Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties

Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties

Journal Pre-proof Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming pro...

8MB Sizes 0 Downloads 5 Views

Journal Pre-proof Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties Guijiang Liang, Wenpu Chen, Xuejiao Qie, Maomao Zeng, Fang Qin, Zhiyong He, Jie Chen PII:

S0268-005X(19)30939-7

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105764

Reference:

FOOHYD 105764

To appear in:

Food Hydrocolloids

Received Date: 4 May 2019 Revised Date:

9 February 2020

Accepted Date: 11 February 2020

Please cite this article as: Liang, G., Chen, W., Qie, X., Zeng, M., Qin, F., He, Z., Chen, J., Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/ j.foodhyd.2020.105764. 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.

Graphical Abstract

1/1

1 2

Modification of soy protein isolates using combined pre-heat treatment and controlled enzymatic hydrolysis for improving foaming properties

3 4 5

Guijiang Liangab, Wenpu Chenac, Xuejiao Qieab,Maomao Zengab, Fang Qinab, Zhiyong He*ab, Jie Chen*ab

6

a

7

214122, China

8

b

9

China

10

c

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi

International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122,

Abbott Nutrition Research & Development (ANRD), Singapore

11 12

*Corresponding author:

13

Professor Jie Chen

14

Professor Zhiyong He

15

State Key Laboratory of Food Science and Technology, School of Food Science and

16

Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China

17 18

Tel.: +86-510-85329032

19

Fax: +86-510-85919065

20

E-mail address: [email protected]; [email protected]

21 22 23 24 25 26 27 28 29 30 31 32 33 1|P a g e

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77

Abstract The foaming properties of soy protein hydrolysates modified with a combination of pre-heat treatment and controlled enzymatic hydrolysis by pepsin were investigated. Degree of hydrolysis, soluble protein percentage, peptide profile, molar mass distribution, surface properties at air-water interface and interfacial rheological properties of hydrolysates were measured to gain insights into the relationships between molecular composition and foaming properties. It was found that pre-heat treatment of soy protein at 55˚C for 30 min promoted the hydrolysis of hydrophilic acidic subunits and increased the ratio of 7S/11S. As result, it resulted in the best interfacial properties and foam capacity as well as stability amongst all treatments. Although pre-heat treatment above 65˚C before soy protein isolate hydrolysis increased degree of hydrolysis, higher proportion of large protein aggregates in the hydrolysates with increasing temperature deteriorated their foaming capacity and stability, which resulted in lower storage modulus (G') of the adsorbed layer and less surface excess concentration (Γ) compared to pre-heat treatment at 55˚C. Without pre-heat treatment, unfolded soy protein did not provide sufficient accessible sites for pepsin hydrolysis. As result, foam capacity and stability were inferior compared to 55˚C pre-heat treatment hydrolysates. This study provided the foundation for the application of soy protein isolate in aerated food products with desirable foaming properties.

Key words: Soy Protein, Pepsin, Hydrolysates, Foaming Capacity, Foaming Stability

2|P a g e

78

1.0.

Introduction

79

Food foams provide desirable textures and unique mouthfeel to many aerated foods,

80

such as meringues, cakes, bread, soufflés and marshmallow in which tiny air bubbles

81

are trapped in the food system (Campbell & Mougeot, 1999; Zeng et al., 2013). Food

82

foam is a thermodynamically unstable system where air phase is dispersed in a liquid

83

matrix, and thus requires external energy to generate and maintain its stability.

84

According to the thermodynamic dictum, phase separation rapidly occurs to minimize

85

the interfacial contact area and free energy in colloid dispersions with two immiscible

86

phases. Typically, surface active species with amphiphilic properties are added to

87

reduce interface tension between continuous and dispersed phases. Two common

88

surface active species, namely small molecule surfactants, such as monoglycerides,

89

and proteins have been used to lower surface tension at air-water interfaces and

90

impact foam stability (Patino, Niño, & Sánchez, 2003). They play a key role in

91

maintaining foam stability and preventing foam destruction caused by liquid drainage,

92

coalescence and disproportionation (Sadoc & Rivier, 2013). Traditionally, egg white

93

proteins and milk proteins e.g. sodium caseinate are commercial agents for the

94

desirable foaming properties (Nicorescu et al., 2011)The behaviors of those proteins

95

at air-water interfaces and their role on foaming capacity and stability have been

96

studied in previous research papers (Murray, 1998; Morris & Gunning, 2008).

