Effect of Barley beta-glucan on Gluten Polymerization Process in

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Effect of Barley beta-glucan on Gluten Polymerization Process in Dough during Heat Treatment Zehua Huang, Yang Zhao, Ke-Xue Zhu, xiaona guo, Wei Peng, and Hui-Ming Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02011 • Publication Date (Web): 03 Jul 2017 Downloaded from http://pubs.acs.org on July 5, 2017

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Journal of Agricultural and Food Chemistry

Effect of Barley beta-glucan on Gluten Polymerization Process in Dough during Heat Treatment

Ze-Hua Huang, Yang Zhao, Ke-Xue Zhu*, Xiao-Na Guo, Wei Peng, Hui-Ming Zhou*

State Key Laboratory of Food Science and Technology, Collaborative Innovation Center for Food Safety and Quality Control, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, PR China

Corresponding authors: Prof. Ke-Xue Zhu Fax: +86 510 85329037 Tel: +86 510 85329037 E-mail: [email protected] Prof. Hui-Ming Zhou Fax: +86 510 85329037 Tel: +86 510 85329037 E-mail: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT: Barley (Hordeum vulgare L.) beta glucan (BBG) is of interest due to its

2

health benefits, but BBG presents significant disrupts on gluten network with a negative

3

impact on food texture. To clarify the interaction between BBG and gluten in dough, the

4

dynamic rheological, thermo-chemical process of gluten and micro-structure of dough

5

with BBG during heating were detected. The results showed that BBG delayed gluten

6

thermal polymerization reaction during heating, and affected polymerization of specific

7

molecular weight protein subunits. These impacts depended on heating temperature and

8

time. When heating under 25~65 °C, tanδ of the dough reached the highest level at BBG

9

concentration of 1%. However, under the temperature of 65~95 °C, tanδ was positively

10

correlated with BBG content (0~3%). The DSC curves revealed that peak temperature

11

(TP) of the two endothermic peaks increased by 3.86 °C and 3.10 °C respectively.

12

SE-HPLC analysis showed that BBG mainly affected the peak area of gliadin and

13

glutenin. Furthermore, after added 3% BBG, the bands of 59.8 and 64.9 kDa in

14

SDS-PAGE patterns delayed vanishing for 120 s when heating at 95 °C. Therefore, BBG

15

delayed the polymerization reaction of specific molecular weight protein subunits rather

16

than all the proteins.

17

Key words: Barley β-glucan, dough, rheology, gluten, heating process, protein

18

polymerization


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19

INTRODUCTION

20

Researches on improving human metabolic function through diet control have been

21

heightened in recent years. It is attributable to the increasing incidence of chronic

22

metabolic diseases such as coronary heart disease and type-2 diabetes. Barley beta

23

glucan (BBG) provides a variety of health benefits such as lower serum cholesterol

24

levels

25

of BBG, U.S. Food and Drug Administration 3 and EFSA 4 have approved health claims

26

for BBG. It was found that BBG content increased from outer to inner of barley grain 5,

27

and the highest concentration was in center of barley endosperm 6. Thus, barley flours

28

could be sieved or air-classified to produce fractions with enhanced levels of beta-glucan

29

up to 25% 7. Recently, some simplified and economic feasible methods to extract BBG

30

were introduced 8, these functional and bioactive barley products rich in beta-glucan

31

could be functional ingredients in food industry.

1

and attenuate postprandial blood glucose 2. Because of the functional activities

32

Incorporation of barley in various food products, including bread, pasta and

33

noodles, has been accomplished with moderate success 9. However, adding BBG to food

34

will inevitably lead to some problems, such as damaging the structure of gluten matrix.

35

As a hydrophilic colloid, BBG significantly changed food texture as well as nutritional

36

and functional properties

37

displayed smaller volume and darker color than those made from pure wheat flour 11.

