Further interpretation of the underlying causes of the strengthening effect of alkali on gluten and noodle quality: Studies on gluten, gliadin, and glutenin

Journal Pre-proof Further interpretation of the underlying causes of the strengthening effect of alkali on gluten and noodle quality: Studies on gluten, gliadin, and glutenin Chuanwu Han, Meng Ma, Man Li, Qingjie Sun PII:

S0268-005X(19)31690-X

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105661

Reference:

FOOHYD 105661

To appear in:

Food Hydrocolloids

Received Date: 27 July 2019 Revised Date:

27 December 2019

Accepted Date: 12 January 2020

Please cite this article as: Han, C., Ma, M., Li, M., Sun, Q., Further interpretation of the underlying causes of the strengthening effect of alkali on gluten and noodle quality: Studies on gluten, gliadin, and glutenin, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/j.foodhyd.2020.105661. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Statement Chuanwu

Han:

Investigation,

Formal

analysis,

Writing-Original

Writing-Review & Editing. Meng Ma: Validation, Investigation, Methodology, Formal analysis. Man Li*: Software, Data Curation, Conceptualization, Funding acquisition. Qingjie Sun: Validation, Supervision.

Draft,

Graphic Abstract

Glutenin

Gliadin

S-S bond

Alkali (K2CO3) treated Hydrophobic interaction

negative charge

1

Further interpretation of the underlying causes of the strengthening

2

effect of alkali on gluten and noodle quality: Studies on gluten,

3

gliadin, and glutenin

4

Chuanwu Han 1a, Meng Ma 1ab, Man Li a*, Qingjie Sun a

5

a

School of Food Science and Engineering, Qingdao Agricultural University, Qingdao,

6

266109, Shandong Province, PR China

7

b

8

Beltsville Agricultural Research Center, United States Department of

Agriculture-Agricultural Research Services, Beltsville, 20705, United States

9

1

Equally-contributing authors

10

*

11

Tel: +86 532 88030448;

12

Fax: +86 532 88030449;

13

E-mail: [email protected] (M., Li)

Corresponding author

1

14

Abstract

15

Alkali significantly enhanced gluten strength and noodle texture. To further

16

understand the underlying mechanisms of the gluten strengthening effect of alkali, the

17

macroscopic rheological properties, microstructure, intermolecular interactions, water

18

mobility, molecular weight distribution (MWD) and structure, and the molecular

19

chain morphology changes of gluten and its subfractions (glutenin and gliadin) were

20

separately investigated. Alkali increased the G' and G'' of gluten and glutenin fractions.

21

Scanning electron microscopy (SEM) images confirmed that alkali induced a more

22

compact structure in all fractions and a membrane-like structure in gluten and glutenin.

23

Quartz crystal microbalance with dissipation (QCM-D) results demonstrated that

24

alkali promoted alkali/protein-protein interactions in gluten and glutenin fractions.

25

Hydrophobic interactions and water-solids interaction were enhanced by alkali in all

26

fractions. Glutenin fraction was shown to play a key role in the protein polymerization

27

of fresh gluten samples in the presence of alkali, while both glutenin and gliadin

28

contributed to the enhanced polymerization during cooking. Atomic force microscopy

29

(AFM) images showed that alkali induced remarkable aggregations of protein

30

molecular chains in gluten system.

31

Keywords:

32

interactions, QCM-D adsorption

alkali,

gluten

subfractions,

2

molecular

structure,

intermolecular

33

1. Introduction

34

The staple food of oriental food culture is generally rice, steamed bread, and

35

noodles (Fu, 2008). Wheat-based noodles have been popular with Asians for

36

thousands of years. Approximately 40% of wheat in China is used for various types of

37

noodle production (Li, Sun, Han, Chen, & Tang, 2018). Now, noodles are a staple

38

food, second only to bread worldwide.

39

Noodle dough is a complex system with proteins, starch, lipids, and additives.

40

And the rheological properties of the dough and the textural characteristics of the

41

noodles determine the quality of the final products. According to the presence or

42

absence of alkaline salts or regular salts, wheat flour noodles can be divided into two

43

categories, known as yellow alkaline noodles and white salted noodles, respectively

44

(Fu, 2008). White salted noodles have been developed in northern China, and the

45

addition of alkaline salts seems to have originated in the south of China. Between

46

them, the alkaline salts have a more significant influence on the color change, flavor

47

and texture improvement of noodles (Rombouts, Jansens, Lagrain, Delcour, & Zhu,

48

2014).

49

Shiau & Yeh (2001) found that alkali can increase the storage modulus and

50

tensile strength of noodles. Fu (2008) reported that alkali can increase the water

51

absorption and texture properties of noodles. Our previous research found that alkali

52

can significantly enhance the stability and resistance of wheat dough as well as the

53

hardness and springiness of cooked noodles, and indicated that these macroscopic

54

quality changes were significantly related with the structural and molecular changes 3

55

of the wheat gluten (Li et al., 2018). So, we assumed that gluten and its subfractions

56

(gliadin and glutenin) may play an important role in the quality enhancement of wheat

57

dough and fresh noodles induced by alkaline salts.

58

The glutenin is a macromolecule formed by disulfide bonds of polypeptide

59

chains and has a relatively broad molecular weight distribution. The gliadin is a

60

single-chain protein formed by hydrophobic polypeptides through disulfide bonds

61

inner the molecule, and its molecular weight is relatively lower (Wrigley, 1996). Both

62

glutenin and gliadin can influence dough stability and noodle texture (Barak, Mudgil,

63

& Khatkar, 2013). The gluten network structure formed by these two proteins makes

64

the dough and noodles viscoelastic; the macromolecular glutenin polymer imparts

65

gluten elastic properties, while the monomeric gliadin imparts gluten viscosity

66

properties (Gianibelli, Larroque, MacRitchie, & Wrigley, 2001). Shiau et al. (2001)

67

suggested that alkali increased the tensile strength and cutting force of extruded

68

noodles by inducing the interchange of sulfhydryl group and disulfide bond,

69

indicating the aggregation of gluten protein may play an important role in the quality

70

of the final products. In a recent report, Deleu, Lambrecht, Vondel, & Delcour, (2019)

71

introduced the effect of alkaline conditions on the chemical cross-links of gluten

72

protein model system, which indicated that wheat gliadins lack free SH groups,

73

dehydroalanine-derived cross-links during heating at alkaline pH after β-elimination

74

reaction of intramolecular SS bonds can occur. In addition, SS bond formation

75

through sulfhydryl oxidation or SH-SS exchange reactions could be favored under

76

alkaline conditions. Our previous study also found that the addition of alkali can form 4

77

a more closed gluten network structure mainly due to the presence of

78

disulfide/sulfhydryl exchange in the noodle system (Li et al., 2018). However, the

79

internal causes underlying the gluten strengthening effect of alkali are still unknown.

