugs, foods or for the treatment of wastes.
From microbiological point of view, it is the processes in any technological
application for bio conversion or modification into a valuable product or even for
biomass production (Shetty et al. 2006).

11.1.2. History
The history of food biotechnology is an attractive one amongst several evolved
since few decades back in 1970s. Its applications have been shown to contribute a
lot of related techniques in biological sciences for the use of mankind. The sectors
of healthcare, agriculture, pharmaceutical, environmental protection, food
production, and chemicals are few of respective sciences. Food biotechnology
started back in ancient times in the field of beverages fermentation when the food
processing was initiated. The conventional techniques are still being used without
losing their traditional essence in every single culture of the world including
processes and production of cheese, ice cream, wine, yoghurt, bread etc. Other
products such as sausage, pickle, vinegar and beer are being manufactured by the
use of microorganisms through biotechnological applications. Bio-yogurts that
include intentional incorporation of bacteria (probiotics, mainly Lactobacillus
acidophilus and Bifidobacterium bifidum) additionally to the fermentative bacteria
for yoghurt manufacture but are not naturally present to provide health benefits
(John Innes Centre 1998).
11. Food Biotechnology 283

In beverage and food industries, vigorous use of enzymes achieved by

immobilization technique; proteases and amylases in brewing industry and rennins
and lactases in dairy industry are the products of biotechnology. High fructose corn
syrup by the use of alpha amylase, glucose isomerase, amyloglucosidase are the
references of important biotechnological applications. In baking industry, yeast
fermentation can improve the digestibility of cereal grain and increase nutrients
availability to the consumers (Gutiérrez-López and Barbosa-Cánovas 2003).

11.1.3. Importance
Food biotechnology finds its solicitations in various major industries including crop
production and agriculture, health care (medical), food and non-food uses of crops,
and other environmental products. It is applied in metabolic engineering of bacteria
for food ingredients that are being used for microbial productions and metabolites
like enzymes, hormones, bioactive components, flavours, carotenoids and in amino
acids production as additives in food uses. It is pragmatic in production of
microbial polysaccharides, genetic engineering of dairy starter cultures, baker’s
yeast and production of citric acid to enhance stress tolerance, development,
improvement and metabolism.
Application of microbial biotechnology for flavor, oils and fats attains a potential
use in the food industry. Biomass production with the purpose of value addition and
academia research cannot be left unattended in the field of food biotechnology.

11.1.4. Role of Food Biotechnology in Food Science

In the last few years, role of biotechnology in food science has significantly grown
up and expectedly with increased trend of its adoption. Value addition at industrial
level for the production of foods with varying benefits for the human health
through, the application of bio-techniques is getting more and more popularity.
Therefore, for several purposes to achieve the targets mentioned in the part of
importance, field of food biotechnology is becoming quite in an organized manner
towards the usage in a domain of marvellous application in food science.

11.2. Food Biotechnology and Fermentation

Early Egyptians were the first to use process of fermentation for production of wine
from grapes, beer production through the aerobic conversion of the alcohol, acetic
acid/ vinegar and leavening of bread. In the innovative application, pectinases and
amylases extracted from microbes are recently being used in production of food.
Pectinases for increasing the juice extraction from tissue of fruit juices and for the
enzymatic modification of starches by amylases are the important examples of
fermentation. The process involves either direct or indirect application of
microorganisms to foods or the components of food. Xanthan gum is produced by
Xanthomonas campestris by the plant pathogenic bacterium used as a thickening
agent, texture modification and increased viscosity in beverages are achieved by the
use of microorganism as an additive. Citric acid is produced from mold Aspergillus
niger since 1920s for application in food and beverage as an acidulant.
284 T. Zahoor, M.Saeed, Salim-ur-Rehman and N. Khalid

