LIFE S C IENC ES GRO UP

ISSN: 2455-815X DOI: https://dx.doi.org/10.17352/ijasft

Received: 13 December, 2021

Research Article Accepted: 04 January, 2022
Published: 05 January, 2022

Optimization of oxidative *Corresponding author: Mahsa Shafiesoltani, Depart-

ment of Food Science and Technology, Islamic Azad

improver’s formulation for University, Shahr-e-Qods Branch, Thehran, No 211,

Goodarzi Alley, Neda joonoobi Street, Azimieh, Karaj,

the wheat flours with different Iran, Tel: 00989125248721; Fax: 00982636679059;
E-mail:

extraction rates ORCID: https://orcid.org/0000-0003-2860-065X

Keywords: Glucose oxidase; Ascorbic acid; Wheat

flour; Bread; Extensograph; Response surface method
Mahsa Shafiesoltani*, Mania Salehifar and Saeed Baeghbali
Copyright License: © 2022 Shafiesoltani M, et al.
Department of Food Science and Technology, Islamic Azad University, Shahr-e-Qods This is an open-access article distributed under the
terms of the Creative Commons Attribution License,
Branch, Thehran, Iran which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

https://www.peertechzpublications.com

Abstract
In this study, the effect of oxidative improvers such as glucose oxidase (10-30 mg/kg) and ascorbic acid (50–150 mg/kg) were compared on the rheological properties
of two sets of flours with different extraction rates (75% and 82%). The optimized formulation via the response surface method revealed that the oxidative improvers have
a different reaction in different types of flours. In flours with a 75% extraction rate, glucose oxidase played the main role, while in the flours with an 82% extraction rate,
ascorbic acid was more effective. Also, this study showed that the effects of both improvers are dose-dependent, for the 75% extraction rate of flour, the optimal dose of
glucose oxidase is 23 mg/kg, and for the 82% extraction rate of flour, the optimal dosage is 90 mg/kg of ascorbic acid. Finally, the effect of the optimal formulation was
investigated on the bread properties and the results were compared with the control sample.

Introduction on flour with different extraction rates is also an important

issue that has not been studied in previous investigations.
Wheat bread is one of the main sources of human energy
in many countries [1]. The most important factor determining In this research, the effects of glucose oxidase and ascorbic
the quality of wheat flour is the gluten network. Gluten retains acid on the gluten network of flour with different extraction
co2 produced by the yeasts in its network and creates a soft rates were studied. Additionally, can a fixed formulation be
plus tender texture in the bread [2]. The quality of wheat used to strengthen the gluten network in different types of
depends on the type of soil and geographical conditions [3]. flours?
To improve the quality of the gluten network, an oxidative
Materials and methods
improver is used to enhance dough stability [4]. In general,
it has been concluded in all papers that oxidative improvers Two sets of wheat flours (Fully automatic milling-Buhler
such as glucose oxidase and ascorbic acid can strengthen the Co, Germany) with different extraction rates (75% and 82%)
gluten network. Despite this, choosing the right ingredients were used. The average value of the moisture, protein (N×5.7),
along with their optimal dose (the formulation) of oxidative ash, gluten content of the flour with a 75% extraction rate were
improvers are important factors affecting the quality of flour 14.12gr, 11.37gr, 0.590gr, 26.7gr /100gr flour, respectively, the
and ultimately in bread. In previous studies, the details of the falling number was 350 s. The average value of the moisture,
formulation of the oxidative improvers have not been studied. protein (N×5.7), ash, gluten content of the flour with 82%
On the other hand, comparing the effect of oxidative improvers extraction rate were 13.95gr, 11.81gr, 0.830gr, 27.5gr/100gr