97

Recently, soy protein isolate (SPI) has been considered as an alternative to or

98

extension of egg white protein and milk proteins because of its excellent nutritional

99

value and functionalities (Anderson, Anthony, Cline, Washburn, & Garner, 1999;

100

Dickinson & Matsumura, 1991). Soy protein isolate with exposed hydrophobic and

101

hydrophilic groups have been used as foaming agents (He et al., 2015). However,

102

intact soy protein isolate with less flexible biological structure limits surface

103

properties and foaming capacity (Wagner & Guéguen, 1999). Flexible protein

104

structure and rapid molecular adsorption at air-water interface are required for soy

105

protein as a good foam agent. Moreover, the formation of a cohesive viscoelastic

106

adsorbed protein layer via intermolecular interactions at air-water interfaces is 3|P a g e

107

necessary to keep foam stability (Dickinson, 2003). The interfacial behaviors of

108

adsorbed protein layer are impacted by many factors such as protein molecular

109

structure, protein size and molar mass, conformational structure, composition and

110

sequence of amino acids, and some environmental factors such as pH and ionic

111

strength (Foegeding, Luck, & Davis, 2006).

112

Soy protein subunits demonstrate diverse interfacial properties due to their

113

inherent structure and amino acids composition. For example, β-conglycinin (7S)

114

shows better foaming and emulsifying properties compared to glycinin (11S) because

115

β-subunit from β-conglycinin has a high proportion of hydrophobic amino acids such

116

as alanine, valine, leucine, and phenylalanine, which have a high tendency to interact

117

with hydrophobic phase at the interface (Nielsen, 1985; Lam & Nickerson, 2013).

118

Basic subunit with higher hydrophobicity in glycinin (11S) are buried and shielded by

119

the hydrophilic acid subunit in aqueous solution (Kuipers & Gruppen, 2008). Hence,

120

emulsifying properties of glycinin (11S) are inferior to β-conglycinin (7S).

121

To improve foaming and emulsifying properties of soy protein isolate, thermal

122

treatment and enzymatic treatment have been used to modify the soy protein structure

123

(Tsumura et al., 2005; Were, Hettiarachchy, & Kalapathy, 1997). Shao, Lin, & Kao

124

(2016) reported that thermal treatment to commercial soy protein isolate improved its

125

foam capacity and stability. According to Cui, Zhao, Yuan, Zhang, & Ren (2013), soy

126

protein hydrolysates made by 0.3% pepsin at pH 2 for 60 min had an improvement in

127

solubility, emulsification and foam stability. For enzymatic modification, the choice

128

of protease and control of degree of hydrolysis have great impacts on foaming

129

properties of SPI hydrolysates. According to Ortiz & Wagner (2002), SPI hydrolysates

130

processed with bromelain (pH 7) had a good foaming property at pH 4.5. As an

131

endopeptidase, pepsin has been used to improve the functionality and structure

132

characteristics of intact soy protein in the controlled condition (Cui, Zhao, Yuan,

133

Zhang, & Ren, 2013; Tsumura et al., 2005). Tsumura et al. (2005) found that SPI

134

hydrolysates with selective digestion of glycinin by pepsin exhibited better

135

whippability than other treatment. However, there are limited studies reported on the 4|P a g e

136

use of a combination of pre-heat treatment at different temperature with controlled

137

enzymatic hydrolysis to manipulate the compositions of hydrolysates. Moreover, the

138

impacts of the compositions of SPI hydrolysates with pre-heat treatment on the

139

surface properties are still unknown.

140

In this study, soy protein isolate was subjected to pre-heat treatment at various

141

temperatures prior to hydrolysis and the effects of pre-heat treatment on controlled

142

pepsin-driven hydrolysates and their interfacial properties were observed. Degree of

143

hydrolysis, soluble protein percentage, protein profile, molar mass distribution,

144

interfacial rheological properties and surface properties of the hydrolysates were

145

studied to gain insights into the relationship of peptides composition and surface

146

properties, which were used to understand their foaming properties. The purpose of

147

this study was to determine the optimum pre-heating temperature condition before

148

pepsin-driven hydrolysis for SPI hydrolysates with desirable foaming properties.

149

2.0 Materials and methods

150

2.1. Materials and chemicals

151

Soybeans were obtained from a local market (Wuxi, China). Pepsin (3000 U/mg) was

152

purchased from Sangon Biotech Co, Shanghai, China. Trinitro-benzene-sulfonic acid

153

(TNBS) solution and β-mercaptoethanol were purchased from Sigma-Aldrich (St.

154

Louis, USA). All chemicals used were of analytical grade unless otherwise specified.

155

Deionized water was used as the ingredient water.

156

2.2. Preparation of SPI hydrolysates

157

Soybeans were crushed and peeled into small particles, then degreased with n-hexane

158

to obtain defatted soy flour. SPI was prepared from defatted soy flour according to the

159

method provided by Puppo et al. (2004) with slight modifications. Soy flour was

160

dispersed into water and adjusted to pH 8 using 2M NaOH. The dispersion was stirred

161

for 2h at room temperature and then centrifuged for 20 min at 10000 x g at 4℃. The

162

supernatant was adjusted to pH 4.5 with 2M HCl and centrifuged (3300 x g, 10 min,

163

4℃) for the sediment. The obtained sediment was re-suspended with distilled water (1:

164

5, v/v) and adjusted to pH 7.0 with 2M NaOH before freeze drying. 5|P a g e

165

The samples above were reconstituted in water to make a solution with 7% (w/v)

166

protein with Biuret method given by Chang (2014). The solution was divided into 6

167

portions for different heat treatment. Portion 1 was not subjected to thermal treatment.

