Journal Pre-proof

Modification of dough characteristics and baking quality based on whole wheat flour
by enzymes and emulsifiers supplementation

Zahra Sheikholeslami, Maryam Mahfouzi, Mahdi Karimi, Mahdi Ghiafehdavoodi

PII: S0023-6438(20)31783-7
DOI: https://doi.org/10.1016/j.lwt.2020.110794
Reference: YFSTL 110794

To appear in: LWT - Food Science and Technology

Received Date: 18 September 2020

Revised Date: 14 December 2020
Accepted Date: 17 December 2020

Please cite this article as: Sheikholeslami, Z., Mahfouzi, M., Karimi, M., Ghiafehdavoodi, M., Modification
of dough characteristics and baking quality based on whole wheat flour by enzymes and emulsifiers
supplementation, LWT - Food Science and Technology, https://doi.org/10.1016/j.lwt.2020.110794.

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Zahra Sheikholeslami: conceptualization, methodology, supervision

Maryam Mahfouzi: formal analysis, investigation, writing, visualization

Mahdi Karimi: software, data curation, visualization

Mehdi Ghiafedavoodi: validation

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1 Modification of dough characteristics and baking quality based on whole

2 wheat flour by enzymes and emulsifiers supplementation

4 Zahra Sheikholeslami a, *, Maryam Mahfouzib, Mahdi Karimia, Mahdi Ghiafehdavoodia

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6 a Agricultural engineering research department. Khorasan Razavi agricultural and natural

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7 Resources research education center, Agriculture Research, Education and Extension Organization

8 (AREEO) AREEO, Mashhad, Iran.

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b department of Food Science and Technology, Ferdowsi University of Mashhad (FUM), Mashhad, Iran
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11 Zahra Sheikholeslami*, Email: [email protected], Tell: + (98) 9153175239 , orcid.org/0000-0001-

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12 9901-1380
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13 Email of other authors: [email protected]

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14 [email protected]

15 [email protected]

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17

18

19

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21

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22 Abstract

23 The objective of the present study was to evaluate the effect of enzymes (xylanase and amylase)

24 and emulsifiers (SSL and DATEM) on the rheological properties of dough and physicochemical,

25 textural, microstructural, nutritional, as well as sensory attributes of whole wheat bread (WWB).

26 Based on the results, the combination of xylanase and emulsifiers had a synergetic influence on

27 the dough rheology and bread attributes due to the interactions among enzyme-emulsifiers and

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28 bread ingredients. Incorporating xylanase, SSL, and DATEM into the whole wheat bread

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29 formulation significantly enhanced the stability, extensibility, total antioxidant activity, and

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general acceptability, while phytic acid content and hardness of the final bread were decreased.

Regarding SEM analysis, a more homogeneous and uniformed texture was observed in the
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32 samples containing xylanase and emulsifiers, compared to the bread without any additives. On
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33 the other hand, amylase demonstrated no strengthening effect on whole wheat bread, this could
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34 be explained by its influence on the starch decomposition. WWB prepared with a combination of
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35 enzymes and emulsifiers was given higher scores in the organoleptic parameters. In conclusion,

36 fortification of whole wheat bread with xylanase, amylase, SSL, and DATEM can play a positive
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37 role in the quality as well as the nutritional value of WWB.

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39 Keywords: Whole wheat bread; Additive supplementation; Antioxidant activity,

40 physicochemical attributes; Microstructure.

41

42

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43 1. Introduction
44

45 During recent years, there has been a general trend toward enhancing the nutritional value of

46 bakery products which are widely consumed in various nations. The demand for consuming the

47 whole wheat bread (WWB) has considerably increased due to its bioavailability, nutritional, and

48 potential health benefits. Whole-grain wheat includes three main fractions: bran, germ, and

49 endosperm. Wheat-bran (WB) is rich in dietary fibers, soluble vitamins, minerals, and bioactive

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50 compounds which have health-promoting properties. In terms of health, phenolic compounds as

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51 a source of antioxidants in WB play a protective role against a large number of diseases such as

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cardiovascular disease, type 2 diabetes, cancer, blood cholesterol, and obesity (Trombini,
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53 Mischan, & Leonel, 2016). However, the presence of high phytic acid content in WB adversely

affected the bioavailability of minerals like iron, magnesium, zinc, and calcium. The strong
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55 phytate-minerals bonds alter the solubility, absorption, digestibility, and functionality of the
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56 minerals (Onipe, Jideani, & Beswa, 2015). In addition, WB weakens the gluten network of bread
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57 and prevents the formation of proper volume in the final product.