80

The rule of dynamic changes of gluten is the premise and basis for accurately

81

controlling the process and extent of its formation. Based on the above analysis and

82

our previous studies, this study further focuses on the key component of gluten as

83

well as its subfractions (glutenin and gliadin), aiming at answering the key question of

84

how are the gluten network and noodle texture gradually formed in the presence of

85

alkali, based on the insight into the behaviors of protein molecular structure and

86

conformation changes, morphology of molecular chains of protein, GMP particle size

87

distribution, as well as the protein molecular interaction forces and water-solids

88

interactions in gluten and its subfractions. We speculate that alkali may have different

89

impacts on glutenin and gliadin subfractions at these levels, which can contribute to

90

the changes in macroscopic qualities of gluten and cooked noodles to different

91

degrees.

92

2. Materials and methods

93

2.1. Materials

94

Wheat gluten was manufactured by the Binzhou Zhongyu FOOD CO. LTD

95

(Binzhou, China) with contents of protein, fat, carbohydrate, and sodium of 80.6, 0.8,

96

12.5, and 0.101 g/100 g gluten, respectively. The ratio of glutenin to gliadin in gluten

97

is 0.91. Xiangxue wheat flour was manufactured by China Oil and Foodstuffs

98

Corporations (Beijing, China) with contents of carbohydrates, protein, and fat of 5

99

73.80, 11.90, and 1.32 g/100 g flour, respectively. All chemicals and reagents used

100

were of analytical grade. Based on our previous experiments on Na2CO3 and K2CO3

101

(Li et al., 2018), and in response to the call for a low sodium diet, K2CO3 was used as

102

the alkaline salt in this study.

103

2.2. Textural analysis

104

The fresh noodles were made using our previously reported method (Li et al.,

105

2018). K2CO3 was first dissolved in water. Fresh noodles were initially cut into

106

strands of 20 cm, and the noodles were cooked to the optimal cooking time. The

107

textural properties of uncooked and cooked noodles were measured using a

108

TA-XTplus Texture Analyser (Stable Micro Systems, London, England). The cooked

109

noodles were measured after 10 min of cooking at 25 °C. Tensile strength was

110

obtained using A/SPR probe at optimal test conditions as follows: initial distance, 50

111

mm; tensile distance, 100 mm; test speed, 2 mm/s. Maximum shear force was

112

obtained using A/LKB probe at optimal test conditions as follows: strain, 75%; test

113

speed, 1 mm/s; induction force, 5 g.

114

2.3. Extraction and separation of gliadin and glutenin

115

Gliadin fraction was extracted from 20 g of gluten with 70% ethanol (400 mL),

116

and placed in a magnetic stirring hot plate at 37 °C for 4 h. The beaker mouth should

117

be sealed with plastic wrap to prevent alcohol volatilization. The stirred mixture was

118

centrifuged at 9500 rpm for 15 min at room temperature (The resulting sediment was

119

further extracted twice with 70% ethanol solution). Combined supernatants after

120

rotary evaporation (gliadin) and sediment (glutenin) were freeze-dried and milled. 6

121

2.4. Dynamic rheological measurements

122

To ensure the complete hydration of gluten fractions, gluten and gliadin samples

123

with alkali (0.5%, 2%) were hydrated with water at 5:3 (w/v) while glutenin samples

124

were hydrated with water at 4:6 (w/v) and reshaped to disks, relaxed for 10 min at

125

25 °C. Samples for dynamic rheological measurements were performed on a

126

controlled stress rheometer (MCR102, Anton Paar, Austria) at 25 °C at the frequency

127

range of 0.1-100 Hz. The gluten/glutenin dough and gliadin slurry were placed

128

between the plates (40 mm diameter, 2 mm gap) and the edge of the sample was

129

coated with a thin layer of paraffin oil to avoid drying during testing. Stress sweep

130

tests at 1 Hz frequency (25 °C) were applied to determine the linear viscoelastic zone

131

(Hu, Wang, & Li, 2017). The sample was allowed to relax for another 5 min during

132

the loading process before starting the measurement. The storage modulus (G′), loss

133

modulus (G′′), damping factor (tanδ (G′′/G′)) data of samples were recorded.

134

2.5. Scanning electron microscopy (SEM) analysis

135

The microstructures of cross-section of gluten (or gliadin, or glutenin) samples

136

were obtained using a scanning electron microscope (JEOL 7500F, Japan) at an

137

accelerating voltage of 2 kV. The sample was soaked in 2.5% glutaraldehyde solution

138

overnight before testing and then rinsed with cold phosphate buffer (0.1 mol/L) for

139

four times (Ma et al., 2019). The lyophilized sample was adhered to the specimen

140

holder with conductive adhesive, and a layer of gold particles was homogeneously

141

coated three times (10 min each). All microstructure of samples was observed at 600×

142

magnification. 7

143

2.6. Zeta potential analysis

144

Zeta potential of gluten, glutenin, and gliadin samples was tested with a

145

commercial Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, U.K.) at

146

25 °C. The sample suspensions were prepared with a solid content of 0.1% and tested

147

using the method described by Chen et al. (2018) to obtain the Zeta potential value.

148

Three runs were carried out for each measurement.

149

2.7. Q-Sense analysis

150

The interaction of gluten and its subfractions with the alkali was measured using

151

a quartz crystal microbalance with dissipation (QCM-D, QE401-F1719, Q-sense,

152

Biolin Scientific, AB, Finland) equipped with one measuring chamber. All

153

experiments were carried out at room temperature using a gold-coated quartz sensor at

154

a constant flow rate of 100 µL/min. The protein samples were diluted to 0.1 mg/mL

155

with 0.5 M acetic acid solution. First, the acetic acid solution was passed 10 mins to

156

obtain a stable baseline, the solution of control and alkali-treated samples was

157

separately added to the measuring chamber to obtain the adsorption curves of samples.

158

The normalized frequency (∆F) and the energy dissipation (∆D) were obtained and

159

the mass and thickness of the adsorbed sample were calculated by Q-sense Dfind

160

software.