38

The deformation quantity, flowability and viscoelasticity of dough under low stress

39

could be increased by BBG

40

hardness of bread crumbs was reduced by the increasing of BBG level 13.

10

. Bread made from mixed wheat flour with barley powder

12

. While the bread structure became rougher, and the



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41

Previously studies on the processing technology of food production process had

42

been carried out, including optimizing the content of BBG 14, selecting the appropriate

43

BBG varieties

44

and addition of additives (carboxy propyl methyl cellulose, etc.)

45

mechanism of BBG weakening the dough or its derivative products quality was still not

46

fully understood. Some studies trying to explain the influencing mechanism of other

47

dietary fibers in food. Lebesi et al. 18 studied the influence of dietary fiber or bran on the

48

porosity formation in cake, and speculated that dietary fiber increased the viscosity of

49

the batter and thus provided more stable and voluminous cakes. It is well known, the

50

characters of the gluten net are important to the quality of wheat products. Indeed, some

51

dietary fibers might interfere with the formation of the gluten net structure mechanically

52

11,19

53

that, in bread dough, surface layer properties of the dough liquor components were the

54

key parameters in gas retention.

11, 13, 15

, as well as preliminary fermentation on whole barley powder

16

17

. However, the

, and cause the destruction of "gas cell" in the dough 20. Primo-Martin

21

suggested

55

Hydrocolloids can strongly affect the secondary conformation of proteins 22 and the

56

hydration properties of gluten 23. As a kind of hydrocolloids, BBG is tightly bound with

57

large amounts of water in dough. BBG competes with protein for water absorption,

58

which is detrimental to the formation of gluten network, and affects the dough formation

59

time and stability

60

cooking

61

buffer fell sharply, which showed that protein polymerized acutely during cooking

62

process

13

, and further affects the food quality and structure in process of

11

. After cooking, the protein extractability in sodium dodecyl sulfate (SDS)

24

. The initial polymerizing temperature of glutenin and gliadin were different.


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63

First, they formed inter- and intra-chain disulfide bond respectively, and when the

64

temperature

65

sulfhydryl/disulphide interchange reactions

66

polymerization processes might be divided into several segments. But there was little

67

systematic information about interaction between BBG and gluten on the protein

68

thermal polymerization procedure during heating process.

reached

a

higher

value,

they

formed

complexes

through

25

. This process indicated that gluten

69

The objective of this study was to determine the effect of BBG on the

70

physicochemical properties of dough and formation procedure of gluten matrix during

71

heat-treatment. Effects of BBG on the rheological property of dough before or during

72

heating treatment were analyzed. Furthermore, component and molecular weight

73

distribution analysis of protein in the dough with or without BBG were performed with

74

size-exclusion high-performance liquid chromatography (SE-HPLC) and sodium

75

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) respectively, to

76

integrate the procedure of gluten thermal polymerization. Meanwhile, the dynamic

77

thermo-chemical change in the dough were determined by DSC. The fluorescence

78

microscope was also applied in this study to intuitively reveal dynamic progress of

79

BBG-gluten interaction at different temperature.

80

MATERALS AND METHODS

81

Materials Barley flour (13.3% moisture, 67.3% starch, 10.1% protein, 2.7%

82

β-glucan, 1.4% lipids, and 0.96% ash) was obtained from Dafeng Dade barley trade Co.,

83

Ltd. (Yancheng, China). Wheat flour (WF, 13.6% moisture, 70.8% starch, 11.3% protein,

84

0.25% β-glucan, 1.5% lipids, and 0.45% ash) supplied by Keming Noodle



85

Manufacturing Co., Ltd. (Yiyang, China). The other reagents were of analytical grade.