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58 To mitigate these problems, integrating emulsifiers and enzymes into WWB may have an

59 important positive effect on dough properties and bread quality. Although a wide range of

60 enzymes is consumed, amylases are one of the most extensively used enzymes during the baking

61 technology (Sanz Penella, Collar, & Haros, 2008). These enzymes are able to hydrolyze the

62 starch which leads to an increase in the bread volume and an improvement in the color

63 characteristics of the crust and crumb of bread and overall acceptability in the final products

64 (Eduardo, Svanberg, & Ahrné, 2014). However, flour containing inappropriate alpha-amylase

65 activity provides a dark and dry bread with lower specific volume and shelf life (Mangan,

66 Szafranska, McKie, & McCleary, 2016). Xylanase (EC 3.2.1.8) is another enzyme which has an

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67 important influence on the formation of gluten network and the improvement of bread elasticity,

68 due to the hydrolysis of un-extractable arabinoxylans (Amiri, Shahedi, & Kadivar, 2016;

69 Caballero, Gómez, & Rosell, 2007).

70 Besides, the structure of gluten protein is chemically modified after treatment with some

71 emulsifiers (Pareyt, Finnie, Putseys, & Delcour, 2011). Emulsifiers can correlate with the

72 hydrophobic surface of protein in the gluten network, results in a better texture, softer crumb,

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73 and a larger volume in the bread (Ribotta, Pérez, León, & Añón, 2004). Among the emulsifiers

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74 utilized in bakery products, sodium stearyol-2-lactylate (SSL) and diacetyl tartaric ester of mono

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and diglycerides (DATEM) have been employed as dough stabilizer and bread softener

(Colakoglu & Özkaya, 2012). SSL with high hydrophilic–lyophilic balance has a positive impact
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77 on the gluten strength in which the hydrophobic tails of the emulsifier bind with hydrophobic
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78 sites in gluten proteins (Ferrer, Gómez, Añón, & Puppo, 2011). Adding DATEM to dough
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79 improves some features such as gas retention capacity, mixing tolerance index, and dough
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80 resistance to collapse (Ribotta et al., 2004).

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81 To the best of our knowledge, the interaction and mechanism of enzymes in combination with

82 emulsifiers, or alone, have not been investigated regarding a traditional Sangak bread produced

83 by whole wheat flour (WWF). The present study aimed to evaluate the chemical and rheological

84 assessment for dough and physicochemical, textural, microstructural, and sensory analysis for

85 bread in order to reply to some complicated questions whether these improvers can prevent the

86 adverse effects of phytic acid on the quality of Sangak bread containing WWF.

87 2. Materials and methods

88

89 2.1. Materials
90

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 91 Wheat flour with an extraction yield of 96% was purchased from Parsan Co, Mashhad, Iran. The

92 wheat flour packages were maintained at 10 °C until chemical analysis. Other bread ingredients

93 including active dry yeast (Razavi Co, Mashhad, Iran), sugar, and salt were carefully supplied

94 from the local market. Then, emulsifiers including Diacetyl Tartaric Acid Ester of Mono-and

95 Diglycerides (DATEM) and Sodium Stearoyl-2-Lactylate (SSL) were provided from Pars

96 Behbood Asia Co, Mashhad, Iran, and enzymes including α-amylase and xylanase were bought

97 from DSM Co, Netherland.

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98 2.2. Chemical composition analysis of whole wheat flour
99

100
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The international methods of American Association of Cereal Chemists (AACC, 2000) were
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101 utilized to estimate the chemical composition of WWF. Method 44-16 for moisture content,
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102 method 46-12 for protein content, method 08-01 for ash content, method 32-05 for dietary fiber

103 content, method 38-11 for wet gluten content, method 38-12 for gluten index, and method 56-
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104 81B for falling number test were used. Next, total antioxidant activity (TAA) and phytic acid
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105 content were assessed by the methods previously described by (Oufnac, 2006) and Thompson
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106 and Erdman (1982), respectively.

107 2.3. Dough formulation and bread preparation

108

109 Sangak bread was produced based on the method of Rahnama, Mohammadzadeh Milani, and

110 Gohari Ardabili (2015) with a modification. The main Sangak bread formula consisted of WWF

111 (1000 g), water (850 g), active dry yeast (5 g), sugar (5 g), and salt (10 g). Then, emulsifiers

112 including SSL and DATEM at concentration of 5 g/kg, and enzymes including α-amylase (5

113 mg/kg) and xylanase (15 mg/kg) were added to the dough and made the different formulations

114 (Table 1). All ingredients were carefully mixed in a spiral mixer (KNS256CDH, Kitchenaid,

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115 USA) and then stored at room temperature for 2 h. At the next step, the dough was divided into

116 500 g pieces, and put on baking sheets with 70 cm in length, 40 cm in width, and 5 mm in

117 thickness and placed in a traditional rotary baking oven at 400 °C for 5 min.

118 2.4. Rheological characteristics of dough

119

120 To assess the dough behavior, the most important dough rheological properties were measured

121 along with Brabender Farinograph (O. H. Duisburg, Germany). According to method 54-21

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122 (AACC, 2000), some parameters of dough such as water absorption (%), dough development

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123 time (min), stability (min), and mixing tolerance index (BU) in various formulations were

124 examined.
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125 2.5. Physicochemical properties
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127 In the present study, some physicochemical features including water activity (aw), moisture,
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128 hardness, extensibility, crust color, and microstructural characteristics of bread affected by WWF
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129 and different emulsifiers and enzymes were evaluated to produce the bread with high quality and
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130 nutritional value.

131 2.5.1. Water activity and moisture

132

133 In order to calculate water activity and the moisture content, methods of American Association

134 of Cereal Chemists (AACC, 2000) were utilized.