161

2.8. Fluorescence spectroscopy analysis

162

Extrinsic emission fluorescence spectroscopy of all samples was determined

163

according to the method of Wang, Zou, Gu, & Yang (2018) using an F-2500

164

fluorescence spectrometer (Hitachi, Tokyo, Japan). The sample (100 mg) was 8

165

dissolved in 20 mL 0.5 M acetic acid solution at 20 °C for 2 h and was centrifuged at

166

10,000 g for 15 min. The supernatant was diluted to 1 mg/mL with the above acetic

167

acid solution. The test conditions were as follows: excitation wavelength, 280 nm;

168

emission wavelength, 290-410 nm; slit width, 5 nm.

169

2.9. Measurement of surface hydrophobicity

170

Surface

hydrophobicity

(So)

of

all

samples

was

determined

using

171

8-Anilino-1-naphthalenesulfonate (ANS) as the fluorescence probe (Gulati et al.,

172

2017). The sample solution was prepared as stated in 2.8 section, and then diluted into

173

several different concentrations. Then 50 µL of ANS solution (8 mM in 0.1 M

174

phosphate buffer, pH 5.8) was added to 10 mL sample solutions and incubated for 20

175

min in dark. The fluorescence intensity was obtained at an excitation wavelength of

176

390 nm and an emission wavelength of 470 nm, and the slit width was set as 5 nm.

177

The initial slope of fluorescence intensity versus protein concentration plot was used

178

as an index of So. Measurements were performed in triplicate.

179

2.10. LF-1H NMR analysis

180

Low-field 1H nuclear magnetic resonance measurements of the samples were

181

performed with a 23 MHz NMR analyzer (NMI20-040V-I, Niumag Co., Ltd., Suzhou,

182

China). The sample formula consisted of 4 g of sample powder and 6 mL of distilled

183

water. Alkali was first dissolved in water. The sample-water mixture was prepared by

184

mixing (stirring by hand with a glass spike) and sealed with preservative film to

185

prevent evaporation and sticking to the NMR glass tube (25-mm diameter) during the

186

experiments (Ritota, Gianferri, Bucci, & Brosio, 2008). The transverse relaxation time 9

187

(T2) was measured by the Carr-Purcell-Meiboom-Gill (CPMG) sequence at 32 °C.

188

2.11. Size exclusion-HPLC analysis

189

Size exclusion-HPLC analysis of samples with or without cooking was

190

performed using an LC system (LC-20AT, Shimadzu, Kyoto, Japan) equipped with a

191

UV detector. For cooking process, the hydrated samples (4 g) were placed in a sealed

192

bag and placed into boiling water bath for 1, 2, and 4 min, respectively. All the

193

samples were freeze-dried and ground. The lyophilized samples (1.5 mg for

194

unreduced profiles and 1mg for reduced profiles) were dissolved in 1 mL sodium

195

phosphate buffer (50 mM, pH 7.0) consisting of 1% SDS (w/v). The sample was

196

vortexed for 20 mins, centrifuged at 10,000 rpm for 10 minutes, and the supernatant

197

was collected and filtered through a 0.45-µm micropore filter. A 10-µL supernatant

198

was injected into a TSK G4000-SWXL analytical column (Tosoh Biosep, Japan) and

199

eluted with sodium phosphate buffer mentioned above (The flow rate was 0.7

200

mL/min). All samples were tested for signal intensity at 214 nm at 30 °C. The

201

solubility and protein molecular weight distribution were calculated from the peak

202

areas (Veraverbeke, Larroque, Békés, & Delcour, 2000). The reducing sodium

203

phosphate buffer contained 1% dithiothreitol.

204

2.12. Glutenin macropolymer (GMP) particle size distribution analysis

205

GMP was isolated by gluten in 1.5% SDS solution (The ratio of gluten to SDS

206

solution is 1:20) and centrifuged at 12,000 g for 30 min at 25 °C as described by Don,

207

Lookhart, Naeem, MacRitchie, & Hamer (2005). A gel layer (GMP) of 1 g was

208

collected from the precipitate and dispersed in a 10-mL 1.5% SDS solution (Liu et al., 10

209

2017), and vortexed for 30 min to form a homogenous opalescent suspension, and the

210

particle size distributions of GMP was measured by an S3500 Bluewave Particle Size

211

Analyzer (Microtrac, Montgomeryville, PA, USA). The instrument parameters setting:

212

refractive index 1.5, flow rate 55%.

213

2.13. Atomic force microscopy (AFM) analysis

214

The pulverized lyophilized samples (1 mg) was dissolved in 0.5 M acetic acid

215

solution, vortexed for 10 min, and centrifuged at 10,000 rpm for 10 min. The

216

supernatant was diluted with the above acetic acid solution to prepare a protein

217

solution with a concentration of about 0.01 µg/mL. A 10-µL of solution was deposited

218

on a freshly cleaved mica substrate. The substrate was placed in a controlled

219

environment and quickly air-dried for 3-5 min to evaporate the solvent (Zhao et al.,

220

2013). AFM images were obtained using a Nanoscope atomic force microscope and

221

the microscope (SPM-9700, SHIMADZU Corp., Japan) was operated in phase mode,

222

the size of the sample stage is 125 µm.

223

2.14. Statistical analysis

224

Statistical analysis was carried out using SPSS 20.0 (SPSS Inc., Chicago, USA).

225

Analysis of variance (ANOVA) was used to determine significant differences between

226

the results and Duncan’s test was used to compare the means with a significant

227

difference at the level of P < 0.05.

228

3. Results and discussion

229

3.1. Effect of alkali on the texture properties of uncooked and cooked noodles

230

Fig. S1 shows the textural parameters of uncooked and cooked noodles enhanced 11

231

by alkali. Alkali significantly increased the tensile strength and maximum shear force

232

of both uncooked fresh noodles and cooked noodle samples (Fig. S1a, b), indicating

233

the enhancement in gluten strength and noodle hardness. Compared with the addition

234

of 0.5% K2CO3, 2% K2CO3 induced a slightly decreased (P > 0.05) tensile strength

235

value for both fresh and cooked noodles, demonstrating that excess alkali addition

236

may not further improve gluten strength and noodle texture. These results were

237

consistent with the conclusion reported by our previous study (Li et al., 2018).

238

3.2. Effect of alkali on the rheological properties of gluten, gliadin, and glutenin

239

fractions

240

The rheological properties reflect the viscoelastic properties of the sample, and

241

the dynamic rheological properties of the gluten component determine the quality of

242

dough and wheat products. In general, gliadin has viscosity and extensibility, and

243

glutenin provides elasticity and strength (Shewry, Tatham, Forde, Kreis, & Miflin,

244

1986). The storage modulus (G') represents the elastic nature of sample and the loss

245

modulus (G'') represents the viscous nature of sample.