Fermentation has become an imperative part of human life with a beneficial process
for enhancement of food’s shelf life. Now-a-days, most of the foods are processed
through fermentation processes that are used for intake with well recognized health
benefits. The typical example of old traditional biotechnological history is of
brewing but the major aims of fermentation remain with the preservation of foods.
Presently, biotechnological process comprises the use of yeast for leavened breads
production, yogurt and cheese production in dairy sector whereas brewing of beer,
wine and sake in beverage industry. Recombinant DNA technology can be used in
biotechnology to modify the baking process with developments in cereal grains,
starter cultures for the desired dairy fermentations, enzymes productions for
processing aids, and application of advanced batch and continuous fermentation
technologies. The ultimate factors to control in fermentation are acceptability,
wholesomeness and overall quality of food product needed to be maintained during
the process. However, food biotechnology is a very lively, sharp and fast growing
area that is absorbing an ever enhancing products and processes through
fermentation techniques. In comparison to any area of biological sciences, the
fermentation technology has a longer history, ties and a bright future for the welfare
of human beings by playing marvelous role in different sciences, medicines and
food industry in particular (Shetty et al. 2006).

11.2.1. Fermentation
Fermentation is a Latin word derived from ‘fervere’ that means to boil. During the
process several products are formed, carbon dioxide is one of those that make
bubbles to appear at boiling. For different stakeholders, fermentations meanings
reflect their purpose of adoption of the process. For Biochemists, fermentation
process is the generation of energy whereas, for food scientists, it is the process of
providing versatility in food conversion to the most acceptable form. For
microbiologist point of view, it is a biomass production. Fermentation of food is
one of the oldest biotechnological processes used for the preservation of food from
food biotechnologist’s point of view. With the passage of time, it has been evolved,
diversified and refined in the recent years. A large number of fermented foods have
been introduced through this technology. Worldwide, fermented foods have
become an important part of human diet. In some countries, because of
development of complementary foods especially in Africa, a process of
fermentation is considered as an important aspect in the nutrition of newly born
babies and young children. It has remained one of the most important processing
methods throughout the human history (Shetty et al. 2006).
A number of benefits have been linked with fermentation process in providing food
preservation and increasing keeping quality, digestibility, food enrichment and
improvement in taste and flavor that varies with consumer’s demand and even
nations. Therefore, it contributes towards human nutrition which is a dire need in
developing countries with over population and limited resources as a major
hindrance to ensure food safety. Generally, fermented food products, particularly
those developed under controlled conditions have been found as a good safety
measure. Important role of fermentation cannot be denied in human lives as many
11. Food Biotechnology 285

of the foods consumed in daily life are fermented as exemplified above (Shetty
et al. 2006).
Advances in biotechnology have been approached with the manipulation of
microorganisms through recombinant DNA technique as experienced by several
scientists (Linko et al. 1997; Linko et al. 1998). Bio-preservation of food is one of
the major objectives of fermentation with the advantages of improved
wholesomeness, nutritious and consumer acceptability (Holzapfel 2002).
11.2.1.1. Types of Fermentation
Fermentation process is principally categorized into two main types: submerged
fermentation and solid-state fermentation. The former has been mostly utilized in
various industrial procedures for the manufacturing of organic acids, alcohol,
enzymes, antibiotics, amino acids and vitamins. It has been utilized in the
development of microbial metabolites by using fungi, bacteria and yeasts.
i. Submerged Fermentation
Submerged fermentation is commonly utilized with a wide range of
microorganisms for the production of a large number of products. Relatively highly
processed ingredients are utilized for the type of process. If asepsis is not
maintained, medium’s higher water activity makes it appropriate for spoilage. High
substrate concentrations may impart rheological problems unless taken as
optimized parameters of temperature, time, and rest of the microbial requirements.
In case of submerged fermentation limitations in diffusion of nutrients is not
encountered. Well-developed bioprocess control of fermentation is possible by
using online sensors (OoijKaas et al. 2000).
ii. Solid-State Fermentation (SSF)
Solid-State fermentation (SSF) is used under the conditions of low moisture content
for bio-productions using microorganisms. Solid substrates (bran, wheat, grain,
rice) are mostly used for this type of fermentation. During solid-state fermentation,
it is important to consider the properties of water absorption of solid substrate to
maintain water activity that is basically a vital for growth (Gervais and Molin
2003). In comparison to submerged fermentation, power requirements are lower in
solid-state fermentation (SSF). Generally, SSF is applied in low value products
with less control and monitoring due to restrictions of nutrients diffusion, metabolic
heat accumulation, improper mixing and unproductive process control. For the
development of highly desired and important products SSF on inert substrate
supports impregnated media (OoijKaas et al. 2000).