001

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

flour, respectively and the falling number was 300 s. L(+)- the ICC method of 114/1, weighting of the dough pieces,
Ascorbic acid (E 300) was purchased from Henan Tech way homogenization and shaping of the dough into a cylindrical
Co, Zhengzhou, China. Glucose oxidase enzyme (E.C.1.1.304) piece, fixing of the sample in the proving cabinet, stretching
derived from Aspergillus oryzae with the activity of 10000 u of the sample until it tears. The force exerted is recorded as
/ g was obtained from DSM Co, Heerlen, Netherlands. The a function of time in the extensogram. Subsequently, the
additives ranges were (50-150 mg/g) for ascorbic acid and (10- extensogram is evaluated by the software program. The
30 mg/g) for glucose oxidase according to their manufacture’s reason for choosing the extensograph device, compared to
recommendations. Used levels of the additives are given in other rheological instruments (Gluten Index, Farinograph,
Mixolab instruments) for measuring the rheological properties
Table 1.
of the dough is because of the improved ability of this device
In this study, six samples of wheat flour were divided into two to measure the quality of flour after adding the improvers
groups (each group contained three flour samples) according because there is a dough resting step in extensograph which
to the milling extraction rates (American classification). The give enough time to improvers for having the better activity.
milling extraction rate is determined by the ash content. The After measuring the rheological properties, the optimal
ash content of the first set was between 0.5- 0.6% (extraction oxidative improver for each flour set was determined by the
rate of 75 %), the ash content of the second set was between Response Surface Methodology [5]. The bread baking process
was done after adding the optimal improver and the results
0.75-0.9% (extraction rate of 82%).
were compared with the control bread sample. In this study,
After preparing two sets of flour, the analytical flour quality all experiments were performed in triplicate and the average
was determined (Table 1). Then the effects of ascorbic acid results of each group were reported.
and glucose oxidase on the dough rheological properties were
Characterization of wheat flours
evaluated by Brabender Extensograph instrument (Brabender
GmbH Co, Duisburg, Germany). The measuring procedure with The moisture, protein, ash, and gluten content were
the Extensograph has been done according to the international determined using AACC methods (Approved Methods of the
standard( AACC 54-10). Basically the procedure is as follows; American Association of Cereal Chemists International, 11th
preparation of the dough in the Brabender farinograph edition, 2000), 44-10.01, 46-13.01, 08-01.01, and 38-12-02
(Brabender GmbH Co, Duisburg, Germany) according to (AACC, 2010), respectively.

Table 1: Central composite design for the additives and Extensograph parameters (responses) in relation to the addition of the additives (glucose oxidase and ascorbic acid).
Flour Type Additives Levels Responses (Depended variables)
Run No Go(mg/kg) AsA(mg/kg) R(BU) Rmax(BU) E(mm) Energy(cm2)
1 20 30 628 650 107 96
2 20 100 523 567 110 86
3 20 100 514 553 116 90
4 10 150 505 544 112 84
5 10 50 540 570 116 90
Flour
6 20 100 518 559 114 88
With
7 20 100 511 577 110 91
75%
8 20 100 513 560 115 89
Extraction
9 35 100 517 559 117 91
Rate
10 30 150 534 564 105 82
11 30 50 594 621 102 87
12 5 100 458 507 121 84
13 20 170 537 589 110 89
14 0 0 340 362 122 64
1 10 50 337 367 118 66
2 30 50 453 480 110 76
3 30 150 408 449 115 74
4 10 150 412 436 106 65
5 20 100 451 469 110 73
Flour
6 20 100 458 479 114 77
With
7 20 50 372 387 117 68
82%
8 20 100 453 475 112 76
Extraction
9 20 100 459 480 113 77
Rate
10 20 100 460 487 112 78
11 5 100 480 520 111 74
12 35 100 540 570 111 77
13 20 170 360 370 107 66
14 0 0 245 249 134 51
-Bu: Brabender unit; Go: Glucose oxidase; AsA: Ascorbic acid; R: Resistance to extension; Rmax: Maximum resistance to extension; E: Elasticity.
-Run number of 14 is control sample.
002

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

Alpha-amylase enzyme activity was analyzed according Sensory evaluation: Sensory evaluation tests were done
to AACC method 56-81-03 by Falling Number (FN) device by ten trained judges including males (5) and females (5)
(Infracont Co, Pomez, Hungary). in the age group of 20-50. External characteristics such as
(volume, crust, color, shred and symmetry of form and crust
Determination of the rheological properties characteristics) and internal characteristics such as (grain,
crumb color, aroma, and taste, chewability, and crumb texture)
A Brabender extensograph was used for determining the
were scored for each loaf according to the bread score method
rheological properties of flours. The following parameters
developed by the American Institute of Baking and reported by
were determined in the extensograph analysis: resistance to
extension (R-value), maximum resistance to extension (Rmax- Matz (1991). These scores were converted into a global concept