246

As shown in Fig. 1, both the G' and G'' of the samples increased with frequency.

247

For gluten sample, the G' was higher than G'', which indicated that the sample was an

248

elastic soft solid. For glutenin sample, G' was much larger than G'', and G'' remained

249

in a low value, showing solid-like behavior. On the contrary, G'' was higher than G' in

250

the gliadin samples, showing liquid-like behavior. The G' value of glutenin sample

251

was lower than that of gluten sample, this may be due to the addition of more water.

252

When the alkali was added, the G' significantly increased for gluten and glutenin 12

253

samples, but showed no significant influence on the gliadin sample. Alkali led to a

254

stronger gluten dough with more solid-like behavior, indicating that the alkali may

255

play an important role in rheological properties by enhancing physical cross-linkings

256

and the free sulfhydryl/disulfide exchange. These results also indicated that alkali

257

treatment has a greater impact on the rheological properties of the glutenin subfraction

258

and this was more likely to be the cause of the changes in macroscopic quality of the

259

dough.

260

In addition, the impact of ethanol extraction procedure on the rheological

261

properties of gluten protein (without separating the supernatant and precipitate) was

262

also investigated and the results showed that both G' and G'' were increased after

263

treated with ethanol (Fig. S3 A, B). This would not be thoroughly discussed here as it

264

does not interfere with the results intending to obtain in this study.

265

3.3. Microstructure changes of gluten, gliadin, and glutenin fractions

266

SEM provides some information on the physical properties of the food

267

components by images of microstructure. According to Wang et al., (2014), gluten

268

protein forms a stable three-dimensional network structure with viscoelastic

269

properties after hydration. The cross-section of all samples was observed using SEM

270

at magnifications of 600. As shown in Fig. 2, porous network structures of all the

271

control samples were formed, and with increasing alkali contents, the number of pores

272

decreased and the degree of network density increased, inducing a more closed

273

network structure. Meanwhile, excess alkali led to a membrane-like structure for

274

gluten and glutenin samples, and the pores almost disappeared. With respect to the 13

275

gliadin samples (Fig. 2C), homogeneous small pores were still observed even after the

276

addition of the 2% of alkali. These changes showed that alkali promoted the formation

277

of a strong gluten network, and glutenin subfraction may contribute more to the shape

278

and strength of the network.

279

3.4. Zeta potential analysis

280

Zeta potential is the potential of charged particles in solution, related to the

281

amount of surface charge on the protein, which could be used to verify the

282

electrostatic interactions in the system. The high absolute value of zeta potential

283

indicates that the protein molecules in the system have more surface charges and

284

strong electrostatic interactions (Chen, et al., 2018).

285

With the addition of the alkali, the surface charges of the three factions gradually

286

changed from positive to negative (Fig. 3). Meanwhile, the absolute value of charge

287

significantly increased as the amount of alkali increased, indicating that a certain

288

amount of alkali can enhance the electrostatic interaction between protein components,

289

which is usually related to changes in the pH environment. As shown in Tab S1, with

290

increasing alkali addition from 0% to 0.5% and 2%, pH values of the gluten

291

suspensions increased from 5.24 to 5.72 and 7.60; pH of glutenin suspensions

292

increased from 5.69 to 6.54 and 7.59, while for gliadin samples from 5.66 to 6.31 and

293

7.90, respectively. These changes in pH value and absolute surface charge indicated

294

that alkali promoted the electrostatic interactions of all the samples. And the surface

295

charge of the gliadin fraction was more sensitive to alkali and may be the key fraction

296

in enhancing the electrostatic interactions of gluten proteins. In addition, changes of 14

297

the absolute value of surface charge of all samples with 0.5% alkali was not obvious,

298

but it also significantly enhanced dough stability (Li et al., 2018) and noodle strength

299

(Fig. S1) and led to a more developed gluten network (Fig. 2), which also indicated

300

that the electrostatic interaction was not the only mechanism in enhancing the gluten

301

strength by alkali.

302

3.5. Q-sense analysis

303

Quartz crystal microbalance with dissipation (QCM-D) can reflect the interaction

304

of molecules by mass changes. A film can form as the continuous adsorption of

305

proteins onto the gold surface of QCM-D, which can be represented by changes in

306

mass and thickness. The adsorption processes of samples were investigated at the

307

same pH value (1.9) and protein concentration. Fig. 4 shows the curves of mass

308

changes as a function of time. The adsorption curve consists of two processes, one is

309

the process of sample adding, and the other is the rinsing process. It can be seen from

310

Fig. 4 that there was no significant change in the sample adsorption curve after the

311

rinsing procedure, indicating that the adsorption process was irreversible adsorption.

312

This irreversible adsorption was caused by the interactions between the surface of the

313

protein and the gold-coated quartz sensor, such as electrostatic attraction, and could

314

not be removed during the rinsing step (Kim, Weber, Shin, Huang, & Liu, 2007).

315

The frequency is the embodiment of the vibration rate of the gold-coated quartz

316

sensor. The decrease in frequency indicated that the vibration rate of the gold-coated

317

quartz sensor was reduced, which was reflected by the increase in adsorption weight.

318

The increase in adsorbed mass indicated an increase in molecular interactions 15

319

between samples. Protein-gold surface interaction predominated during initial protein

320

adsorption, followed by protein-protein interactions becoming important and slowing

321

down the adsorption process as surface coverage increased. Cantarutti et al. (2018)

322

also explained the same adsorption mechanism. After washing with acetic acid

323

solution, for gluten samples (Fig. 4A), the samples with 0.5% and 2% alkali added

324

had an adsorption mass of 440 and 530 ng per cm2, respectively, which were

325

significantly higher as compared with the control (370 ng per cm2). The glutenin

326

subfraction (Fig. 4B) also showed the same trend, the adsorption mass increased

327

(from 320 ng per cm2 of the control) to 407 and 480 ng per cm2 respectively for 0.5%

328

and 2% alkali samples. However, there was no significant mass change in the gliadin

329

samples (Fig. 4C). These results indicated the enhanced alkali-protein and

330

protein-protein interactions, this promotion effect was mainly manifested in glutenin

331

fraction, and the interaction between glutenin and gliadin fraction may further

332

enhance this effect (gluten system).