11.3. Genetically Modified Organisms (GMOs)

The universal rule to develop a genetically modified organism is to interpose the
DNA of an organism's genome. In 1972 Paul Berg produced first recombinant
DNA. In 1973 Herbert Boyer and Stanley Cohen, introduced the concept of direct
transfer of DNA from one organism to another through the process of genetic
engineering. With the passage of time, developments permitted scientists to control
286 T. Zahoor, M.Saeed, Salim-ur-Rehman and N. Khalid

and supply genes to a number of dissimilar organisms to attain broader varieties

with unique effects (Lee and Gelvin 2008).
Previously in 1976, the technology has been used in many foods and medicine
companies producing and selling genetically modified products consisting of
adding genetic material, mutating and deleting practices. Recombinant DNA of an
organism and addition of genetic material from different species results in
microorganism is called a transgenic organism. The transgenic organism obtained
after using different genetic engineering techniques is referred as genetically
modified organisms (GMO) (Park 2007).
GMO consists of yeast, bacteria, fish, plants, mammals and insects and are
considered as the basis of genetically customized foods broadly used in production
of foods, medicine, pharmaceutical drugs and in scientific and biological research
(Lai et al. 2002). A fluorescent protein called GFP is obtained from jellyfish can be
physically related as well as articulated with mammalian genes to recognize the
position of the protein encoded by the GFP-tagged gene in the mammalian cell.
Biologists use such method in different research procedures, fundamental
biological processes, prokaryotic cells or eukaryotic cells as well as in the
mechanisms of human diseases (Baur et al. 2005).

11.3.1. Importance of GMOs

Food production is very imperative in international and national scale. Changes in
the quality and quantity of food cause certain variations in the market regarding
supplying and demanding. Industries and institutions requisite to link up in food
chain system encountering the nutritional needs for technology, quality and
production for safe and healthy food for mankind. Modern agriculture is illustrated
by greater productivity of genetically modified organisms (GMOs) and products
round the globe. In the most overpopulated states in the world, biotechnology and
economy, is based on GMO agriculture, a field of interest of all domains of
sciences (Knezevic et al. 2012).
Recombinant DNA technology for the production of increased cow’s lactation by
injecting recombinant bovine somatotropin, Flavr Savrtomato with extended
freshness, cheese obtained with synthesized enzyme chymotrypsin, golden rice and
insect-resistant BT corn which contains more carotene and iron, etc. are the good
examples of genetic modifications (Knezevic et al. 2012). The technology has
played a role in the manufacturing of specific protein in bacteria making it
conceivable to execute the gene transfer from unrelated, evolutionary distant
species into another. This occurs without causing conversion of one organism to
another but only desirable trait of gene of interest is expressed. Various agricultural
plant species including soybeans, cotton, corn, sugar beets, pumpkin, banana,
potato are the good examples of genetic modification for the desired traits (Drinic-
Mladenovic et al. 2006).
11. Food Biotechnology 287

11.3.2. Bio-fortification
A process of upgrading nutritional quality of food by using biological resources is
referred as bio-fortification. It is different from traditional fortification as it targets
to enhance nutrients level during plant growth in crops instead of adding them
during processing. Bio-fortification can improve the nutritional status of the staple
foods which are already consumed by poor people, bestowing a relatively
economical, profitable, viable, enduring resource of providing higher
micronutrients. The approach does not only lessen the sum of sternly malnourished
people requiring treatment by harmonizing interferences, but will also help in
maintaining enhanced nutritional standing. Furthermore, bio-fortification delivers a
reasonable means of reaching malnourished pastoral in habitants who have
restricted admittance to commercially marketed supplements and fortified foods
(Bouis et al. 2011).