86

Extraction of Barley β-glucan. BBG was extracted using the method given by

87

Kim and Sayar et al 26, 27 with some modifications showed as follow: (1) pretreatment of

88

barley flours with 85% (v/v) ethanol refluxing for 2 h at 85 °C. (2) BBG extraction was

89

carried out by dispersing exactly weighted 100.0 g of pretreated barley flour in 1.0 L of

90

distilled water (pre-heated to 55°C), and the pH of the mixture was adjusted to 7.0 by 2

91

mol/L NaOH and 2 mol/L HCl. The mixture was held at 55°C and stirred mildly for 2h.

92

Supernatant was collected after centrifuged at 7690 × g for 30 min. (3) Contaminating

93

starch was hydrolyzed by thermostable α-amylase (preheated to 95°C and kept for

94

30min, Jiangsu Ruiyang Biotech Co. LTD., Wuxi, China) at 10U/mL, pH 6.5,

95

temperature 95°C for 30min. (4) Contaminating proteins were removed by pancreatin

96

from hog pancreas (Sigma-Aldrich Shanghai Trading Co Ltd, Shanghai, China) at

97

0.05mg/ml, pH4.5, 40°C for 3h. (5) BBG was obtained by precipitating with 95% (v/v)

98

ethanol, then the sediments were washed twice with 95% (v/v) ethanol and freeze-dried.

99

Beta-glucan contents and the molecular weight were determined by AACC method

100

(Approved Method 32-23, AACC 2000) and size-exclusion high-performance liquid

101

chromatography (SE-HPLC) method 28. The contents and molecular weight of purified

102

BBG samples were 91.63% and 1.74×106 Da.

103

Preparation of BBG/WF mixed dough. BBG/WF ratio of the dough was 0/100,

104

0.5/99.5, 1/99, 3/97. Accurately weighted BBG was dispersed in distilled water and

105

stirred mildly in water bath at 85 °C until completely dissolved. After cooling to room

106

temperature (25 °C), the BBG solution was adjusted to a certain volume to make sure


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107

that every dough was prepared at a constant water absorption of 60% (v/w, basis on the

108

total weight of BBG and WF). The dough was obtained by mixing BBG solution and

109

WF in a pin mixer (Beijing Dongfu Jiuheng Instrument Technology Co. Ltd., Beijing,

110

China) at 104±5 r/min for 4.5 minutes. The dough above was prepared for rheological

111

and DSC measurements, and freeze-dried dough was ground into powder for SE-HPLC,

112

SDS-PAGE and fluorescence microscope tests.

113

Rheological Measurements. Rheological properties of wheat dough were

114

measured using a DHR3 rheometer

115

piece was cut from the wheat dough and loaded in the plate and plate geometry

116

(diameter of 40 mm, and gap of 1 mm). The wheat dough was equilibrated for about 5

117

min, before rheological determination. The storage modulus (G′) and loss modulus (G″)

118

and tanδ of the wheat dough were recorded within the linear viscoelastic area of wheat

119

dough. The analysis was performed in duplicate.

120 121

29

(TA instruments, West Sussex, U.K.). A small

(1) The frequency scanning tests were performed with the frequency sweep changed from 0.01 to 10 Hz, at a constant strain of 0.2%.

122

(2) The temperature scanning tests were performed with strain of 0.2%. The edge of

123

the dough piece was covered with silicone oil. The dough piece was heated from 25 °C

124

to different temperature (55, 65, 75, 85, 95 °C) at a rate of 5 °C/min, and held at 95 °C

125

for 0, 60, 180, 300 s respectively. After heated to different level, the dough pieces were

126

freeze-dried and grinded for SE-HPLC, SDS-PAGE and fluorescence microscope tests.

127

Differential Scanning Calorimeter Analysis. Thermal properties of the wheat

128

dough were determined by differential scanning calorimeter (DSC8500, PerkinElmer,



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129

Waltham, MA) in N2 flow after calibration with indium and tin. The measurement was

130

performed according to the method of Sozer

131

equilibrating the dough for 5 min, sample (9~10 mg) was accurately weighed into

132

aluminium sample pans. An empty pan was used as reference. The pans were sealed and

133

heated from 25 to 110 °C at a rate of 10 °C/min. The onset temperature (To), peak

134

temperature (TP), conclusion temperature (Tc), and enthalpy (∆H) were recorded.