333

3.6. Fluorescence spectroscopy analysis

334

The intrinsic fluorescence spectrum provides valuable information about the

335

microenvironments of fluorescent amino acid (mainly due to the tryptophan residues),

336

which can be used as a sensitive indicator to characterize proteins based on their

337

conformation, dynamics, and intermolecular interactions (Wang et al., 2017). Fig. 5A

338

shows the alkali induced fluorescence spectrum changes of gluten, glutenin, and

339

gliadin samples, the samples were investigated at the same pH value (1.9). The

340

fluorescence emission maximum around 335-338 nm was conferred by tryptophan 16

341

residues located in hydrophilic area of proteins, which suggested that most of the

342

tryptophan residues in the gluten, glutenin, and gliadin were located in a polar

343

environment (Stanciuc, Banu, Bolea, Patrascu, & Aprodu, 2018). Moreover, the

344

intensity of the fluorescence emission peak for all the samples treated with 0.5% and

345

2% alkali decreased. This result showed that the tryptophan residues were buried or

346

masked upon the folding process because of protein aggregation caused by the alkali

347

treatment.

348

3.7. Surface hydrophobicity (So) analysis

349

ANS as a hydrophobic fluorescent probe that specifically binds to the exposed

350

hydrophobic regions in the sample. Surface hydrophobicity (So) was used as a probe

351

for protein conformational changes, indicating differences in the aggregation and

352

folding of protein molecules under different processing conditions (Li, Zhu, Zhou, &

353

Peng, 2012). This value can characterize the hydrophobic interaction of the samples.

354

As shown in Fig. 5B, compared with the control, the So value of all alkali treated

355

samples significant decreased, indicating a decrease in the hydrophobic regions of the

356

sample. This phenomenon indicated that alkali treatment resulted in the

357

polymerization of the protein, encapsulating the hydrophobic regions inside. In

358

addition, the So of the gliadin sample was higher than that of other samples because

359

the gliadin fraction was highly hydrophobic (Wang et al., 2014).

360

3.8. LF-1H NMR spectroscopy

361

The T2 relaxation time represents the diffusion and chemical exchange process

362

between water molecules and biopolymers (like gluten protein in this system) or other 17

363

solutes (Ritota et al., 2008). T2 relaxation time also exhibits multi-component

364

behavior, in which individual components can be interpreted as different waters

365

domains (Assifaoui, Champion, Chiotelli, & Verel, 2006). In general, the length of the

366

relaxation time can represent the strength of the interaction between water and solids

367

(short times represent a close bond between water and solids, and also demonstrate

368

strong water-solid interaction), and these interactions can affect the food stability. Sai

369

Manohar & Haridas Rao (2002) found that the rheology and machinability of the

370

dough were affected by the water distribution.

371

In our study, the water distribution in gluten, glutenin, and gliadin after alkali

372

treatment was investigated based on the same water content, and the T2 relaxation

373

time spectrums are shown in Fig. 6A, B, and C. The T2 relaxation times of all

374

alkali-treated samples were left shifted, which should be assigned to the lower

375

mobility, indicating that the alkali enhanced the interaction of water molecules and

376

protein (Kontogiorgos, Douglas Goff, & Kasapis, 2008). The T2 relaxation time range

377

of the gluten sample was 1.65-34.28 ms, and the T2 range of glutenin sample was

378

0.16-32.14 ms. The gliadin sample was shown with a higher T2 relaxation time range

379

of 4.64-65.34 ms, which may be due to the high hydrophobicity of gliadin. This was

380

also consistent with the results of surface hydrophobicity; high hydrophobicity

381

indicated more hydrophobic moieties were exposed, resulting in higher water mobility

382

(Wang et al., 2014). These results indicated that the strong interactions of solids and

383

water molecules were enhanced by alkali addition. Moreover, during the experiment,

384

it was found that glutenin fraction has strong binding ability to water and can absorb 18

385

more water. Compared with gluten and gliadin samples, the lower T2 relaxation time

386

range of glutenin indicated that water-glutenin interactions were stronger.

387

3.9. Molecular weight distribution of gluten, gliadin and glutenin fractions

388

SE-HPLC is a technique for measuring the molecular weight distribution of

389

wheat protein, which can indicate the degree of cross-linking. According to Wang et al.

390

(2018), the profiles of soluble proteins can be divided into four peaks of known gluten

391

components, which refer to large glutenin polymers (the first peak), medium glutenin

392

polymers (the second peak), monomeric proteins (the third peak), peptides and amino

393

acids (the last peak). The elution profiles of uncooked and cooked gluten, glutenin,

394

and gliadin samples with different levels of alkali were shown in Fig. 7 and Fig. S2.

395

For uncooked gluten samples (Fig. 7A), the peak area decreased with increasing

396

alkali contents, indicating that the protein was polymerized to some extent; for

397

glutenin samples, it is noticeable that the addition of alkali markedly decreased the

398

peak area, specifically in the first peak fraction. However, there is no significant

399

change in the peak area of the uncooked gliadin samples. These results indicated that

400

the alkali treatment led to the polymerization of the gluten protein, mainly by

401

polymerizing the large glutenin polymers to form glutenin macropolymers (GMP,

402

insoluble in SDS). This polymerization may be due to the dissolving promoting effect

403

of alkali which led to the exposure of the hydrophilic free sulfhydryl groups that

404

continue to crosslink, resulting in an increased degree of polymerization (Batey &

405

Gras, 1984).

406

As shown in Fig. 7E, F, and G, during cooking, all samples were significantly 19

407

polymerized. For gluten sample, the peak area significantly decreased with the

408

extension of heating time. Moreover, the peak area decreased with increasing alkali

409

content at the same cooking time. A similar trend was observed for glutenin and

410

gliadin samples. An interesting phenomenon was found that there was no significant

411

change in the peak area of the 2% alkali treated gliadin samples with increasing

412

heating time, indicating an immediate polymerization of gliadin during cooking.

413

Moreover, the polymerization caused by cooking occurred at the third peak position,

414

indicating that cooking led to more aggregations of monomeric proteins. In reduced

415

profiles

416

gluten/glutenin/gliadin samples with or without alkali; during cooking, only samples

417

with 2% alkali gradually decreased in peak area with increasing cooking time, the

418

decreasing degree was much lower as compared with non-reduced profiles. These

419

findings indicated that the polymerization caused by alkali and cooking was mainly

420

through disulfide bonds cross-linking. Deleu, Lambrecht, Vondel, & Delcour, (2019)

421

also indicated that disulfide (SS) bond formation through sulfhydryl oxidation or

422

SH-SS exchange reactions could be favored under alkaline conditions. The decrease

423

in peak intensity of the 2% alkali samples during cooking also suggested the presence

424

of other possible cross-links (such as lanthionine or lysinoalanine cross-link).