11.4. Product Development and Food Biotechnology

Over the years, behavior towards biotechnological foods has progressively become
more favorable as people realize the environmental, economic, and nutritional
benefits they can impart. It has been recognized that the safety of food products
with respect to Human health and the environment has already been visualized due
to application of biotechnology. Regardless of intermittent resistance from certain
environmental groups, the mounting food and bio-fuel demands world-wide are
quickening the broader acquiescence of biotech foods in the market. An increasing
trend of bio-products processed through biotechnology has been approved for sale,
any stigmas related to biotechnology continue to lessen, as awareness increases and
consumers reap the rewards of these enhanced crops and foods.
A huge diversity of food needs microbial activity as an essential feature of their
production. It is simply an empirical observation that certain ways of food storage
affect desirable changes in its original characteristics (Fox et al. 2004). A food
prepared with microbial activities through a fermentation process includes dairy
products (yoghurt, cheese) vegetables (pickle, kimchi, sauerkraut, olives) meat, fish
and poultry etc. Several fermented food products with application of biotechnology
are detailed below:

11.4.1. Dairy Products

11.4.1.1. Yoghurt
Food biotechnology plays a key role in the production of fermented milk products
mainly yoghurt, cheese (commercial) and butter, dahi and lassi etc. (traditional)
while using lactic acid bacteria for their specific functionalities of texture and
flavor. Yogurt can be prepared from all types of milk including skim milk or
fortified milk however, in developing countries the milk sources of milk are cow,
buffalo, sheep and goat. For the preparation of yoghurt, before addition of starter
cultures, milk is preheated at 80-90°C for 30 min to prevent from all heat sensible
microbes over and above to heat resistant spores that can compete with starter
288 T. Zahoor, M.Saeed, Salim-ur-Rehman and N. Khalid

culture cells and enhance ability of milk to serve as growth media for the started
cells by inactivating immunoglobulins that causes hurdles in proper functioning of
culture. The process of inactivation is completed by removal of oxygen to maintain
microaerophilic conditions in milk based product via the release of sulfhydryl
group at the same moment. Another benefit of heating is to make interactions
between protein level especially casein of serum and whey that promotes viscosity
of yoghurt by stabilizing gel herby reducing syneresis (separation of whey)
(Aziznia et al. 2008). After heat treatment, milk is cooled to 40-43°C known as
fermentation temperature and considered as optima of two specific starters i.e.,
Streptococcus thermophilus and Lactobacillus bulgaricus grow best at 39 and 45°C
respectively, for curdle formation. Normally, 2% starter culture on the basis of
volume is added to provide an initial level of 109-10 CFU/mL.
Standardized milk (3.5% Fat, 8.5% SNF)

Homogenization (55-65°C)

Cooling to inoculation temperature (40-43°C)

Addition of yoghurt culture (2-3%)

Packing (250 mL cup)

Incubation (40°C, 4.5 hrs)

Cooling storage (at 4-6°C)

Fig. 11.1 Flow Diagram of Yoghurt Manufacturing

Fermentation can be performed in container (stirred yoghurt) to produce for

commercial level in bulk or in retail pack to impart firm continuous coagulum (set
yoghurt) for individual commercial packs. Fermentation takes 4-5 hours during
which bacteria is responsible to produce lactic acid by converting lactose (milk
sugar) by lowering the pH at 6.3-6.5 (Fox et al. 2004). The isoelectric point of
casein at pH 4.6-4.7 causes the protein to aggregate and produce a continuous gel
with ability to entrap the components having very little or no whey off potential
(Fig. 11.1). At the last stage of fermentation, total acidity becomes in the range of
0.9-0.95 percent. However, the population of both starter cultures is in balance at
the level of 1x108-9 CFU/mL cell count (Fox et al. 2004). In dairy products, di-
acetyl is the main flavoring component at lower level of 0.5 mg/Kg and thought to
contribute specific yoghurt flavor. Whereas, production of ethanol as acetaldehyde
is important to impart yogurt flavor (23-41 mg/Kg) in pH range of 4.2-4.4 which is
essential for yoghurt manufacture. Both Streptococcus thermophilus and
11. Food Biotechnology 289