135

30

with slight modifications. After

SE-HPLC Analysis. According to the method of Guo

31

and Lagrain

32

, under

136

nonreducing conditions, raw wheat flour and freeze-dried wheat dough samples

137

(containing 1.0 mg protein) were accurately weighted and dispersed in 1.0 mL of sodium

138

phosphate buffer (PBS, 0.02 M, pH 6.8) containing 2% sodium dodecyl sulfate (SDS).

139

After vortex oscillation 30min and centrifugation (10 min, 5220 × g), the supernatant

140

was filtrated over polyethersulfone (Millex-HP, 0.45 µm, polyethersulfone).

141

The SE-HPLC analysis was performed using LC-2010 system (Shimadzu, Kyoto,

142

Japan). A sample of 50 µL was loaded on a TSK G4000-SWXL analytical column

143

(Tosoh Biosep, Tokyo, Japan). The elution solvent was PBS (0.02 M, pH 6.8) containing

144

2% SDS at the flow rate of 0.7 mL/min. The elution curve was monitored at 214 nm, at

145

the column temperature of 30 °C. To calculate the protein extractability in SDS buffer

146

(SDSEP) content, the peak area under the SE profile is integrated with LCSolution Lite

147

software (Shimadzu, Kyoto, Japan).

148

SDS-PAGE Analysis. SDS-PAGE tests were performed according to the method of

149

Wang

150

freeze-dried BBG/WF dough were extracted separately. The extraction buffer was 0.05

33

, included three parts. The non-reduced protein and reduced protein of


Page 9 of 32


151

mol/L Tris-HCl of pH 6.8, containing 10 % (w/v) SDS, 10 % (v/v) glycerol, and 0.1 %

152

(w/v) bromophenol blue. All samples (containing 1.0 mg of protein) were dispersed in 1

153

mL of the extraction buffer and shaken for 30 min. Then the dispersion was centrifuged

154

at 7690 × g for 15 min, non-reduced SDS-extractable protein was contained in the

155

supernatant, which was used in the tests of part (1). Take 0.5 ml of the supernatant, and

156

an equal volume of 4% (w/v) dithiothreitol (DTT) was mixed under the protection of N2

157

with the supernatant for 15 minutes, the mixture was centrifuged and the supernatant

158

was used as reduced SDS-extractable protein in the tests of part (2). For part (3), the

159

residue in part (1) was dispersed in the buffer above of 1 mL with 2% (w/v) DTT added.

160

After shaken and centrifugation again, the reduced SDS non-extractable protein was

161

contained in the supernatant. The protein extracts in (1) ~ (3) were heated for 3 min at

162

100 °C, and then centrifuged for 10 min at 7690 × g.

163

Separating gel (12%, pH 8.8) and stacking gel (5%, pH 6.8) were prepared by

164

premixed gel‐casting buffers of 29:1 Acrylamide/Bis solutions, 4 × Tris-HCL/SDS

165

buffer, pH 8.8 and 4 × Tris-HCL/SDS buffer, pH 6.8 (Sangon Biotech, Shanghai, China).

166

Electrophoresis was performed in a vertical electrophoresis cell (DYCZ-28A, Beijing

167

Liuyi Biotechnology Co., Ltd.), with running buffer of 0.025 mol/L Tris-glycine buffer

168

(pH 8.3), and sample volume of 15 µL for (1) or (3) and 30 µL for (2), and running

169

voltage of 100 V. The molecular weight markers as follows: Rabbit phosphorylase B (97

170

400 Da), bovine serum albumin (66 200 Da), rabbit actin (43 000 Da), bovine carbonic

171

anhydrase (31 000 Da), trypsin inhibitor (20 100 Da), hen egg white lysozyme (14 400

172

Da).