(Fig.

S2),

no

significant

changes

were

detected

for

uncooked

425

Furthermore, gliadin was more sensitive to temperature in the presence of large

426

amounts of alkali. It could be concluded that glutenin may be the key fraction

427

determining the protein polymerization of uncooked gluten samples in the presence of

428

alkali and play an important role in the strengthening effect of alkali on fresh dough 20

429

and noodles. Moreover, both glutenin and gliadin contributed to the alkali enhanced

430

gluten polymerization during cooking and may decide the texture of cooked alkaline

431

noodle products.

432

The molecular weight distribution profiles of gluten protein before and after

433

ethanol extraction were also compared and no obvious difference was detected (Fig.

434

S3C).

435

3.10. GMP particle size distribution

436

Glutenin macropolymer (GMP) is a glutenin polymer insoluble in SDS, it's

437

content and particle size distribution are closely related to dough characteristics, and it

438

is the most important determinant of wheat storage protein (Wang, Zhao, & Zhao,

439

2007). The distribution ranges of GMP particle size of the three samples (alkali and

440

non-alkaline treated gluten samples) are shown in Fig. 8. For non-alkaline samples,

441

the volume percentages of the small GMP (particle size < 10 µm), medium GMP

442

(10-100 µm), and large GMP (> 100 µm) were 24.82%, 69.62%, 5.56%, respectively.

443

And the GMP particle size of 0.5% and 2% alkali treated samples were significantly

444

higher than the non-alkaline sample. The percentages of medium GMP with 0.5% and

445

2% alkali treated sample were increased by about 10% (76.09%) and 20% (78.54%),

446

and the content of large GMP with 2% alkali treated sample was increased by three

447

times (17.72%). This result suggested that the polymerization degree of glutenin

448

macropolymer was increased in alkali treated sample, which was also consistent with

449

the results of SE-HPLC.

450

3.11. Morphology of molecular chains 21

451

Atomic force microscopy can reflect the surface characteristics of any polymer

452

on a nano-scale, which was used to characterize the molecular chain size and

453

morphology of the samples in this study. The properties of protein molecular chains

454

could potentially indicate their physicochemical function. The changes in molecular

455

chain morphology could significantly affect the mechanical properties of gluten

456

networks (Chichti, George, Delenne, Radjai, & Lullien-Pellerin, 2013). Similar

457

studies have also reported the use of AFM to characterize gluten molecular chain

458

morphology (Zhang et al., 2015; Zhao, Liu, Hu, Li, & Li, 2016).

459

Fig. 9 shows AFM 3D images of gluten, glutenin, and gliadin samples with or

460

without alkali treatment. The average molecular chain height and width of all samples

461

were calculated by SPM-9700 software. For gluten samples (Fig. 9A), compared with

462

the control (The molecular chain height range was distributed at 4-7 nm, and the peak

463

width was about 0.15 µm), the peak height and width of alkali treated samples was

464

significantly increased; the peak height and width of gluten molecular chains with 2%

465

alkali was 27.6 nm and 0.49 µm, respectively. The glutenin samples had a larger peak

466

height and peak width (about 11.28 nm and 0.27 µm, respectively) while the gliadin

467

sample showed a slightly smaller peak height and width. Fig. 9 showed that the size of

468

molecular chains of the glutenin and gliadin fractions with alkali addition also

469

increased, but were less obvious as compared with gluten samples. This indicated that

470

the interaction of the two fractions may play an important role in the molecular chain

471

aggregation in gluten system with the presence of alkali.

472

In summary, rheological measurements showed that alkali significantly increased 22

473

the G' of gluten sample, leading to an enhanced gluten dough; glutenin fraction

474

contributed more to the rheological enhancement of gluten in the presence of alkali.

475

SEM images indicated that alkali induced a more closed gluten network structure

476

which was highly related to a strong gluten network, and the glutenin subfraction

477

contributed more to the shape and strength of the network. Zeta potential analysis

478

demonstrated that alkali promoted the electrostatic interactions in gluten system and

479

changes of gliadin fraction were more sensitive to alkali and may be the key fraction

480

in enhancing the electrostatic interactions of gluten proteins. QCM-D results indicated

481

alkali significantly increased the adsorption mass of gluten and glutenin samples,

482

indicating the promoted alkali/protein-protein interactions, and glutenin fraction

483

contributed more to the alkali induced promotion effect; the interaction between

484

glutenin and gliadin fraction may further enhance this effect (gluten system). In

485

addition, the fluorescence intensity and surface hydrophobicity for all alkali-treated

486

samples were decreased, demonstrating that alkali enhanced the hydrophobic

487

interactions in all the fractions. LF-1H NMR spectroscopy suggested that alkali

488

enhanced the water-solids interaction in all the fractions and water-glutenin

489

interactions were much stronger. In SE-HPLC profiles and GMP particle size

490

distribution curves, glutenin was shown to be the key fraction in the alkali induced

491

protein polymerization of uncooked gluten samples, which may play an important role

492

in the strengthening effect of alkali on fresh dough and noodles; however, both

493

glutenin and gliadin contributed to the enhanced gluten polymerization during

494

cooking, which may finally decide the texture of cooked noodles; the polymerizations 23

495

were formed mainly through the SH/S-S exchange. AFM images demonstrated that

496

alkali increased molecular chain size of all samples, and both glutenin and gliadin

497

contributed to the aggregation of molecular chains in gluten system.

498

4. Conclusion

499

In conclusion, the addition of alkali significantly improved gluten strength and

500

noodle texture. This study further revealed the underlying causes of the strengthening

501

effect of alkali on wheat gluten and noodle quality, from the perspective of changes in

502

gluten and its subfractions. Physical and chemical properties of glutenin and gliadin

503

changed to varying degrees in the presence of alkali, which determined the change of

504

gluten strength. Alkali led to a more closed network structure and enhanced

505

alkali/protein-protein interactions, which are mainly contributed by glutenin fraction.

506

Meanwhile, alkali enhanced electrostatic interactions in gluten system, which were

507

mainly contributed by gliadin fraction. In addition, both glutenin and gliadin

508

contributed to the alkali enhanced hydrophobic interactions, water-solids interactions,

509

and molecular chain aggregation in gluten. Significant protein polymerizations were

510

caused by alkali during cooking mainly through disulfide bonds cross-linking.

511

Glutenin was concluded to be the key fraction in the protein polymerization of

512

uncooked gluten samples in the presence of alkali, which may play an important role

513

in the strengthening effect of alkali on fresh dough and noodles; on the other hand,

514

both glutenin and gliadin contributed to the enhanced gluten polymerization during

515

cooking, which may finally decide the texture of cooked noodle products.