135 2.5.2. Textural analysis

136

137 In the present research, textural parameters of bread with WWF such as hardness and

138 extensibility were measured by using a texture analyzer (CNS Farnell, Hertfordshire, UK)

139 equipped with a cylindrical probe 25 mm in diameter (the cross speed: 50 mm/min, penetration

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140 depth: 30 mm, trigger point: 0.05 N, and target value: 30 mm). During the puncture experiment,

141 the peak force required to penetrate into the middle of the bread samples (10* 10 cm) was

142 regarded as hardness.

143 In terms of extensibility, the bread was subjected to the texture analyzer and the penetration of

144 the probe into the bread core was measured until the bread was torn. The stretch length of the

145 bread to rupture was reported as the extensibility (Sheikholeslami, Karimi, Komeili, &

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146 Mahfouzi, 2018).

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147 2.5.3. Image processing
148

149
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During recent decades, image processing has been applied to visualize the crust color of bakery
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150 products. To take the images of bread slices (20*20 cm2), a flatbed scanner (Scanjet G3010, HP,
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151 China) with a resolution of 600 DPI was used and then the images were saved as JPEG format.
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152 Color components including lightness (L*), red-green (a*), and yellow-blue (b*) were measured

153 through Image J software by activating the LAB Plugin (Fathi, Mohebbi, & Razavi, 2011).
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154 2.5.4. Scanning electron microscopy (SEM)

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156 In order to evaluate the effect of emulsifiers and enzymes on the microstructure of the bread

157 prepared by WWF, SEM can be useful. Firstly, slices of bread (10*10 mm2) were prepared by

158 the method of freeze-drying and then mounted on metal stubs and coated with a layer of gold (2

159 min, 2 mbar). A scanning electron microscope (MIRA3, TESCAN, USA) with a vacuum of 2 ×

160 10-5 torr and an accelerating voltage of 10.0 kV was used to obtain the SEM images with a

161 magnification level of (1000 ×).

162 2.6. Total phenolic content (TPC)

163

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164 The WWF and WWB solutions were prepared by dissolving 5 g of each sample in 50 mL

165 methanol 80%, then, centrifuged (Heraeus Labofuge 200, SEPATECH, Germany) at 4600 g for

166 20 min and the supernatant was collected. To calculate TPC, 1.5 mL of Folin-Ciocalteu reagent

167 and 1.5 mL of sodium carbonate were mixed with the prepared methanolic solutions. After

168 maintaining away from the light source for 90 min, the absorbance was monitored at a

169 wavelength of 750 nm using a spectrophotometer (Pharmacia Biotech, Novaspec II, LKB,

170 England). Based on a calibration curve of Gallic acid, TPC was expressed as µg Gallic acid per g

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171 sample (Oufnac, 2006).

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174 The total antioxidant activity of WWF and WWB was measured by employing the radical DPPH
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175 (2,2-diphenyl-1-picrylhydrazyl) scavenging capacity method (Oufnac, 2006). 2 mL of extract

176 (prepared according to the method described in the section 2.6) was added to 2 mL 0.1 mM
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177 DPPH solution (dissolved in ethanol) and the absorbance was read at 750 nm by using a
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178 spectrophotometer. Subsequently, inhibition percentage values were specified by the following
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179 Equation:

180 Percentage of free radical inhibition (%) = Abs blank- Abs sample/ Abs blank × 100

181 2.8. Sensory evaluation

182

183 To assess the organoleptic attributes of WWB, some parameters such as texture, crumb color,

184 crust appearance, flavor, crust color, odor, overall appearance, and general acceptability were

185 analyzed by a five-point hedonic scale. To this purpose, 20 panelists were provided with a glass

186 of mineralized water as flavor neutralizer. Each evaluator was instructed to consume water

187 before each analysis to differentiate samples from each other carefully (Sheikholeslami et al.,

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188 2020). In this step, all analyses were performed six times. In other words, all panelists were

189 given 6 pieces (the different parts of each bread) from all bread for more accuracy in the

190 analysis.

191 2.9. Statistical analysis

192

193 A completely randomized design with Tukey's Multiple Range Test was carried out using

194 Minitab software (version 17) at a significance level of P<0.05. To draw the curves, MS-Office

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195 Excel 2013 was applied. All the experiments were done in triplicate and the results were reported

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196 as the average and standard deviation.

197 3. Results and Discussion

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199 3.1. Chemical composition measurement of whole wheat flour

200

Table 2 exhibits the chemical components of WWF with an extraction yield of 96%. To better
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202 understanding, the results of the previous research for wheat flour with an extraction yield of
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203 82%, conducted by Sheikholeslami et al. (2018), are also given in Table 2. As displayed in Table
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204 2, while there was no distinct difference in the moisture content for two kinds of flour, protein,

205 ash, and dietary fiber content of WWF were remarkably higher than refined wheat flour,

206 indicating the higher nutritional value of WWF than that of refined wheat flour. A progressive

207 increase in ash content confirms that the minerals are mainly concentrated in the external layers

208 of the grains (Yeung & Vasanthan, 2001). Dietary fiber is defined as edible parts of plants

209 including cellulose, hemicellulose, lignin, and other useful substances which has several

210 potential health benefits (Almeida, Chang, & Steel, 2013). Phytic acid, as myo-inositol hexa-

211 phosphate, is mainly distributed in the outer layers of the cereals. Phytic acid content ranges

212 from 600 –1000 mg/100 g in WWF, while is less than 400 mg/100 g in the refined wheat flour.