173

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Fluorescence microscopy The images were filmed according to the method

174

described by Silva

175

(CM1850 UV, Leica, German), then stained with Calcoflour White (0.01%, v/v,

176

preferentially stain BBG), and a solution of Fluorescein 5-isothiocyanate (FITC, 0.25%,

177

w/v, preferentially stain starch) and Rhodamin B (0.025%, w/v, preferentially stain

178

protein). The stained samples were observed through a fluorescence microscope (Axio

179

Vert A1, Carl Zeiss Microscopy GmbH, Jena, German) using the LED filter set with

180

excitation/emission wavelengths at 410/455 for Calcoflour White, 488/518 nm for FITC,

181

and 568/625 nm Rhodamin B, respectively. The samples were observed with a 40 ×

182

objective lens coupled with an AxioCam MRC Zeiss camera and Zen 2012 software.

34

and Sikora

35

. The samples were sliced by freezing microtome

183

Statistical Analysis. Significant differences of evaluated parameters among

184

different samples were analyzed with SPSS statistical software (Version 22.0, SPSS Inc.,

185

Chicago, IL, USA), by the method of one-way-analysis of variance (ANOVA). p < 0.05

186

was considered to be significant.

187

RESULTS AND DISCUSSION

188

Effect of BBG on dynamic rheological and thermomechanical properties of

189

dough

190

As shown in Fig. 1a and 1b, both storage (G′) and loss (G″) moduli were increased

191

with BBG. And at all the frequency range examined, the dough showed the elastic

192

behavior. These results were in agreement with previous researches

193

frequency ranges, the tanδ changed positively with the BBG content. While within high

194

frequency range, the tanδ increased to a maximum value at 1% BBG concentration and


13, 36

. At low

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195

then decreased (Fig 1c). These results illustrated that the dough tended to be solid-like

196

behavior when deformed slowly, but liquid-like when sheared at high rate

197

shear rate was slow enough that the dough was always close to mechanical equilibrium

198

37

199

tanδ indicated the formation of soft and sticky dough by adding BBG. Water may be

200

present in dough as large regions of bulk water, dispersed in the protein network as small

201

drop, as well as implanted or surround in the starch granules 38. BBG tightly bind large

202

amounts of water and changed the moisture distribution in the dough

203

competitive adsorption of water on the large regions of bulk water

204

shear rate and content of BBG made the dough more solid-like.

36

. When

, therefore, the tanδ at low frequency ranges implied the dough structure, and the rising

11

. And BBG

36

.Thus, the higher

205

The dynamic rheological Temperature scanning had been shown in Fig. 1d, 1e, and

206

1f. Under 25~55 °C, G′ and G″ of the doughs were slightly lowered. In the temperature

207

ranged from 55~95 °C, G′ and G″ both reached the peak value in the temperature range

208

of 65~75 °C, the tanδ reached maximum value around 65 °C, and then G′, G″, tanδ

209

decreased. These results indicated that protein polymerization and starch gelatinization

210

in the dough started around the temperature of 55 °C.

211

The impact of BBG content on dynamic rheological parameters varied with

212

different heating period. At low temperature, with BBG level increased, G′, G″, tanδ

213

showed a similar trend to the frequency sweep of shear rate at 1 Hz (Fig 1a and 1b).