516

Notes 24

517 518

The authors declare no competing financial interest. Acknowledgments

519

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

520

(Grant No. 31601522), and the Special Funds for Taishan Scholar Projects of

521

Shandong Province (No. ts201712058).

25

522

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30

630

Figure captions

631

Fig. 1 Effect of alkali on the dynamic rheological properties of gluten, glutenin, and

632

gliadin fractions. Gluten + 0.5% alkali represents the addition of 0.5% alkali to gluten,

633

the same below.

634

Fig. 2 The images of scanning electron microscope. A1, A2, and A3 represent the

635

cross-section of gluten, gluten + 0.5% alkali, and gluten + 2% alkali dough,

636

respectively; B1, B2, and B3 represent the cross-section of glutenin, glutenin + 0.5%

637

alkali, and glutenin + 2% alkali dough, respectively; C1, C2, and C3 represent the

638

cross-section of gliadin, gliadin + 0.5% alkali, and gliadin + 2% alkali dough,

639

respectively.

640

Fig. 3 Zeta potential of gluten, glutenin, and gliadin samples added by alkali in

641

deionized water. Glu, gluten; Glu 0.5%, gluten + 0.5% alkali; Glu 2%, gluten + 2%

642

alkali; Gin, glutenin; Gin 0.5%, glutenin + 0.5% alkali; Gin 2%, glutenin + 2% alkali;

643

Gli, gliadin; Gli 0.5%, gliadin + 0.5% alkali; Gli 2%, gliadin + 2% alkali. The same

644

below.

645

Fig. 4 Time course of the adsorbed mass changes obtained from the QCM-D

646

measurement. A1, A2, and A3 represent gluten, gluten + 0.5% alkali, and gluten + 2%

647

alkali, respectively; B1, B2, and B3 represent glutenin, glutenin + 0.5% alkali, and

648

glutenin + 2% alkali, respectively; C1, C2, and C3 represent gliadin, gliadin + 0.5%

649

alkali, and gliadin + 2% alkali, respectively.

650

Fig. 5 Fluorescence spectrum (A) and surface hydrophobicity (B) of gluten, glutenin,

651

gliadin samples. 31

652

Fig. 6 The spin-spin relaxation time (T2) changes of water molecules in gluten (A),

653

glutenin (B), and gliadin (C) samples.

654

Fig. 7 Size-exclusion HPLC chromatogram of gluten, glutenin, and gliadin samples

655

during cooking. A, B, and C represent uncooked gluten, glutenin, and gliadin samples,

656

respectively; E, F, and G represent gluten, glutenin, and gliadin samples at different

657

times of cooking.

658

Fig. 8 The distribution of glutenin macropolymer (GMP) particles in gluten samples.

659

Fig. 9 AFM morphology 3D images of gluten (A1), gluten + 0.5% alkali (A2), gluten

660

+ 2% alkali (A3), glutenin (B1), glutenin + 0.5% alkali (B2), glutenin + 2% alkali

661

(B3), gliadin (C1), gliadin + 0.5% alkali (C2), gliadin + 2% alkali (C3).

32

220000

240000

200000

220000 200000 180000 160000

Gluten Gluten+0.5% alkali Gluten+2% alkali

180000 160000 140000

G''/Pa

G'/Pa

140000 120000 100000

120000 100000 80000

80000

60000

60000 40000

40000

20000

20000

0 0.1

Gluten Gluten+0.5% alkali Gluten+2% alkali

1

Frequency (HZ)

10

0 0.1

100

1

Frequency (HZ)

10

100

55000 55000 50000 45000 40000

50000

Glutenin Glutenin+0.5% alkali Glutenin+2% alkali

45000 40000 35000

G''/Pa

35000

G'/Pa

30000 25000

30000 25000 20000

20000 15000

15000

10000

10000

5000

5000

0 0.1

Glutenin Glutenin+0.5% alkali Glutenin+2% alkali

1

Frequency (HZ)

10

0 0.1

100

1

10

100

Frequency (HZ) 180000

120000

100000

160000

Gliadin Gliadin+0.5% alkali Gliadin+2% alkali

140000

Gliadin Gliadin+0.5% alkali Gliadin+2% alkali

120000

G''/Pa

G'/Pa

80000

60000

100000 80000 60000

40000

40000 20000

20000 0

0.1

1

10

Frequency (HZ)

100

0 0.1

1

Frequency (HZ)

10

100

Fig. 1 Effect of alkali on the dynamic rheological properties of gluten, glutenin, and gliadin fractions. Gluten + 0.5% alkali represents the addition of 0.5% alkali to gluten, the same below.

A1

A2

A3

B1

B2

B3

C1

C2

C3

Fig. 2 The images of scanning electron microscope. A1, A2, and A3 represent the cross-section of gluten, gluten + 0.5% alkali, and gluten + 2% alkali dough, respectively; B1, B2, and B3 represent the cross-section of glutenin, glutenin + 0.5% alkali, and glutenin + 2% alkali dough, respectively; C1, C2, and C3 represent the cross-section of gliadin, gliadin + 0.5% alkali, and gliadin + 2% alkali dough, respectively.

30

Zeta potential (mV)

20 10 0 -10 -20 -30 Glu Glu 0.5% Glu 2% Gin Gin 0.5% Gin 2% Gli Gli 0.5% Gli 2%

Fig. 3 Zeta potential of gluten, glutenin, and gliadin samples added by alkali in deionized water. Glu, gluten; Glu 0.5%, gluten + 0.5% alkali; Glu 2%, gluten + 2% alkali; Gin, glutenin; Gin 0.5%, glutenin + 0.5% alkali; Gin 2%, glutenin + 2% alkali; Gli, gliadin; Gli 0.5%, gliadin + 0.5% alkali; Gli 2%, gliadin + 2% alkali. The same below.