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213 In the present study, the content of phytic acid in WWF was 864.46 mg/100 g, meaningfully

214 higher than the refined flour with an extraction yield of 82% (53.82 mg/100 g). Our data

215 manifested that the total antioxidant activity value of WWF was 28.29 % (Table 2). It is believed

216 that the antioxidant property of WWF has a positive correlation with the phenolic compounds in

217 flour (Emmons, Peterson, & Paul, 1999).

218 3.2. Rheological properties of dough

219

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221 in different mixture ratios. Our data demonstrated that the xylanase alone and with combination

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223 degradation of un-extractable arabinoxylans. There is a competition between the gluten and
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224 arabinoxylans for adsorbing the water during dough preparation. Therefore, with degrading the

225 un-extractable arabinoxylans to their extractable form with lower molecular weight, gluten is
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226 able to absorb the further amount of water and form a better gluten network. In fact, a widening
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227 network of pentosan–protein is happened by means of this alternation, thus releasing the water
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228 and results in further hydration of the gluten (Hardt, Boom, & van der Goot, 2014). It is worth

229 noting that the addition of hydrolyzing enzymes like xylanase causes a reduction in required

230 water in bread formulation. The formulation containing xylanase, SSL, and DATEM (X)

231 revealed the maximum dough development time (DDT). Compared to the control sample (I),

232 xylanase with emulsifiers significantly raised this parameter, while amylase and emulsifiers

233 alone or with combination together (III, V, VI, XI, and XII) had no influence on DDT (Table 3).

234 An enhancement in this feature may contribute to the interactions between emulsifiers and gluten

235 proteins, allowing a longer DDT. Although the presence of enzymes alone in whole wheat dough

236 had no notable effect on stability and mixing tolerance index (MIT), emulsifiers alone or with

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237 combination of enzymes (formulation V to XIV) resulted in an augment in stability and a

238 decrease in MIT (Table 3). The reason for this occurrence has been hypothesized that connecting

239 the hydrophobic tails of the emulsifier (SSL) with lyophilic sites of dough proteins leads to a

240 strong dough (Ferrer et al., 2011). Adding DATEM to wheat flour caused considerable

241 improvement in some rheological parameters of dough such as mixing tolerance, stability, and

242 resistance of the dough to collapse (Ribotta et al., 2004). The results are in line with those

243 reported by Liu, Brennan, Serventi, and Brennan (2017), in which they displayed that xylanase

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244 increased the water absorption, DDT, stability, and MTI of wheat dough, while amylase

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245 meaningfully reduced the stability and MTI.

246 3.3.
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Chemical characteristics of whole wheat bread
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247
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248 Table 4 expresses the results of moisture, water activity (aw), protein, dietary fibers, and phytic

249 acid content for different bread formulations affected by enzymes (xylanase and amylase) and
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250 emulsifiers (SSL and DATEM), alone or mixed forms. No notable impact was perceived in
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251 moisture and protein content of bread after adding the mentioned additives. However, emulsifiers
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252 and enzymes considerably affected other parameters including water activity, dietary fiber, and

253 phytic acid content. Compared to the control sample, no change was detected in water activity

254 after adding enzymes or emulsifiers lonely, while a combination of enzymes and emulsifiers

255 reduced this chemical parameter (Table 4). It is noteworthy that formulations including SSL and

256 enzymes (VIII & XI) had no impact on this parameter, due to the capability of SSL in interacting

257 with protein network in bread and in maintaining the water activity. Regrading to dietary fiber

258 and phytic acid, the addition of xylanase and emulsifiers alone or as a combination led to a

259 remarkable decrease in the content of these parameters. The dietary fiber content in bread

260 including amylase alone (formulation III) and a mixture of amylase and SSL (formulation XI)

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261 showed no significant difference compared to the control sample. These bread formulations (III

262 & XI) had a higher content of phytic acid than other formulations. This may be related to the fact

263 that phytic acid inhibits alpha-amylase activity (Li, Alli, & Kermasha, 1993). The highest

264 reduction in phytic acid content took place when xylanase with emulsifiers were added into the

265 bread formulation (formula x). It is anticipated that the effect of xylanase and emulsifiers on

266 WWB leads to the separated phosphoric bonds in phytic acid structure, thus preventing the

267 formation of covalent bonds with minerals. In this regard, the nutritional value of bread produced

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268 with the high extraction yield will be enhanced compared to the bread sample without any

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269 additives.
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270 3.4. Textural parameters
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271
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272 Parameters associated with the texture of WWB including hardness and extensibility are

273 illustrated in Fig 1. The influence of emulsifiers and enzymes on reducing the hardness and on
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274 enhancing the extensibility of WWB was significant (Fig. 1). The obtained observations
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275 indicated that the gluten structure was modified by enzyme-emulsifier treatment, means that it
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276 prevented the negative intervention of phytic acid in forming the gluten network. Our

277 observation was in agreement with the data reported by Gómez, Ferrer, Añón, and Puppo (2013).