value), energy, and dough extensibility (E-value). determined as: very good (>90), good (80–90), regular (70–
80), and detestable (<70) (Shafiesoltani, et al. 2013).
Bread baking
Statistical analysis
The bread baking was performed according to the straight
dough method according to AACC method 10-09.01 (Approved The effects of oxidative improvers on the dough rheological
Methods of the American Association of Cereal Chemists properties were determined by Response Surface Methodology
International, 11th edition, 2000), the protocol used was: (RSM) using CCD (Khuri and Mukhopadhyay 2010). Response
flour 100%, compressed yeast 2%, salt 2%, sugar 4%, fat Surface Methodology can be regarded as a collection of statistical
3%, and water as much as necessary for reaching the optimal and mathematical techniques. The independent variables were
consistency of 500 BU. The dough was prepared from 300g ascorbic acid (50- 150 mg/kg) and glucose oxidase (10-30 mg/
of flour in a Farinograph (Brabender GmbH Co, Duisburg, kg). The dependent variables were: resistance to extension
Germany). Dough dividing and roll shaping were made by hand (R-value), maximum resistance to extension (Rmax-
and after the standard proofing time, the pieces of dough were value), energy, and dough extensibility (E-value). A central
baked at 204°C for 25 minutes. Composite Design (CCD) with five coded levels (- 1.41, -1, 0,
+1, and +1.41) was used for studying the effect of independent
Bread Tests variables on the dough properties. According to this design,
the total number of treatment combinations was 13 (Table 2).
Specific volume: Specific volume was determined by the
The CCD and statistical analysis of the data were done with
seed displacement method (AACC method 10-05.01) and was
the Design-Expert software package (version 11, State-Ease
calculated as the ratio (v/m). Specific volume determination
Inc., Minneapolis, MN, USA). Coded models were generated to
was carried out on bread samples one hour after baking in
relate independent variables to the dough. Analysis of variance
triplicate.
(ANOVA) was performed to evaluate significant differences and
Shape ratio: The height and width of the central slice of the check the adjusted and predicted coefficient of determination
bread were measured using a pachymeter and the shape was (R2 values). The significant test level was set at 5% ( p<0.05).
determined by the height/width ratio. A ratio of 0.5 indicates a
For comparing the results of bread characteristics before
regular roll shape; a ratio above 0.5 indicates a spherical shape,
and after adding the additives, ANOVA and the T-test method
whilst a ratio below 0.5 indicates a flat shape (Shafiesoltani, et
were used by SPSS software.
al. 2013).

Table 2: Coded models and coefficient for extensograph parameters as a function of the quantities of the glucose oxidase and ascorbic acid.
Flour
Intercept A B AB A² B² R2
type
R 515.8 20.804 -27.961 -6.25 -12.087 35.412 0.98
p-values < 0.0001 < 0.0001 0.1287 0.0032 < 0.0001
Flour
E 113 -3.3321 0.40533 1.75 1.75 -3.5 0.66
With
p-values 0.0529 0.7853 0.4162 0.2920 0.0567
75%
R max 563.2 18.067 -21.158 -7.75 -15.475 27.775 0.97
Extraction
p-values 0.0002 < 0.0001 0.0625 0.0006 < 0.0001
rate
Energy 88.23 0.61243 -2.6124 0.35
p-values 0.6075 0.0472
R 455.6 23.764 13.896 -30 21.247 -67.079 0.97
p-values 0.0006 0.0184 0.0013 0.0011 < 0.0001
Flour
E 112.0 -9.7644 -2.8742 4 0.86
With
p-values 1.0000 0.0006 0.0004
82%
R max 477.2 23.6471 14.368 -25 28.824 -69.911 0.97
Extraction
p-values 0.0005 0.0154 0.0034 0.0002 < 0.0001
rate
Energy 75.98 2.76471 0.1251 -0.25 -0.1780 -5.65728 0.8
p-values 0.0226 0.9115 0.8618 0.8565 0.0030
A: Glucose oxidase; B: Ascorbic acid; R2: Regression coefficient; R: Resistance to extension; Rmax: Maximum resistance to extension; E: Elasticity.
P-value colors: P<.05 P>.05
003

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

Results and discussion in flour with 75% extraction. Less than 100 mg/kg leads to a
decrease in the dough stability. Thus, finding the optimal dose
Resistance to extension of the flours with low extraction of ascorbic acid by Extensograph device at the laboratory before
rate adding it to flour by microfeeder at the product line is important
economically because adding values higher than optimal dose
Resistance to extension (R-value) and the maximum are not statistically significant. Ascorbic acid in the presence
resistance to extension (Rmax-value), characterize the force of oxygen and the ascorbic acid oxidase enzyme converts to
counteracting stretching so this factor indicates the strength of dehydroascorbic acid, which is the oxidative factor in the dough.
the dough (AACC-10-54, 2012). As shown by the results of the Ascorbic acid in the absence of oxygen acts as a reductive agent
Extensograph test in Table 1, the R-value and the Rmax-value and reduces the strength of the dough [6]. However, this study
of the control sample are 340 Bu and 362Bu (Brabender unit) showed that ascorbic acid acts as a reductive agent (at dosages
respectively, indicating a weak flour. less than 100 mg/kg) in the presence of glucose oxidase enzyme.
The reason can be attributed to the competition between
Statistical analysis of the results showed that glucose glucose oxidase and ascorbic acid for oxygen consumption.
oxidase and ascorbic acid had a significant effect (p<0.05) Ascorbic acid cannot compete with glucose oxidase and behave
on this parameter, so a coded and graph model was obtained as a reductive agent in the dough at concentrations of less than
(Table 2, Figure 1a,b). According to Figure 1a and Figure 1b, 100 mg/kg [7]. Increasing the dose of ascorbic acid (greater
the addition of ascorbic acid up to 100 mg/kg has reduced the than 100 mg/ kg) enhances its ability to compete with glucose
strength of the dough (R-value, Rmax value), while the values oxidase (Figure 1c). Hence, ascorbic acid is able to act as an
higher than 100 mg/kg are not statistically significant. This oxidative agent in the dough at doses above 100 mg/kg along
study showed that for increasing the dough stability, ascorbic with glucose oxidase. Reduction of glucose oxidase activity in
acid should be used in higher doses (more than 100 mg/kg) higher doses (Figure 1d), and the ascorbic acid beginning to act