Lactobacillus bulgaricus have ability to produce acetaldehyde on the basis of

glucose portion of lactose through pyruvate via the process of threonine aldolase.
On the completion of fermentation, yoghurt is subjected to cool down (15-20°C)
before addition of other ingredients (fruits, flavors, nuts etc.) The product is further
cooled down to 5°C to keep for about three weeks that result in gradual increase in
acidity during storage period (Fox et al. 2004).
11.4.1.2. Cheese
Cheese is a consolidated curd of milk solids in which milk fat is entrapped by
coagulated casein because of coagulation to a greater limit as a result of proteolysis.
Large amount of water content is removed in the form of whey. Typically, the yield
of cheese from milk is classified as 10 percent of the original milk taken. A variety
and larger quantities of cheese with little modification using different types of milk
is also produced for taste and acceptability. Mainly, cheese is classified on the basis
of its moisture content, fat content and further subdivision can be made on the basis
of milk type and a variety of microorganisms used for fermentation and ripening
(Kindstedt 2012).
Cheddar cheese, for its smooth texture and good keeping quality, is famous and
produced throughout the world. The pasteurization in cheddar is usually applied at
the initial processing stage making surety about its safety and fermentation
reliability. Whereas, cheeses made from raw unpasteurized milk is claimed to
acquire an enhanced flavor. In pasteurized one, milk is cool down to about 30°C.
Mesophilic starters (Lactococcus lactis or its subspecies) and thermophilic starters
(casei, Lb. Lactobacillus helveticus, Lb. lactis, Lb. bulgaricus, Strep. thermophilus
and delbrueckii subsp) are applied as a starter to initiate fermentation process. The
organism plays crucial and a complex function in developing flavor and texture of
cheese that are considered as the main sensory parameters (Ravisankar et al. 2014).
However, mainly lactose sugar is converted into lactic acid resulting in lower pH
and higher acidity to add shelf-life and safety with quick and fresh flavor to the
curd. The stability of casein (colloidal suspension) is also weakened resultantly
releasing calcium from the micelles that improves the action of chymosin. The acid
thus produced, aids in moisture removal for curd formation with resultant texture of
the cheese. Citrate fermentation to diacetyl in several cheese varieties require
starter bacteria of Lactococcus lactis subsp. lactis or Leuconostoc cremoris that
produces with characteristic structure due to the production of carbon dioxide like
Dutch and mold treated blue-veined cheeses (Flasarova et al. 2015).

11.4.2. Vegetables
11.4.2.1. Sauerkraut and Kimchi
The usage of microorganisms as a part of biotechnology cannot be overlooked in
fruits and vegetables. To preserve most of the fruits and vegetables, particularly
cabbage, cucumbers, carrots, and olives, lactic acid fermentation is required.
Although small amounts of carrots, celery, cauliflower, okra, onions, hot peppers
and green tomatoes are also fermented for their intended use through said
technology. Kimchi is composed of fermented vegetables, which is abundantly
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eaten with meals in Korea. The main substrates are cabbage and radish along with
garlic, peppers, onions and ginger. Surveys have shown its importance in the
Korean diet. It has been reported to comprise 12.5% of the total daily food intake or
a daily adult consumption of 50-100g in summer increasing to 150-200g in winter
(Jung et al. 2012). In Pakistan, traditional pickles are made using various spices in
vegetables after a fermentation process as taste of the consumers but comparatively
lesser spice than Kimchi.
It is thought that Tartars have brought sauerkraut production to Europe from China
with a simple process like a number of other traditional and commercial
fermentation technologies. After removal of outer leaves, the cabbage is shredded,
salted and then packed into containers for the fermentation phase with the requisite
amount of salt about 2-3percent weight by weight (Kim et al. 2015). During
fermentation period, micro-organisms produces adequate amount of acid (pH value
below 4), thus inhibiting the competing microbes. The fermentation is started by
Leuconostoc mesenteroides which grows faster during early stages. As the pH falls
owing to acid making in a faintly buffered intermediate, the Leuconostoc is
introverted and interchanged by heterofermentative lactobacilli. At the end of
fermentation, the total acidity of the product reaches 1.7-2.3%, articulated as lactic
acid, with a ratio of volatile to non-volatile acid of around 1 to 4. Defects of
sauerkraut arises typically as outcome of yeast and mold growth yielding off-odor,
loss of acidity, a creepy, softened product as a result of pectolytic action, or a pink
discoloration due to the progression of yeast Rhodotorula (Jung et al. 2012).
11.4.2.2. Olives
Since 3000 BC, Olives have been cultivated in eastern Mediterranean region, being
native to this area. At present, 98 percent of the world’s cultivation of olives is this
region; most of which is utilized for the production of olive oil. Substantial amount
(4600 000 tons per annum) is processed into table olives and a little is preserved in
cans similar to those of other foods in brine and the product becomes stable due to
fermentation. Pickled olives have a complex taste which frequently needs to
acquire considerable application (Grattan et al. 2006).
The unripe fruits are first treated with lye (1.0-2.6% NaOH solution) in the
production of Spanish-style green olives to hydrolyze the glucoside oleuropein that
imparts a bitter flavor and also restrains lactic acid bacteria. The process continues
for up to 10h during which the lye enters the flesh between a half and three quarters
of the way to the stone. The lye is then rinsed off with water for several hours and
fruits are placed into the brine (5-6% salt) initially and then increased in strength
(8%) during the fermentation process.
Lactobacillus plantarum have been reported amongst the complex sequences of
bacteria for fermentation. Numerous other LAB have been reported that includes an
early growth phase of Leuconostoc mesenteroides particularly depending upon salt
concentration. The increasing acidity, decreasing pH and the salts in combination
replaces the natural microflora, dominated by Gram-negatives with some lactic acid
bacteria and yeast. The process of fermentation continues for several weeks and
terminates in a product (1% lactic acid, pH of 3.6-4.2) as described by Blana et al.
(2014).
11. Food Biotechnology 291