278 They evaluated the effects of SSL and amylase on the quality of pan bread and then claimed that

279 these ingredients had positive effect on reducing the hardness of pan bread. Also, the present

280 finding was in line with the results of previous observation conducted by Kornbrust, Forman, and

281 Matveeva (2012), which demonstrated an improvement in the crumb structure and bread volume

282 by adding amylase and xylanase, respectively. It is believed that amylases are unique enzymes in

283 softening the bread structure and in enhancing the elasticity and edibility aspects of WWB

284 (Gómez et al., 2013). Amylases produce fermentable saccharides which have the positive effect

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285 on gas retention capacity in WWB. This activity leads to an increase in the volume of bread loaf

286 as well as a decrease in the crumb hardness. Furthermore, it was mentioned that xylanases can be

287 utilized for all types of bread in combination with emulsifiers. They are able to modify the

288 elasticity of the gluten network (Caballero et al., 2007). Xylanases are well known to improve

289 the structural characteristics of bread, it is contributed to their effect on producing water-

290 extractable arabinoxylans in WWB. Altering the water-unextractable pentosanes to their

291 extractable form and transferring released water from pentosanes located in the wall cell to the

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292 gluten network by means of xylanase provide the softer texture and more extensibility in bakery

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293 products (Ghoshal, Shivhare, & Banerjee, 2013). The role of emulsifiers as bakery additives in

294
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softening the crumb and in providing longer crumb freshness has been explained by their ability
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295 to bind to gluten proteins and starch (Colakoglu & Özkaya, 2012). It could be hypothesized that
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296 SSL can interact with gliadin and prohibit it from participating in cross-linking interactions.
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297 Therefore, the internal structure of bread would be formed softer (Van Steertegem, Pareyt, Brijs,

298 & Delcour, 2013). Additionally, It has been previously reported that some emulsifiers such as
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299 SSL and DATEM are responsible for softening the WWB but not white bread (Armero & Collar,
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300 1997).

301 3.5. Image processing

302

303 The color quality of WWB with various formulations was estimated by image processing (Fig 2).

304 As shown in Fig 2.a, a considerable augment in the brightness (L*) of the bread fortified with

305 xylanase and SSL was seen. On the contrary, the presence of amylase and DATEM expressed no

306 brightening effect on the WWB. The results are consistent with those reported by Ghoshal et al.

307 (2013). Their observation signified that higher brightness value was progressively impacted by

308 adding xylanase. Higher L* values of WWB with xylanase supplementation may attribute to its

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309 capability to convert the insoluble arabinoxylans to their soluble form, after which the gluten

310 network can abstract the released water. It is suggested that bakery products with higher water

311 absorption seem brighter. Further, it is speculated that SSL can interact with phenolic

312 compounds in WWB and decrease the poly-phenol-oxidase activity, thereby preventing the

313 darkness process (Niu, Hou, Kindelspire, Krishnan, & Zhao, 2017). The clear discrepancy in a*

314 and b* of different formulations was linked with amylase, due to the involvement of this enzyme

315 in hydrolyzing the starch polymer and in producing the saccharides with lower molecular weight,

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316 which are able to participate in the fermentation and Maillard browning.

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318
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319 In order to assess the role of different enzymes and emulsifiers on the microstructural properties
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320 of WWB, SEM was used (Fig 3). As displayed in Fig. 3, the control sample (I) demonstrated a

321 less uniformity compared to samples containing additives. It can be easily identified that phytic
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322 acid prohibited a regular interaction between gluten and starch. A homogeneous and uniform
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323 texture under the influence of xylanase is noticeable in Fig 3 (II). This could be due to the
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324 influence of xylanase on destructing the pentosanes and reducing the viscosity. By decreasing the

325 viscosity, the possibility of aggregation is increased, due to the fact that starch granules become

326 closer easier. In addition, xylanase could interact with gluten protein and increase the stability

327 and firmness (Gómez et al., 2013). Regarding WWB formulation including amylase (III), it

328 presented a more porous structure as compared to the control sample. The samples including

329 SSL showed a greater level of connectivity in the gluten network of WWB, which was in line

330 with the results of stability in Table 3. This is due to a fact that SSL has an amphiphilic property,

331 thereby propelling starch-protein interaction and generating a more compact structure. It is

332 believed that SSL could combine with amylose in starch granules and bind with the gluten

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333 network, thus increasing the protein-starch interactions and also raising the cohesiveness value

334 (Van Steertegem et al., 2013).