214

However, when protein polymerization reaction continued, a high strength of dough

215

texture was formed

216

with the control (0%), the dough with BBG was more fluidic, and after heat-treatment

39, 40

.Thus, during 65~95 °C, tanδ was reduced quickly. Compared



Page 12 of 32

217

accomplished, tanδ was positively correlated with BBG level (0.5~3%). An explanation

218

for this result might be that BBG affected the thermo-polymerization behavior of gluten,

219

and damaged food texture.36

220

Effect of BBG on DSC thermodynamical curves of dough

221

As showed in Fig 2, there were 2 endothermic peaks in the DSC curve of the wheat

222

dough at 65 °C and 85 °C, marked as P1 and P2 respectively. In heat-treatment process,

223

dough endothermic peaks indicated that starch gelatinization and protein polymerization

224

at 65 °C and 85 °C

225

addition of BBG of 3%, peak temperature of P1 and P2 were increased, from 65.04 °C

226

and 85.26 °C to 68.90 °C and 88.36 °C respectively. Large amounts of water tightly bind

227

to BBG in dough, reduced the available water amount of protein and starch in dough

228

system, and made it harder for physicochemical reactions to be started

229

possibility was the proportion of components in the dough such as protein was changed

230

due to the addition of BBG. Furthermore, the net formed by BBG intercrossed in the

231

structure of gluten matrix, these changed the route of chemical progresses in the dough

232

system during thermal treatment.

41

, which was consistent with the results of rheological tests. With

36

. Another

233

For further clarifying, the reasons for these phenomena above and the interactions

234

between BBG and protein in the dough, the changes of protein composition and content

235

during heat-treatment were investigated by SE-HPLC and SDS-PAGE tests.

236

Effect of BBG on SDS solubility of proteins during heat treatment

237

The protein extractability in SDS buffer (SDSEP) was a good indicator of protein

238

thermal-polymerization 31 42. As shown in the SE-HPLC curves, compare with the dough


Page 13 of 32


239

without BBG, obvious changes were observed in peak areas of glutenin and gliadin by

240

addition of 3% BBG to the dough, but there was little change in the peak areas of

241

albumin and globulin (Fig. 3). When the temperature was higher than 75 °C, the loss in

242

glutenin extractability in SDS buffer was significant both in dough with and without

243

BBG, while the loss in gliadin extractability in SDS buffer was less significant. The

244

aggregation of protein led to a decrease in the protein extractability in SDS buffer

245

Therefore, the results indicated that glutenin was more sensitive to heat-treatment than

246

globulin. In addition, adding BBG in dough increased glutenin extractability in SDS

247

buffer, but decreased that of gliadin. This phenomenon suggested that BBG showed

248

different effects on glutenin and gliadin during heating processes. It was reported that

249

glutenin was the major components that confer elasticity to the dough

250

impart viscous properties 45, and the amount and properties of the high molecular weight

251

glutenin polymers was related to dough strength. Therefore, a less aggregation of

252

glutenin (less elasticity & lower G′) and more aggregation of gliadin (more viscous &

253

higher G″) made the dough more fluidic. These confirmed the result of rheological tests,

254

which showed BBG weakened the dough and increased the ratio of tanδ (tanδ=G″/G′)

255

after heat-treatment.

43

.

44

, the gliadins

256

Effect of BBG on electrophoretic profiles of proteins in dough

257

To evaluate changes in polymerization of gluten in the heat treatment process,

258

unreduced SDS-PAGE (Fig.4a) and reduced SDS-PAGE (Fig. 4b) of SDS extractable

259

protein in dough of 3% BBG were analyzed. As the temperature increased, the intensity

260

of high molecular weight aggregates bands were found an obvious decrease on the top of



261

separation gel. The profile showed very faint bands that would correspond to high

262

molecular weight glutenin between 43.2 kDa and 117.1 kDa. While bands could be

263

assigned to α-, β- or γ-gliadins of 31.7, 33.1, 34.6, 37.6 kDa were clear, and also clear

264

bands that would correspond to albumin and globulins from 13.9~27.7 kDa (Fig 4a) 22.In

265

the reduced electrophoretic profile (Fig. 4b), we observed dark bands that would

266

correspond to high molecular weight glutenin subunits (HMW-GS, 67~103.2 kDa) and

267

low molecular weight glutenin subunits (LMW-GS, 30~45 kDa) 46. The high molecular

268

weight protein subunits (ranging from 40.4 kDa up to 103.2 kDa) presented a tendency

269

for intensity of bands to decrease with temperature increase. However, whether in the

270

reduced or unreduced SDS extractable protein electrophoretic profiles, the intensity

271

changes of bands from 32.3 kDa to 37.6 kDa in the profiles were not obvious. The

272

different changing trends of high molecular weight subunits and low molecular weight

273

subunits in SDS-PAGE were consistent with the results of the SE-HPLC tests, in which

274

different effect trends of BBG on different proteins were observed during heating.