11.4.2.3. Cucumbers
Previously, cucumbers were preserved by lactic acid fermentation and then pickling
in the brine whereas, fresh pack techniques have been evolved since 1940s that do
not require fermentation to attain stability. The first technique is based on
acidification directly by using acetic acid followed by pasteurization. Recently,
pasteurization has been replaced by refrigeration. Cucumber fermentations can be
distributed essentially into two diverse types: high-salt and low-salt fermentations.
High salt cucumbers are fermented in brine with 5-8% salt till the product becomes
stable by transformation of all the fermentable sugars to organic acids and other
products. Later on, it has been observed that Leuconostoc mesenteroides cannot
endure elevated salt levels promoting the growth of LAB and some yeast while
inhibiting other organisms under such conditions imparting their role in bio-
fermentation (Franco et al. 2012).

11.4.3. Fermented Meat, Poultry and Fish

Fermented sausages were originated in Mediterranean region with a higher
demand, although developed independently in different regions of China and
Southeast Asia. Fermentation in sausages of meat is carried out for promoting and
keeping meat stability. Mainly lactic acid and salt is used in its fermentation
processes to produce a stable product. Fermented sausages are categorized as dry
sausages ( > 35% moisture content ) and semi-dry sausages ( upto 50% moisture) as
stated by Bacus (1986). The spices are used as an antimicrobial agent to retard
microbial growth in meat but to stimulate the growth of lactic acid bacteria.
Traditionally, casing of gastrointestinal tract of animal’s collagen are produced
from animal source.
Natural fermentation is also carried out in fermented sausages to determine the
dominancy of heterogeneous microflora based on selectivity of starting components
of the heterogeneous microflora. The nitrate-reducing and lactic acid bacteria are
the principal components of commercial starters in the fermentation process. Some
other starters include yeast (P. nalgiovense, Debaryomyces hansenii, Candida
famata) and mold (mold Penicillium spp.). Whereas, LAB mostly important in the
natural fermentation are Lactobacillus plantarum, Pediococcus acidilactici and P.
pentosaceus (Bacus 2005).
The type of fermentation being used for sausages can be conducted on range of
temperature between 15 to 40°C for 20-60h depending upon the type and
concentration of starter being used however, the relative humidity during this
process is an essential step to control to ensure slow drying of the product.
Production of acid causes decrease in pH lower than 5.2 promoting coagulation of
meat protein that further helps in removal of moisture during specific flavor and
texture development of food stuff. The step on the other side imparts better
microbial stability and product safety. In the final stage of process, that consists of
about 6 weeks, moisture is reduced up to a constant level of low relative humidity
(65-85%) and temperature (7-15°C) as stated by the workers (Tabanelli et al. 2012).
Thai fermented sausage (nam) differs from other fermented foods in the form of
European fermented sausages. In ‘nam,’ as the process of fermentation proceeds,
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pH gradually decreased owing to water removal that trapped in active packaging

material which is the indication of food product aging (Rungrassamee et al. 2012).
Fish sauce and pastes are normally prepared from numerous varieties of small fish
packed in jars or tanks with brine or dry salt in a ratio as three parts of fish must be
preserved by one part of salt for improved flavor. The method is considered as
accurate to perform on saturation with water activity <0.75 that diminish the
growth of spoilage causing microbes. Halophiles (anaerobic salt loving) are known
for having resistance against high salt conditions (Anihouvi et al. 2012)