335 With regard to a mixture of emulsifiers and xylanase, micrographs (Fig 3, VIII, IX, and X)

336 displayed that the granules are mingled to the neighboring granules, presenting a more

337 monotonous texture. This occurrence is most likely attributed to the ability of these improvers to

338 bind tightly with protein network and to decrease the repulsing charges on the protein surface,

339 allowing an increase in the cohesiveness and integrity values in WWB. In the sample containing

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340 all emulsifiers and enzymes, the SEM image (XIV) illustrated a continuous surface. Our results

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341 suggested that the additives used in the present study could be linked with WWB compounds,

342
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hence hindering the improper interaction of phytic acid and gluten network and avoiding the
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343 uneven structure.
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344 3.7. Total phenolic content and total antioxidant activity

345
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346 The antioxidant activity has a direct correlation with phenolic compounds in a food system, due
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347 to their capability for free radical quenching. The evaluation of the total phenolic content and
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348 antioxidant capacity of WWF, WWB, and WWB affected by different enzymes (amylase and

349 xylanase) and emulsifiers (SSL and DATEM) was displayed in Fig 4. Different interactions can

350 be distinguished between the phenolic compounds and free radicals, based on their chemical

351 structure (López-Perea et al., 2019). Baking and fortifying the food products alter the chemical

352 structure of phenolic ingredients, thus differing the quenching capacity and also affecting the

353 elimination of free radicals. TPCs were 66.93 and 67.41 µg Gallic acid per g of samples for

354 WWB and samples containing enzymes and emulsifiers, respectively. It appeared that

355 interactions between enzymes/emulsifiers and phenolic compounds led to the increased TPC,

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356 thus enhancing the antioxidant capacity. As far as the DPPH method manifested, an increase was

357 observed in the antiradical capacity of WWB as treated by additives (Fig 4).

358 3.8. Sensory evaluation

359

360 In Table 5, the information about the organoleptic evaluations of WWB under the influence of

361 xylanase, amylase, SSL, and DATEM is observable. As shown, the highest score for texture is

362 related to WWB including xylanase lonely or with combination of enzymes (II, VIII, X). It can

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363 be hypothesized that this combination resulted in a homogenous and uniformed structure in

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364 which starch granules were in a close association with the gluten network. Table 5 exhibited that

365
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WWB formulations including the amylase had a higher score in the crust appearance, flavor,
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366 crust color, and odor than those of other formulations. The main objective for using amylases in
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367 bread is their role in improving some parameters such as crust color, crumb color, texture, and

368 flavor (Eduardo et al., 2014). After adding enzymes and emulsifiers as a combination, a
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369 significant upgrade in the score of overall appearance and general acceptability of WWB was
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370 distinguished. On the other hand, DATEM appeared no notable influence on the organoleptic
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371 attributes of WWB (Table 5).

372 4. Conclusion
373

374 Enhancing the nutritional value of foods with natural resources is one of the most challenging

375 issue in the world. Producing whole wheat bread provides nutritional and health benefits for

376 human. Producing the bread by wheat flour with an extraction yield of 96% was performed by

377 using enzymes (xylanase and amylase) and emulsifiers (SSL and DATEM). Based on the results,

378 addition of enzymes and emulsifiers dramatically affected the characteristics of whole wheat

379 bread. The rheological behavior, softness, extensibility, brightness, structure, antioxidant

16
380 activity, and sensory evaluations of whole wheat bread were positively correlated with additives

381 supplementation. Additionally, a uniformed texture was observed in the SEM images of samples

382 containing xylanase and emulsifiers, which may be directly attributed to the chemical bonds

383 between xylanase and SSL with gluten network and starch granules. Adding xylanase and

384 emulsifiers led to a remarkable reduction in phytic acid content, hence preventing from the

385 negative impact of phytic acid on the bread quality. In conclusion, fortification of whole wheat

386 bread with enzymes and emulsifiers supplementation improved the chemical compositions,

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387 textural, nutritional, and functional features of the final product.

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19
Table 1
Different formulations of whole wheat bread including enzymes and emulsifiers.
Formulations Flour/Enzymes/Emulsifiers
I Dough prepared by whole wheat flour without additives (Control Sample)
II Dough + xylanase (15 mg/kg)
III Dough + amylase (5 mg/kg)
IV Dough + xylanase (15 mg/kg) + amylase (5 mg/kg)
V Dough + SSL (5 g/kg)
VI Dough + DATEM (5 g/kg)
VII Dough + SSL (5 g/kg) + DATEM (5 g/kg)
VIII Dough + xylanase (15 mg/kg) + SSL (5 g/kg)

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IX Dough + xylanase (15 mg/kg) + DATEM (5 g/kg)
X Dough + xylanase (15 mg/kg) + SSL (5 g/kg) + DATEM (5 g/kg)

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XI Dough + amylase (5 mg/kg) + SSL (5 g/kg)
XII Dough + amylase (5 mg/kg) + DATEM (5 g/kg)
XIII
XIV
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Dough + amylase (5 mg/kg) + SSL (5 g/kg) + DATEM (5 g/kg)
Dough + amylase (5 mg/kg) + xylanase (15 mg/kg) + SSL (5 g/kg) + DATEM (5 g/kg)
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 Table 2
Proximate chemical composition of whole wheat flour.

Dietary Wet Falling

Moisture Protein Ash Gluten Phytic acid
Flour type fiber gluten number TAA (%)
(g/100g) (g/100g) (g/100g) index (mg/100 g)
(g/100g) (g/100g) (s)
Wheat flour
with an
13.5±0.24 12±0.28 1.23±0.1 6.84±0.57 28.5±0.21 0.73±0.02 363±4.49 864.46±18.4 28.29±1.13
extraction yield
of 96% (WWF)
Wheat flour
with an
extraction yield
13.5±0.37 10.1±0.16 0.73±0.01 2.61±0.04 27±1.84 - - 53.82±3.23 -

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of 82%
(Sheikholeslami
et al., 2018)

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Table 3
The rheological parameters of whole-wheat dough affected by enzymes and emulsifiers in different
formulations.