A B

C D

Figure 1: Response surface graphs for flour with 75% extraction rate showing (a and b) the effect of glucose oxidase and ascorbic acid on the R and Rmax values,
respectively (c) the effect of one factor of AsA on the R-value (d) the effect of one factor of Go on the R-value.
AsA: Ascorbic Acid; GO: Glucose oxidase; R: Resistance to extension.

004

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

as an oxidative agent in the dough confirms the competition of of hemicellulose gel in the dough. This gel competes with the
these two factors in the dough. gluten network in absorbing water and prevents the formation
of the gluten network [14-16]. Thus, the addition of glucose
Regarding the addition of the glucose oxidase enzyme, oxidase enzyme at low concentration in flours with an 82%
as shown in Figure 1a,b there is a direct correlation between extraction rate had low effect on the dough strength. This is
the level of glucose oxidase and the R-value, showing that an because the high extraction rate of flour lead to a high quantity
increase of the dose of glucose oxidase enhances the strength of pentosanes and frolic acid in response to high quantities of
of the dough. This is attributed to the formation of hydrogen bran in the flour [17]. In flours with a 75% extraction rate, the
peroxide as by-products of glucose oxidation due to the use of glucose oxidase had a significant effect at any concentration,
glucose oxidase [8,9], hydrogen peroxide are formed is unstable but in flours with 82% extraction rate, adding it had less of an
and rapidly converts to water and free radicals of oxygen. Free impact at doses lower than 20 mg/kg.
radicals of oxygen then form disulfide bonds between glutenin
and gliadin, thus increasing the strength of the dough [10]. Another indirect effect of pentosans could be explained by
their ability to form a network that can limit the movement
On the other hand, the subunits of glutenin are tyrosine- of glutenin. They connect to glutenin proteins covalently, thus
rich; these amino acids are catalyzed by peroxidases and hindering the formation of gluten [18]. At the concentration
participate in the formation of dityrosine [11]. Dityrosines are of higher than 20 mg/kg, glucose oxidase caused an increase
products of tyrosine oxidation that contribute to the structure in the stability of the dough (Figure 2c) due to overcoming the
of the gluten network and act as a kind of stabilizer in the formation of hemicellulose gels in response to the reduction of
gluten structure [11]. free pentosans in the dough [18].