600

600

600

A2

A3 500

400

400

400

acetic acid rinsing

200

acetic acid

100 0 0

500

1000

1500

acetic acid rinsing

300 200

acetic acid

100

gluten solution

2000

2500

3000

500

1000

2500

3000

3500

acetic acid rinsing acetic acid

500

1000

1500

400

acetic acid rinsing

200

acetic acid

3500

3500

0

500

1000

1500

2000

2500

acetic acid rinsing 300 200

acetic acid

100

glutenin+0.5% alkali solution

0 3000

3000

2

300

0 2500

2500

B3

400

100

2000

2000

Time (s)

500

glutenin solution 1500

0

500

Mass (ng/cm )

300

1000

gluten +2%alkali solution

600

2

Mass (ng/cm )

2

3000

0 3500 0

500

glutenin+2% alkali solution

1000

1500

Time (s)

2000

2500

3000

3500

4000

Time (s)

Time (s) 600

600

600

C1 500

C3

C2 500

500

acetic acid rinsing

300 200

300 200

acetic acid 100

acetic acid

100

gliadin solution

0

500

1000

1500

2000

Time (s)

2500

3000

3500

acetic acid rinsing

300 200

acetic acid 100

gliadin + 0.5% alkali solution

gliadin + 2% alkali solution

0

0

0

400

2

400 2

acetic acid rinsing

Mass (ng/cm )

400

Mass (ng/cm )

Mass (ng/cm )

400

2

2000

B2

500

Mass (ng/cm )

1500

600

500

acetic acid

Time (s)

B1

0

200

0

0 3500 0

600

100

300

100

gluten + 0.5% alkali solution

Time (s)

200

acetic acid rinsing

2

2

300

Mass (ng/cm )

500

Mass (ng/cm )

500

2

Mass (ng/cm )

A1

0

500

1000

1500

2000

Time (s)

2500

3000

3500

0

500

1000

1500

2000

Time (s)

2500

3000

3500

Fig. 4 Time course of the adsorbed mass changes obtained from the QCM-D measurement. A1, A2, and A3 represent gluten, gluten + 0.5% alkali, and gluten + 2% alkali, respectively; B1, B2, and B3 represent glutenin, glutenin + 0.5% alkali, and glutenin + 2% alkali, respectively; C1, C2, and C3 represent gliadin, gliadin + 0.5% alkali, and gliadin + 2% alkali, respectively.

Fluorescence intensity (AU)

1000

Gluten Gluten+0.5% alkali Gluten+2% alkali Glutenin Glutenin+0.5% alkali Glutenin+2% alkali Gliadin Gliadin+0.5% alkali Gliadin+2% alkali

A

900 800 700 600 500 400 300 200

70

B 60

Surface hydrophobicity (So)

1100

50

40

30

20

100 0

280

300

320

340

360

380

400

420

Glu Glu 0.5%Glu 2%

Gin Gin 0.5%Gin 2%

Gli Gli 0.5% Gli 2%

Emission Wavelength (nm)

Fig. 5 Fluorescence spectrum (A) and surface hydrophobicity (B) of gluten, glutenin, gliadin samples.

70

90 80

A

60

Gluten Gluten+0.5 % alkali Gluten+2 % alkali

70

B

Glutenin Glutenin+0.5% alkali Glutenin+2% alkali

50

Intensity

50 40 30

40 30 20

20

10

10 0 0.1

1

10

100

0 0.1

1000

1

Time (ms)

10

100

Time (ms)

100 90

C

Gliadin Gliadin+ 0.5 % alkali Gliadin+ 2 % alkali

80 70

Intensity

Intensity

60

60 50 40 30 20 10 0 0.1

1

10

100

1000

Time (ms)

Fig. 6 The spin-spin relaxation time (T2) changes of water molecules in gluten (A), glutenin (B), and gliadin (C) samples.

1000

140000

A

E

120000

120000

Gluten Gluten+0.5% alkali Gluten+2% alkali

100000

Intensity (mv)

Intensity (mv)

100000 80000 60000

Gluten 1 min Gluten 2 min Gluten 4 min Gluten+0.5% alkali 1 min Gluten+0.5% alkali 2 min Gluten+0.5% alkali 4 min Gluten+2% alkali 1 min Gluten+2% alkali 2 min Gluten+2% alkali 4 min

80000 60000

40000

40000

20000

20000

0

0 8

10

12

14

16

8

18

10

25000

Intensity (mv)

Intensity (mv)

10000

5000

20000 15000

18

10000 5000 0

0 8

10

12

14

16

18

8

10

Time (min) 300000

14

16

18

G

300000

Gliadin 1 min Gliadin 2 min Gliadin 4 min Gliadin+0.5% alkali 1 min Gliadin+0.5% alkali 2 min Gliadin+0.5% alkali 4 min Gliadin+2% alkali 1 min Gliadin+2% alkali 2 min Gliadin+2% alkali 4 min

250000

Intensity (mv)

Gliadin Gliadin+0.5% alkali Gliadin+2% alkali

200000

12

Time (min)

C

250000

Intensity (mv)

16

Glutenin 1 min Glutenin 2 min Glutenin 4 min Glutenin+0.5% alkali 1 min Glutenin+0.5% alkali 2 min Glutenin+0.5% alkali 4 min Glutenin+2% alkali 1 min Glutenin+2% alkali 2 min Glutenin+2% alkali 4 min

25000

Glutenin Glutenin+0.5% alkali Glutenin+2% alkali

15000

150000

200000 150000

100000

100000

50000

50000

0

14

F

30000

B

20000

12

Time (min)

Time (min) 30000

0 8

10

12

14

Time (min)

16

18

8

10

12

14

Time (min)

16

18

Fig. 7 Size-exclusion HPLC chromatogram of gluten, glutenin, and gliadin samples during cooking. A, B, and C represent uncooked gluten, glutenin, and gliadin samples, respectively; E, F, and G represent gluten, glutenin, and gliadin samples at different times of cooking.

12 Gluten Gluten + 0.5% alkali Gluten + 2% alkali

10

Channel(%)

8

6

4

2

0 1

10

100

Particle diameter (µm)

1000

Fig. 8 The distribution of glutenin macropolymer (GMP) particles in gluten samples.

A3

A1

A2

B1

B2

B3

C1

C2

C3

Fig. 9 AFM morphology 3D images of gluten (A1), gluten + 0.5% alkali (A2), gluten + 2% alkali (A3), glutenin (B1), glutenin + 0.5% alkali (B2), glutenin + 2% alkali (B3), gliadin (C1), gliadin + 0.5% alkali (C2), gliadin + 2% alkali (C3).

Highlights Alkali induced changes in both gluten and its subfractions were explored in-depth A new method (QCM-D) was firstly used to explain alkali/protein-protein interaction Alkali induced a membrane-like structure in gluten and glutenin fractions Glutenin was the key fraction for alkali-induced fresh gluten polymerization Gliadin contributed more to the hydrophobic interactions and heat-polymerization

Conflict of Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected] Signed by all authors as follows: Chuanwu Han, Meng Ma, Man Li, Qingjie Sun