Dough
Mixing tolerance
Formulations Water absorption (%) development time Stability (min)
index (BU)
(min)
I 62.83±0.306bc 3.57±0.062de 2.44±0.068g 161.67±2.89a
II 65.70±0.458a 3.84±0.083abcd 2.61±0.032fg 166.67±2.89a
III 63.40±0.265bc 3.34±0.085e 2.45±0.050g 158.33±2.89a
IV 63.66±0.208b 3.44±0.133abc 2.46±0.028g 163.33±2.89a
V 63.16±0.306bc 3.58±0.076de 3.00±0.100cdef 110.00±10.0bcd
VI 63.26±0.208bc 3.95±0.100e 3.21±0.057cd 103.33±5.77cd
63.53±0.351b 4.00±0.132abc 3.83±0.057a 96.67±5.77cd

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VIII 65.23±0.351a 4.06±0.125ab 3.11±0.028cde 116.67±5.77bc
IX 65.26±0.351a 3.98±0.076abc 3.75±0.050ab 113.33±11.55bcd

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X 65.40±0.265a 4.10±0.050a 3.86±0.076a 103.33±5.77cd
XI 62.93±0.152bc 3.80±0.100bcd 2.73±0.068ef 126.67±5.77b
XII
XIII
63.06±0.252bc
62.53±0.404c
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3.76±0.152cd
3.90±0.050abc
2.93±0.057cdef
3.36±0.511bc
120.00±10.0bcd
113.33±5.77bcd
63.73±0.208b 3.96±0.028abc 2.83±0.152def 116.67±5.77bc
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Note: The different letters in the same column reveal significant differences (P <0.05) among means.
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Table 4
Chemical characteristics of whole wheat bread affected by enzymes and emulsifiers.
Bread Moisture Protein Dietary fiber Phytic acid
aw
formulations (g/100g) (g/100g) (g/100g) (mg/100 g)
I 27.60±0.854a 0.793±0.008a 12.23±0.169a 6.68±0.113a 755.57±3.286cd
II 27.83±0.235a 0.791±0.007a 12.11±0.271a 4.95±0.057f 637.10±0.871g
III 27.80±0.725a 0.803±0.016a 12.06±0.249a 6.80±0.072a 825.34±2.299a
27.53±0.740a 0.797±0.018a 12.08±0.154a 5.39±0.098e 730.16±1.690e

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IV
V 28.16±0.249a 0.799±0.020a 12.10±0.216a 6.25±0.082b 731.11±0.847e
VI 28.26±0.249a 0.780±0.007ab 12.13±0.124a 5.81±0.078cd 729.47±0.977e

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VII 28.20±0.216a 0.773±0.008abc 12.20±0.216a 5.62±0.061cde 716.07±0.796f
VIII 28.46±0.418a 0.770±0.007abc 12.10±0.216a 4.46±0.049g 606.32±1.373h
IX
X
27.93±0.249a
28.40±0.216a
0.733±0.018c
0.726±0.010c
-p
12.03±0.262a
12.13±0.188a
4.06±0.075h
3.70±0.059i
602.25±2.489h
590.48±7.562i
28.43±0.169a 0.757±0.011abc 12.10±0.216a 6.70±0.049a 776.09±3.445b
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XII 28.33±0.124a 0.737±0.012bc 12.10±0.294a 5.85±0.036c 765.08±4.326bc
XIII 27.60±0.244a 0.743±0.012bc 12.03±0.262a 5.56±0.047de 752.74±3.094d
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XIV 28.26±0.478a 0.726±0.012c 12.10±0.282a 4.19±0.061h 734.55±3.397e

Note: The different letters in the same column reveal significant differences (P <0.05) among means.
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Table 5
Organoleptic evaluation of whole wheat bread as affected by various enzymes and emulsifiers.
Crust Overall General
Formula Texture Crumb color Flavor Crust color Odor
appearance appearance acceptability
I 3.50±0.548b 3.16±0.408cd 2.66±0.516d 2.83±0.753c 2.66±0.516c 3.16±0.408c 2.83±0.408d 3.00±0.632d
II 4.66±0.516a 4.33±0.516cd 3.16±0.408cd 3.33±0.516bc 3.33±0.516bc 3.66±0.516abc 3.66±0.516bcd 4.00±0.000abc
III 3.16±0.408b 2.66±0.516abc 3.83±0.408abc 4.33±0.516ab 3.83±0.408ab 4.50±0.548a 3.66±0.516bcd 3.50±0.548bcd
IV 3.50±0.548b 3.50±0.548bcd 3.33±0.516cd 4.33±0.516ab 4.16±0.408ab 4.50±0.548a 3.66±0.516bcd 4.00±0.000abc
V 3.83±0.408ab 3.16±0.408cd 3.66±0.816abcd 4.33±0.516ab 3.83±0.753ab 3.50±0.548abc 3.66±0.516bcd 3.83±0.408abcd

f
VI 3.50±0.548b 4.16±0.408abc 3.00±0.632cd 3.00±0.632c 3.33±0.516bc 3.33±0.516bc 3.33±0.516cd 3.16±0.408cd