To enhance the stability of the flour with a 75% extraction There is an optimal dose of ascorbic acid to the flour with
rate, glucose oxidase has a greater effect than ascorbic 82% extraction rate (Figure 2d). Doses less than 20 mg/kg can
acid. Also, according to the ANOVA, the effect of adding the increase dough strength. But at a concentration greater than
combination of the ascorbic acid and glucose oxidase in flour 100 mg/kg (Figure 2d), ascorbic acid induced a decreased effect
with a 75% extraction rate was insignificant (p-value>0.05) on the dough stability. The reason attribute to the presence
(Table 2). of the glucose oxidase enzyme. At low doses of ascorbic acid,
due to the lack of oxidative activity of glucose oxidase enzyme,
Extensibility: Extensibility (E-value) showed the stretching ascorbic acid has no competitor and acts as an oxidizing agent
properties of the dough. Analysis of variance showed that there in the dough, but above 100 mg/kg, due to the activation of
was not a significant difference between the extensibility glucose oxidase, ascorbic acid becomes a reductive agent in
results of the different flours with a 75% extraction rate the dough. Glucose oxidase in high doses inhibits the activity
(P-value > 0.05) (Table 2). of ascorbic acid in the dough and ascorbic acid due to lack of
oxygen in the environment (consumption of oxygen by glucose
Energy: The energy parameter describes the work applied oxidase) becomes a reductive agent and reduces the strength of
for stretching the dough. According to ANOVA, there was not a the dough [19].
significant difference between the energy results of the flours
with a 75% extraction rate (p-value>0.05) (Table 2). The mechanism of ascorbic acid action in increasing dough
stability can be attributed to the presence of glutathione in
Extensograph parameter of the flour with a high extrac- flour [19]. This attributes to the presence of glutathione in
tion rate flour. Glutathione is a tripeptide composed of the three amino
acids glutamine, cysteine and, glycine. This tripeptide is
Resistance to extension: The results in Table 1 show that the
naturally present in wheat flour. Glutathione attenuates the
R-value and Rmax values of the control sample are 245 BU and
gluten network due to the presence of a sulfhydryl bond on the
249 BU, respectively which indicates the weakness of the flour.
amino acid cysteine, as it normally tends to bind to the glutenin
Analysis variance of the results showed that glucose oxidase polymer [6,7]. Gluten, which is the principal protein in wheat
and ascorbic acid had a significant effect on these parameters. flour, is made up of two components, glutenin and gliadin. The
R-value and Rmax-value can be explained mathematically polymer of glutenin and the monomer of gliadin form bonds
from the variation of ascorbic acid and glucose oxidase (Table through sulfur bonds in the structure of both components
2) and a response surface figure was obtained (Figure 2a,b). and form the gluten network [20]. The gluten network retain
According to Figure 2c, the addition of glucose oxidase to the the co2 formed as a result of fermentation, thereby creating
flour with an 82% extraction rate resulted in an increase in the a tender texture in wheat bread. Increasing the number of
dough stability at doses greater than 20 mg/kg. At doses lower bonds between glutenin and gliadin leads to the greater
than 20 mg/kg of glucose oxidase, changes in glucose oxidase strength of the gluten network [21]. Thus, any factor that
doses had no affect on the strength of the dough (Figure 2c). inhibits the binding of glutenin and gliadin weakens the gluten
This is possibly due to the flour with a high extraction rate, network. Ascorbic acid converts to dehydroascorbic acid in the
where hydrogen peroxide from the glucose oxidase oxidizes presence of oxygen and ascorbic acid oxidase enzyme, which
pentosanes instead of the gluten network [12,13]. Also, hydrogen oxidizes glutathione. In this way, it prevents the weakening
peroxide from the glucose oxide causes the formation of a bond of gluten indirectly [6]. However, the effect of ascorbic acid
between frolic acid and pentosanes, resulting in the formation is dose-dependent. At a concentration greater than 100 mg/
005

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

(a) (b)

(c) (d)

(e) (f )
Figure 2: Response surface graphs for flour with 82% extraction rate showing (a and b) the effect of glucose oxidase and ascorbic acid on the R and Rmax values,
respectively (c) the effect of one factor of AsA on the R-value (d) the effect of one factor of Go on the R-value (e and f) the effect of glucose oxidase and ascorbic acid on
the E and Energy values, respectively.
AsA: Ascorbic Acid; GO: Glucose oxidase; R: Resistance to extension; E: Elasticity.

kg, ascorbic acid induced a diminishing effect on the dough Elasticity: The results of statistical analyses indicated that
stability. The reason for this is the activation of glucose oxidase ascorbic acid had a significant effect on dough elasticity, but
enzyme at higher concentrations. Glucose oxidase enzyme can the addition of glucose oxidase had no significant effect on
limit ascorbic acid oxidative activity [6]. This is because, in the this parameter (Table 2). The elasticity of the dough would
presence of glucose oxidase due to the competition for oxygen diminish if ascorbic acid was used (Figure 2e). This is due to
consumption, ascorbic acid reacts as a reducing agent and the reduction of sulfhydryl bonds due to the oxidizing effect
lowers the strength of the dough. of ascorbic acid because sulfhydryl bonds are one of the

006

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

factors that can increase dough extensibility [4,22]. Due to of ascorbic acid in flours with a low extraction rate is minimal.
the formation of bonds between sulfhydryl and glutenin in The concentration of glucose oxidase suggested is 15-30 mg/
the oxidation reaction, the number of sulfhydryl bonds in the kg.
dough decrease that result in the reduction of dough flexibility
RSM method showed that the use of 23 mg/kg glucose
[23,24].
oxidase can create the highest dough strength and its use is
Energy: Statistical analyses of energy indicated that only more economical than other formulations. This formulation
the addition of glucose oxidase affected this parameter (Table enhances the dough strength up to 711BU in comparison to
2). Figure 2f shows that the dough energy increased by adding the control sample with 340 BU. It would cause an acceptable
increase in the strength of the dough.
the glucose oxidase, which was due to the rise in the strength
of the dough. Analysis of variance showed that ascorbic acid did
In the case of flour with 82% (Figure 3b) in order to achieve
not have a significant on this parameter. the highest dough strength, it is necessary to use the largest
dose of glucose oxidase enzyme while the concentration of
Optimization
ascorbic acid must be at least 90 mg/kg. Thus, in the flours with
Selecting the optimal samples: Optimal samples were a high extraction rate, the use of glucose oxidase at minimum
selected in both sets of flours based on the rheological results by dosages is not effective and causes a waste of resources.
RSM. Graphs of numerical optimization by RSM for flour with a
According to the combination suggested by the response
75% extraction rate are presented in Figure 3a demonstrating
surface method, the most suitable and economical additive
that the maximum recommended dose for the use of ascorbic is ascorbic acid and the optimal dose is 90 mg/kg, which
acid is 30 mg/kg, which is the minimum quantities in the range increased the maximum strength of the dough, in comparison
as recommended by the supplier. This indicates that the effect to the control sample with the dough strength of the 649 BU.