275

As shown in Fig. 4b, the two bands of 59.2 kDa and 64.2 kDa vanished at the

276

temperature of 95 °C (in parentheses ‐), and similarly, the bands of 42.0 kDa and 43.9

277

kDa vanished in heat-process of 95 °C 180 s (in parentheses ‐). These results would

278

suggest there was a temperature threshold effect in thermal-polymerization reactions of

279

these subunits, when reached critical temperature thermal-polymerization reactions

280

occurred quickly. In Fig. 4c and 4d, the two bands of 59.8 kDa and 64.9 kDa (in

281

parentheses ‐) could be considered as the same protein subunits as in Fig. 4b marked in

282

parentheses ‐ with slight shift. With BBG added, the two bands vanished at 95 °C 180 s


Page 14 of 32

Page 15 of 32


283

(in parentheses ‐), compared with control (in parentheses ‐), the vanished time of the

284

two bands was delayed by 120 s. These phenomena indicated that BBG delayed the

285

protein polymerization.

286

During heat-treatment, the polymerization of protein formed macropolymers, which

287

cannot be extracted in SDS buffer 43. Thus, changes of the polymerization substrate can

288

be reflected through SDS-extractable protein, and changes of polymerization products

289

can be reflected by SDS non-extractable protein. After extracting SDS-extractable

290

protein with SDS buffer, the SDS non-extractable protein was contained in the residue.

291

And a part of high aggregated protein could be extractable again when the residue was

292

treated by DTT. Though thermo-polymerization in the dough system mainly occurred

293

between glutenin and gliadin through disulfide bond

294

formed through non-disulfide bonds could not be extracted by SDS buffer. As in Fig. 4c

295

and 4d, with the increase of temperature, the intensity of the protein bands increased first

296

and then decreased. These suggested that during heating treatment, disulfide

297

polymerization occurred first, as heating temperature and time increase, and then

298

non-disulfide polymerization formed, which were possibly resulted in the decrease of

299

the intensity of bands

300

incorporation of BBG in dough, followed by the change of polymerization reaction

301

product and protein net structure, finally affected the senses of food. Furthermore, since

302

the polymerization of specific protein subunits was delayed by BBG, if these protein

303

subunits could be removed or modified, the negative effects of BBG on food texture

304

might be avoided.

25

, however, the macropolymers

31

. This protein polymerization procedure was delayed by the



305 306

Fluorescent photomicrographs analysis of different heat-treatment segments of dough with or without BBG

307

In the fluorescent photomicrographs of the dough, the blue zones represented BBG,

308

the green zones represented starch, and the red zones represented protein. The

309

multicolored image on the right side of each row was the combination of the three single

310

colored images, in which yellow for starch-protein overlap, and purple for BBG-protein

311

overlap.

312

At 25 °C, the starch was not swelled with clear particle outlines. The shape of

313

gluten was like cloud without net structure, while BBG distributed as dots. With the

314

rising of temperature, starch was getting to swell at 55~75 °C, the volume of starch

315

granules expanded, and the outlines of starch granules obscured. The punctate BBG

316

turned to forming reticular structure, the melting process of BBG gel was occurred.

317

Meanwhile, the network structure of protein was built by thermal polymerization

318

preliminarily. When the temperature continued rising to 95 °C and maintained for 300 s,

319

the starch particles swelled and pasted furtherly. The net of BBG and gluten began to

320

interpenetrate, and in the multicolored image, the purple area stood for the compound

321

net of gluten and BBG gel could be clearly observed.