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VII 4.16±0.408ab 3.33±1.211bcd 3.83±0.408abc 4.00±0.632abc 4.50±0.548a 3.16±0.408c 4.16±0.408abc 4.16±0.408ab
VIII 4.66±0.516 a 3.83±0.753abcd 3.83±0.408abc 4.00±0.632abc 4.50±0.548a 3.66±1.033abc 4.66±0.516a 4.33±0.516ab

pr
IX 4.00±0.632ab 4.83±0.408a 3.50±0.837bcd 3.00±0.632c 4.33±0.516ab 3.66±0.516abc 4.00±0.000abc 4.00±0.000abc

e-
X 4.66±0.516a 4.50±0.548ab 3.50±0.548bcd 4.00±0.632abc 4.66±0.516a 3.66±0.516abc 4.33±0.516ab 4.16±0.408ab
XI 3.50±0.548b 3.33±0.516bcd 4.66± 0.516a 4.66±0.516a 4.16±0.408ab 4.50±0.548a 4.33±0.516ab 4.33±0.516ab

Pr
XII 3.16±0.408b 4.00±0.632abc 3.83±0.408abc 4.33±0.516ab 4.16±0.408ab 4.50±0.548a 3.83±0.408abc 4.16±0.408ab
XIII 3.50±0.548b 3.83±0.753abcd 4.50±0.837ab 4.66±0.816a 4.33±0.516ab 4.33±0.516ab 4.16±0.408abc 4.33±0.516ab
3.66±0.516ab 4.16±0.408abc 4.66±0.516a 4.66±0.516a 4.66±0.516a 4.33±0.516ab 4.33±0.516ab 4.50±0.548a

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Note: The different letters in the same column reveal significant differences (P <0.05) among means.

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a)

100

90 a

80
b
70 b
c
Hardness (N)

c c c c
60 c
50 d d
d d

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d
40

30

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20

10

0
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I II III IV V VI VII VIII IX X XI XII XIII XIV
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b)

14
na

12 ab ab ab ab
bcde abcd abc
cde cde abc
e de e
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10
f
Extensibility (mm)

8
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0
I II III IV V VI VII VIII IX X XI XII XIII XIV

Fig 1. Effect of enzymes and emulsifiers on whole wheat bread’s textural properties: a) Hardness and b)
Extensibility. The different letters reveal significant differences (P <0.05) among means.
 L*
XIV ab
XIII abcd
XII e
XI abc
X ab
IX cd
VIII a
VII abc
VI e
V bcd
IV de
III e

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II cd
I e

ro
50 52 54 56 58 60 62 64 66

a* -p a
re
XIV
a
XIII
a
XII
lP

XI a
X b
IX b
na

VIII b
VII b
VI b
V b
ur

a
IV
a
III
Jo

II b
I b
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
 b*
XIV a
XIII abc
XII a
XI ab
X cd
IX d
VIII bcd
VII d
VI cd
V cd
IV a
III a

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II bcd
I cd

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0 2 4 6 8 10 12 14 16 18

Fig 2. Color parameters of whole wheat bread including enzymes and emulsifiers. The different letters
-p
reveal significant differences (P <0.05) among means.
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Fig 3. Microscopic analysis of whole wheat bread produced by enzymes and emulsifiers.
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 80 a
b b
70

60

50

40
a
30
b b
20

10

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WWF WWB WWB with additves

TPC (µg Gallic acid per g of sample) TAA (%)

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Fig 4. Total phenolic content (TPC) and total antioxidant activity (TAA) in whole wheat flour (WWF),
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whole wheat bread (WWB), and whole wheat bread affected by enzymes and emulsifiers. The different
letters reveal significant differences (P <0.05) among means.
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• The properties of WWB were dramatically affected by supplementation of additives.
• WWB containing xylanase and emulsifier demonstrated higher nutritional value.
• Enriching WWB prevented from the negative effect of phytic acid on gluten network.

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 Conflicts of Interest Statement

Manuscript title:
modification of dough charasteristics and baking qiality based on whole wheat flour by
enzymesa and emulsifiers supplementation

The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any
organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus;
membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing
arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in

of
the subject matter or materials discussed in this manuscript.

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Author names:
zahra sheikholeslami, maryam mahfouzi, mahdi karimi, mahdi ghiafe davoodi

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The authors whose names are listed immediately below report the following details of affiliation or involvement in an
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organization or entity with a financial or non-financial interest in the subject matter or materials discussed in this manuscript.
Please specify the nature of the conflict on a separate sheet of paper if the space below is inadequate.

Author names: zahra sheikholeslami, maryam mahfouzi, mahdi karimi, mahdi ghiafe davoodi
This statement is signed by all the authors to indicate agreement that the above information is true and cor-
rect (a photocopy of this form may be used if there are more than 10 authors):

Author's name (typed) Author's signature Date

zahra sheikholeslami 9/14/2020

maryam mahfouzi 9/14/2020

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mahdi karimi 9/14/2020

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mahdi ghiafeh davoodi -p 9/14/2020
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