(a)

(b)
Figure 3: Graphs of numerical optimization for flour with 75% extraction rate (a) and for flour with 82% extraction rate (b).
007

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

This study showed that the type of oxidative improver The reason for the improved crumb color is the oxidation of
for strengthening the gluten network is different for wheat bread pigments due to hydrogen peroxide, which is produced
flours with different extraction rates. At low extraction rates of by glucose oxidase [4]. The improvement of bread crumbs and
flours, glucose oxidase was a more effective additive while at their chewability is due to the increased retention of gases
high extraction rates of flours, adding ascorbic acid was more resulting from the activity of yeasts in the gluten network after
economical as glucose oxidase should be used at higher doses adding oxidative agents [26,27].
(greater than 30 mg/kg). Further, since glucose oxidase is more
expensive than ascorbic acid, it is not economical to add it at However, according to the judges, if an oxidizer improver
high doses to achieve the result ascorbic acid can produce. is used, the symmetry of forms in both groups of the bread
Thus, attention should be paid to the type of flour before using samples, as well as the crust color of bread baked by the lower
the improver. In this study, for the low extraction rate of flour, ash content flour, will decrease significantly (T-test, sig-value
the best oxidative improver was glucose oxidase at 23 mg/kg. 0<0.05) compared to control. The negative viewpoint of judges
On the other hand, for the high extraction rate of flour, it was for symmetry of forms was due to the flinty crust of bread
better to use 90 mg/kg ascorbic acid. After selecting the best (crust breaks like an eggshell), which can be the result of using
oxidative additives, the optimal oxidative dose was added to too much oxidizing improvers [28,29].
the high and low extraction rate flours separately, and after
the bread baking process, the properties of the bread were In the case of using bread with low ash, the color of the
analyzed with results compared to the control bread. bread crust was pale according to the judges. This color occurs
due to the use of glucose oxidase enzyme, which leads to the
Bread tests production of hydrogen peroxide.

Special volume: The results of the specific volume according

The overall score for the bread made with oxidized flour
to Table 3 show that the specific volume of bread prepared
was higher than that of the control bread (Table 3) and in
with the oxidized flour was significantly higher than the
general, according to the judges, better bread can be made with
control sample (T-test, sig- value <0.05). The use of oxidizers
oxidized flour.
improved the gluten network quality, which resulted in better
gas retention in the dough and increases the volume of bread Conclusion
as well as its subsequent volume subsequently [21,25].
In this study, for the 75% extraction rate of flour, the
Shape ratio: As shown in Table 3, the bread containing the optimal improver is glucose oxidase at the doses of 23 mg/kg,
optimal dose of oxidized additives has the highest shape ratio and for the 82% extraction rate of flour, the optimal improver
number (above 0.5), indicating their spherical shape, which is
is 90 mg/kg of ascorbic acid.
due to the increase in the height of loaves due to their stronger
gluten network. On the other hand, the samples of control The results of this study showed that glucose oxidase and
bread had a shape ratio below 0.5, suggesting that the bread ascorbic acid have an optimal dose but excessive use is not
surface is flat due to the weak gluten network. Weak gluten economical.
cannot retain the fermentation gases in its network and the
bread surface would collapse in the oven [25]. Another finding was the reduction of ascorbic acid activity
in the presence of glucose oxidase. This point is important to
Sensory evaluation: To evaluate the sensory attributes, one obtain the optimal formulation for an oxidative improver.
of the most comprehensive methods were used to evaluate all
bread characteristics (Table 3). Bread tests showed a higher specific volume and shape
ratio for bread containing oxidative improvers compared to the
The results of this evaluation showed that the addition of
control sample.
oxidants did not have a significant effect on characteristics
such as grain texture, taste, and smell of the bread (T-test, Regarding the sensory attributes, adding oxidative
sig-value>0.05) while exerting a significant positive effect improvers had a significant positive effect on most attributes,
(T-test, sig-value 0<0.05) on properties such as bread volume, i.e. volume, color, chewability, and texture of bread crumbs,
crumb color, texture, and chewability in both sets of flours. but it did not have a significant effect on grain, aroma, and

Table 3: Influence of the optimal additive on the parameters of bread.