322

This suggested that, before heated, due to the strong water bonding capability, BBG

323

absorbed plenty of free water and formed agglomerate gel particles in the gaps between

324

protein and starch granules. Meanwhile, BBG, starch and protein took differentiated

325

zones in the dough, without overlap. This could be seen from the multicolored image, in

326

which blue, green and red zones were independent of each other. When at the


Page 16 of 32

Page 17 of 32


327

heat-process of 55~75 °C, yellow area appeared in the multicolored image clearly,

328

suggesting that starch and protein overlapped in more zones. This was due to the initially

329

pasted starch encroached in the net of gluten and BBG gel. Finally, after the

330

heat-treatment, gluten and BBG formed complex network, wrapped gelatinized starch,

331

all the materials formed the food texture.

332

In conclusion, according to the results of SDS-PAGE, SE-HPLC and DSC, BBG

333

delayed protein thermal polymerization reaction during heating, and affected

334

polymerization of specific molecular weight protein subunits. These impacts depended

335

on heating temperature and time. This conclusion was supported by the results of

336

dynamic rheological parameters and flourescence micrographs. Since protein

337

polymerization reaction in dough were hindered by BBG, wheat product quality could

338

be improved by optimizing processing technology and formulation. For example,

339

superior heating methods could be used to promote protein thermal-crosslinking.

340

Meanwhile, natural gluten fortifiers such as vitamin C, lipoxidase, glucose oxidase etc.

341

could also be compounded to enhancing gluten structure. Then, the enormous health

342

benefits of BBG could be popularized in wheat products with satisfying flavor and

343

texture.

344

Abbreviations Used

345

BBG, barley beta-glucan; DSC, Differential Scanning Calorimeter; TP, peak temperature;

346

SE-HPLC, size-exclusion high-performance liquid chromatography; SDS-PAGE,

347

sodium dodecyl sulfate–polyacrylamide gel electrophoresis; EFSA, European food

348

safety authority; WF, wheat flour; G′, storage modulus; G″, loss modulus; PBS, sodium



349

phosphate buffer; SDS, sodium dodecyl sulfate; SDSEP, protein extractability in SDS

350

buffer; DTT, dithiothreitol.

351

Corresponding Authors

352

*Fax:

+86 510 85329037; Tel: +86 510 85329037; E-mail: [email protected]

353

*Fax:

+86 510 85329037; Tel: +86 510 85329037; E-mail: [email protected]

354

Funding

355

This work was financially supported by the National Natural Science Foundation of

356

China (Grant No. 31571871), Qing Lan Project, and the National Key Technology R&D

357

Program (Grant No.2013AA102201) and the Jiangsu province "Collaborative

358

Innovation Center for Modern Grain Circulation and Safety" industry development

359

program.

360

Notes

361

The authors declare no conflicts of interest.

362

Acknowledgements

363

The authors gratefully acknowledge Dr. Kun Yu, Dr. Jin-Rong Wang for their valuable

364

comments. Furthermore, we want to express our gratitude to Dr Abdellatief Sulieman

365

and Amr M. Bakry for improving the use of English in the manuscript.


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FIGURE CAPTIONS Fig. 1. Dynamic rheological (a, b, c) and thermomechanical (d, e, f) curves of wheat dough with or without BBG at different frequency and heating time. Labels presented in the graphics represent different substitution levels of BBG and the heating rate was 5 °C/min from 25 °C to 95 °C, held at 95 °C for 0, 60, 180, 300 s respectively.

Fig. 2. Effect of BBG on the thermal properties of dough during heating process, the lowercase letters indicate significant differences of the peak temperature in the inset graph marked P1 and P2 (p

Effect of Barley beta-glucan on Gluten Polymerization Process in

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