11.4.4. Cocoa and Coffee

Cocoa and coffee are two important beverages that rely on a microbial curing
process or fermentation for flavor development. Worldwide popularity of these
products is due to their distinctive flavors and aroma. Fermentation of cocoa
imparts essential for flavor development whereas, with coffee the curing process is
less crucial to flavor and more important for the removal of pulp. Fermented cocao
varies considerably from country to country and in many instances even adjacent
farms due to different curing methods. A substantial amount of research has been
dedicated to fermentation practices. Cocoa beans are fermented in any convenient
container such as fruit boxes, baskets, plastic buckets and fertilizer bags, or when
these are not readily available; the beans are simply piled on a sheet and covered
with any handy material. Although, mainly homo-fermentation process is carried
out but hetero-fermentative lactic acid bacteria also occur in cocoa fermentations
(Minifie 2012).
Fermentation of coffee causes the breakdown of the pulp layer surrounding the
bean in order to aid in processing of fruit to get a desirable finished product in
addition to removal of mucilage with enzymes, chemical treatment (sodium
hydroxide), and mechanical force. Natural fermentation (different microorganisms)
is the primary method used to remove the mucilage. Natural fermentation involves
mold, yeast, several species of lactic acid bacteria, coliform and other gram
negative bacteria. Since pectic substances are a large portion of the mucilage, the
microorganisms responsible for colonization and utilization of this material must be
capable of producing pectinases. Coffee fermentation studies have demonstrated
that the highest microbial activity occurs during the first 12 to 24 h after the beans
are harvested (Lopez 1986).
Fermentation begin immediately after the beans are removed from the pods, as they
are contaminated through microorganisms from the pod surface, knives, labors
hands and the containers used for transportation. The pulp surrounding the cocoa
undergoes microbial fermentation. Chemical changes take place within the bean as
a result of the fermentation of the pulp. The testa of the bean acts as a natural
barrier between microbial fermentation activities outside and chemical reactions
within the bean. Migration of ethanol, acetic acid, lactic acid and water of microbial
origin occurs from outside to inside of the bean. Soluble bean components leach
through the skin and are lost in the draining. The pulp is consisting of about 85%
water, 2.7% pentosans, 0.7% sucrose, 10% glucose and fructose, 0.6% protein,
0.7% acids, and 0.8% inorganic salts, makes it a rich substrate for microbial
11. Food Biotechnology 293

growth. The concentration of sucrose, glucose and fructose in the beans are
influenced by the age of the pod (Minifie 2012).
During fermentation, yeast, lactic acid bacteria, and acetic acid bacteria develop in
succession. At the onset of fermentation, a pH of 3.4 to 4.0, a sugar content of 10 to
12% and a low oxygen tension favor the growth of yeast. Yeast utilizes the
carbohydrates in the pulp under aerobic and anaerobic conditions and may account
for 40 to 65% of the micro-flora when the fermentation begins. The yeast phase
lasts 24 to 48h, while population increases to 90% of the total microflora. Some
yeast produces various pectinolytic enzymes that degrade the cocoa pulp, thereby
aiding in the drainage of juices. Furthermore, metabolizing sugar produces ethanol,
yeasts utilize citric acid, causing the pH to increase. Between 48 and 72h of
fermentation, the yeast population begins to decrease so that, by the third day, it
reduces rapidly to 10% of the total microbial population firstly due to rapid
metabolism of sucrose, glucose, and fructose in the pulp to form carbon dioxide
and ethanol, causing a reduction in energy source. Secondly, the production of
ethanol produces toxic environment suppressing yeast growth (Pereira 2012)