Bread Tests Sensory Evaluation
Specific
Shape crust crumb crumb aroma chew Total
Volume volume form crust grain
Ratio color color texture test ability score
(ml/100gr)
Flours with 75% Control 2.8 0.48 60 71 75 77 77* 80 63 62* 69 71
extraction rate
23 GO (mg/kg) 3.2 0.55 69 75 71 67 79* 86 68 77* 80 80

Control
Flours with 82% 2.5 0.45 59 70 74 76 83 67 61 69* 68 66
extraction rate
90AsA (mg/kg) 2.9 0.51 65 76 59 66 69 74 67 71 *
79 74

* Insignificant at the 0.05 level (T-test); Go: Glucose oxidase; AsA: Ascorbic Acid

008

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

taste. It also had a negative significant effect on the crust color wheat dough as affected by different concentrations of ascorbic acid. J Agric
Food Chem 51: 4948–4953. Link: https://bit.ly/3EMbbUs
and bread symmetry of form.
8. Decamps K, Joye IJ, Courtin CM, Delcour JA ( 2012) Glucose and pyranose
In general, the total sensory evaluation scores of the bread oxidase improve bread dough stability. Journal of Cereal Science 55: 380-384.
with the improver were higher than those of the control bread Link: https://bit.ly/332NM41
sample.
9. Tilley KA, Benjamin RF, Bagorogoza K, Okot-Kotber BM, Prakash O, et al.
Declarations (2001) Tyrosine crosslinks: Molecular basis of gluten structure and function.
J Agric Food Chem 49: 2627–2632. Link: https://bit.ly/3FX1Quu
Availability of data and materials: The data that support
10. Pourmohammadi K, Abedi E (2021) Enzymatic modifications of gluten protein:
the findings of this study are available from the corresponding Oxidative enzymes. Food Chemistry 356: 129679. Link: https://bit.ly/331RfQk
author, upon reasonable request.
11. Peña E, Bernardo A, Soler C, Jouve N (2006) Do tyrosine crosslinks contribute
Authors’ contributions: All authors (Mahsa Shafiesoltani, to the formation of the gluten network in common wheat (Triticumaestivum L)
Mania Salehifar and Saeed Baeghbali) have participated in dough? Cereal Chemistry 80: 52–55. Link: https://bit.ly/3FWFyc0 .

(a) conception and design, or analysis and interpretation of 12. Hoseney RC, Faubion JM (1981) A mechanism for the oxidative gelation of
the data; (b) drafting the article or revising it critically for wheat flour water soluble pentosans. Cereal Chemistry 58: 421-424. Link:
important intellectual content; and (c) approval of the final https://bit.ly/32YvMYy
version.
13. Wang M, Hamer RJ, Van Vliet T, Oudgenoeg TG (2002) Interaction of water
extractable pentosans with gluten protein: Effect on dough properties and
Research highlights:
gluten quality. Cereal Science 36: 25–37. Link: https://bit.ly/3t0hzFh

 Oxidative improvers have different behavior in different

14. Joye IJ, Lagrain B, Delcour JA ( 2009) Use of chemical redox agent and
types of flours. exogenous enzymes to modify the protein network during bread making a
review. Journal of Cereal Science 50: 11–21. Link: https://bit.ly/3ERQxSS
 Glucose oxidase is more effective than ascorbic acid at
flours with 75% extraction rate. 15. Martin CP, Wang M, Lichtendonk WJ, Plijter JJ, Hammer RJ (2005) An
explanation for the combined effect of xylanase-glucose oxidase in
 Ascorbic acid is more effective than Glucose oxidase at dough systems. Science of Food and Agriculture 85: 1186–1196. Link:
flours with 82% extraction rate https://bit.ly/3pSeANp

16. Maa BM, Suna QJ, Lia M, Zhub K (2020) Deterioration mechanisms of
 Ascorbic acid and glucose oxidase are dose-dependent
high-moisture wheat-based food – A review from physicochemical,
improvers. structural, and molecular perspectives. Food Chemistry 318: 126495. Link:
https://bit.ly/3Hvr1EE
Acknowledgment
17. Banu I, Georgeta S, Violeta I, Aprodu I (2012) Effect of the addition of bran
This work was supported by the Ard Tak Karaj Company. stream on dough rheology and bread quality. Food Technology 36: 39-52.
Thanks are due to the quality control unit of the mentioned Link: https://bit.ly/3mUyCEX
company for their skillful assistance in testing the quality of
18. Wong C M, Wong K H, Chen X D ( 2008) Glucose oxidase: natural occurrence,
flours and enzymes activity.
function, properties and industrial applications. Applied Microbiology
Biotechnology 78: 927-938. Link: https://bit.ly/3mXfWnZ
References
19. Kornbrust B A, Forman T, Matveeva I (2012) 19 - Applications of enzymes in
1. Laskowski W, Górska-Warsewicz HRK, Czeczotko M, Zwolińska J (2019) How
Bread making. Bread making 470- 498. Link: https://bit.ly/3pRjSsf
important are cereals and cereal products in the average polish diet? Nutrients
11: 679. Link: https://bit.ly/3FUSL5k
20. Almeida EL, Chang YK ( 2012) Effect of the addition of enzymes on the quality
of frozen prebaked French bread substituted with whole wheat flour. LWT 49:
2. Baratto C, Becker NB, Neves Gelinski JML, Silveira SM (2016) Influence
64-72. Link: https://bit.ly/3FUSiQC
of enzymes and ascorbic acid on dough rheology and wheat bread quality.
African Journal of Biotechnology 15: 55-61. Link: https://bit.ly/3eM6PCj
21. Xu X, Luo Z, Yang Q, Xiao Z, Lu X ( 2019) Effect of quinoa flour on baking
performance, antioxidant properties and digestibility of wheat bread. Food
3. Kouassi-Koffi JD, Kouassi KH, Yapi A, Ahi AP, Muresan V, et al. (2015) Wheat
Chemistry 294: 87–95. Link: https://bit.ly/3FUSiA6
bread dough rheological properties. Journal of Texture Study 46: 475-486.

22. Ooms N, Jansens KJA, Pareyt B, Reyniers S, Brijs K, et al. (2018) The
4. Kouassi-Koffi JD, Sturza A, Paucean A, Man S, Muresan AE, et al. (2019)
Effect of glucose oxidase addition on the textural characteristics of impact of disulfide bond dynamics in wheat gluten protein on the
wheat-maize dough and bread. Food Science and Technology 39. Link: development of fermented pastry crumb. Food Chemistry 242: 68-74. Link:
https://bit.ly/3FXIJjO https://bit.ly/3eTopUE

5. Khuri A , Mukhopadhyay S (2010) Response surface methodology. WIREs 23. Dahiya S, Bajaj BK, Kumar A, Tiwari SK, Singh B ( 2020) A review on
Computer Statistics 2: 128-149. Link: https://bit.ly/3pPCU2j biotechnological potential of multifarious enzymes in bread making. Process
Biochemistry 99: 290–306. Link: https://bit.ly/3FWs3sV
6. Koehler P (2003) Effect of Ascorbic Acid in Dough: Reaction of oxidized
glutathione with reactive thiol groups of wheat glutelin. J Agric Food Chem 51: 24. Steffolani ME, Ribotta PD, Pérez GT, León AE (2010) Effect of glucose oxidase,
4954–4959. Link: https://bit.ly/3eToKXq transglutaminase, and pentosanase on wheat proteins: relationship with
dough properties and bread-making quality. Journal of Cereal Science 51: 366-
7. Koehler P (2003) Concentrations of low and high molecular weight thiols in 373. Link: https://bit.ly/3sVeZAh

009

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137
 https://www.peertechzpublications.com/journals/international-journal-of-agricultural-science-and-food-technology

25. Shafisoltani M, Salehifar M, Hashemi M (2014) Effects of enzymatic treatment historical and modern spring wheat. Cereal Chemistry 95: 226–238. Link:
using Response surface methodology on the quality of bread flour. Food https://bit.ly/3zmS3eB
Chem 148: 176-183. Link: https://bit.ly/3mTDni5
28. Miś A, Nawrocka A, Dariusz D (2016) Identification of baking expansion phases
26. da Silva CB, Almeida LE, Chang YK ( 2016) Interaction between xylanase, of leavened dough using an experimental approach. Food and Bioprocess
glucose oxidase and ascorbic acid on the technological quality of whole wheat Technology 9: 892–903. Link: https://bit.ly/3JAW0kJ
bread. Food Technol 46: 2249-2256. Link: https://bit.ly/3mV5PQt
29. Ooms N, Delcour JA (2019) How to impact gluten protein network formation
27. Malalgoda M, Ohm JB, Meinhardt S, Simsek S ( 2018) Association between during wheat flour dough making. Current Opinion in Food Science 25: 88–97.
gluten protein composition and breadmaking quality characteristics in Link: https://bit.ly/32NbEc2

010

Citation: Shafiesoltani M, Salehifar M, Baeghbali S (2022) Optimization of oxidative improver’s formulation for the wheat flours with different extraction rates. J
Agric Sc Food Technol 8(1): 001-010. DOI: https://dx.doi.org/10.17352/2455-815X.000137