11.5. Probiotics and Prebiotics

11.5.1. Therapeutic Role in Food
Probiotic (which means "for life") bacteria have long been considered to influence
general health and well-being via their commensal association with the
gastrointestinal tract (GIT) and its normal microbiota. Probiotics are live microbial
cell that, when applied or ingested in certain numbers, exert a beneficial effect on
health and well-being. Probiotic microorganisms designed for delivery in food or
dairy products, via supplementation or fermentation, are usually members of the
Lactobacillus or Bifidobacterium genus (Bhadoria and Mahapatra 2011). The
concept of probiotics was first popularized at the turn of the century by the Russian
Nobel laureate, Elie Metchnikoff. He proposed that a normal, healthy,
gastrointestinal microbiota in humans and animals provide resistance to
"putrefactive" intestinal pathogens. It was reported that infants subsisting on breast
milk develop a characteristic intestinal flora of rod- and bifid-shaped bacteria, and
then called Bacillus bifidus now known to represent Lactobacillus and primarily,
Bifidobacterium species (Saini and Saini 2009).
Non-pathogenic microorganisms (yeast, enterococci, Enterobacteriaceae) that
occupy important niches in GIT or tissues are also used as human and animal
probiotics. Important role of the intestinal microbiota in health and resistance to
disease is well recognized. Clinical investigators using well characterized cultures
has gathered evidences supporting the primary claim that probiotics exert a
beneficial influence on the intestinal environment and offer protection against
gastrointestinal infections and inflammatory bowel disease (Sharma and Devi
2014).
Probiotic cultures appear to affect the microbial composition and the associated
metabolic and enzymatic activities of resident harmful or developing pathogenic
294 T. Zahoor, M.Saeed, Salim-ur-Rehman and N. Khalid

bacteria. The probiotic cultures, via their intimate association with the intestinal
mucosa, their cellular components, and their effects on the associated microbiota,
appear to improve immunological function in the GIT and the integrity of the
mucosal barrier. Improvement of the immune system appears to be focal
mechanisms that impact inflammatory responses, anti-carcinogenic activity and
resistance to colonization (Holmes et al. 2012).
The use of prebiotics to stimulate the growth and activity of beneficial bacteria in
an individual's intestinal microbiota is a logical and effective approach to extend
probiotic benefits. Food ingredients classified as prebiotics generally exhibit the
characteristics of limited hydrolysis and absorption in the upper GIT, selective
growth stimulation of beneficial bacteria in the colon, potential to repress
pathogens and limit virulence via a number of processes of attenuation virulence,
immune-stimulation, and stimulation of a beneficial flora promoting colonization
resistance. The best-known prebiotics are fructo-oligosaccharides derived from
food sources. The largest natural source is inulin recovered from the chicory root
by water extraction. Inulin can also be found in edible plants such as onions,
asparagus, bananas, wheat, and Jerusalem artichokes. Most prebiotic compounds
are bifidogenic in nature. Studies with a variety of bifidogenic factors and
prebiotics are providing evidence of health-related effects occurring via the
prebiotic and its stimulated microbiota in the areas of colonization resistance,
reduction of colon cancer markers in animals, reduction of serum triglyceride
levels, and enhanced adsorption of minerals such as calcium, magnesium, iron, and
zinc (Tannock 1999).
11.6. Conclusions
Food biotechnology (one of the oldest biotechnological process) is the branch of
science that has been started since decades back and is effectively used for the
preservation of food. Food biotechnology: is a science to develop living systems
and organisms to make useful products as exemplary GMO foods, the best
indicator and still needs its adaption worldwide. The technology integrates different
branches of science to provide various benefits to mankind in several ways. With
the application of biotechnology in food sector, the industrialists touch production
of innovative food items, novel food, value addition and behavioral functional food.
Food products (Dairy: Yoghurt, Cheese, Fruit and Vegetable: Sauerkraut, Kimchi,
Olives, Cucumbers, Meat, Poultry and Fish, Cocoa and Coffee) are the key
examples being served now a days. The utilization of Probiotics and Prebiotics, role
of food in therapeutic behavior and functional foods are important contour of
biotechnology in food products. Future prospects in the field of Food
Biotechnology still need to work more for developments in food productions for
preserving and securing food for the mankind to meet needs of increasing
population.
11. Food Biotechnology 295

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