168

Portion 2 was subjected to 55˚C for 30 min. Portions 3-6 were subjected to 65˚C,

169

75˚C, 85˚C, and 95˚C for 10 min. After thermal treatment, all the samples were

170

chilled in ice slurries, then were adjusted to pH 2.0 using 2M HCl and incubated at

171

37˚C for 30 min. Pepsin was added to each portion at enzyme: substrate ratio of 0.3%

172

(w/w) to initiate hydrolysis. Each portion was incubated at 37˚C for 60 min.

173

Hydrolysis was terminated by adjusting to pH 7 with 2M NaOH. The hydrolysates

174

were centrifuged (10000 x g, 10 min, 25℃) and the supernatant was sterilized for 20s

175

at 120˚C before freeze drying. The hydrolysates of portion 1-6 was referred as

176

control,SPH55, SPH65, SPH75, SPH85 and SPH95 respectively.

177

2.3. Soluble protein determination

178

All the hydrolysates were centrifuged at 10000 x g for 10 min and the content of

179

soluble protein in the supernatant was determined with Biuret method (Chang, 2014).

180

The percentage of soluble hydrolysates was calculated according to the equation

181

given below.

182 Soluble protein content in hydrolysate(g/mL)Volume of hydrolysate(mL) 183

× 100% (1)

Soluble protein %= Total protein(g)

184 185

2.4. Degree of hydrolysis (DH)

186

DH of the hydrolysates was determined with TNBS method given by Spellman,

187

McEvoy, O’cuinn, & FitzGerald (2003), with slight modifications. L-Leucine with

188

different concentrations was used to generate a standard curve at absorbance at 420

189

nm for the standard nitrogen content. The absorbance at 420 nm of the samples before

190

and after hydrolysis was measured. The values were substituted into the standard

191

curve to calculate the amino nitrogen content of the protein substrate.

192

DH values were calculated using the following formula (Harnedy et al., 2018)

193 DH% =

AN2 - AN1 Npb

6|P a g e

× 100%

(2)

194

where AN1 is the amino nitrogen content of the protein substrate before hydrolysis

195

(mg/g protein), AN2 is the amino nitrogen content of the protein substrate after

196

hydrolysis (mg/g protein). Npb is the nitrogen content of the peptide bonds in the

197

protein substrate (mg /g protein). A value of 109.2 was used for soy protein.

198

2.5. Molar mass distribution

199

The molar mass distribution of the hydrolysates was measured by HPLC equipped

200

with a gel permeation chromatographic (GPC) column (Shodex Protein KW-84

201

column; 8 mm I.D X 30 cm, Shodex Co., Tokyo, Japan) and a Waters 2487 dual λ

202

absorbance detector (Waters Co., USA). The elution buffer consisted of 50mM

203

phosphate (pH 7.0) with 0.3M NaCl (flow rate: 1.0 mL/min). Before testing, all

204

samples were dissolved in ultrapure water and centrifuged to remove insoluble

205

portion (10000 x g, 25 ˚C, 10 min). After that, the protein concentration in all samples

206

was adjusted to 1% for further test. Bovine thyroglobulin (669 kDa), amylase (200

207

kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29

208

kDa) and cytochrome c (12 kDa) were used as markers.

209

2.6. Sodium dodecyl sulphate-polyarylamide gel electrophoresis (SDS-PAGE)

210

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was

211

performed according to the method reported by Liu, Xiong, & Butterfield (2000),

212

with slight modifications. The hydrolysates were run using a mini-protein

213

electrophoresis system (Bio-Rad Laboratories, Hercules, Calif., U.S.A.). SDS-PAGE

214

was conducted on a discontinuous buffered system with 12% separating gel and 4%

215

stacking gel. Samples (2 mg/mL in buffer containing 0.0625M of Tris-HCl, 10%

216

glycerin, 2% SDS and 0.0025% bromophenol blue) were incubated for 1h at room

217

temperature with and without β-mercaptoethanol (5%, v/v). After incubation, all

218

samples were heated at 100℃ for 5 min. Aliquots (20 µL) of the prepared samples

219

were loaded onto the gels. All gels were scanned by a computing densitometer

220

(Molecular Imager ChemiDocXRS+, Bio-Rad, USA) Image Lab software (Bio-Rad,

221

USA) was used to integrate the intensities of bands.

222

2.7. Surface properties measurement 7|P a g e

223

Dynamic interfacial tension (γ) of the hydrolysates at air-water interface was

224

determined using a DCAT21 automated surface tension meter (Dataphysics, Berlin,

225

Germany). The critical micelle concentration (CMC) was automatically calculated

226

from surface tension (γ) of the samples. The surface excess concentration (Γ) and the

227

area occupied by each molecule (A) were calculated according to the Gibbs

228

adsorption in Equation: Γ=

-1



RT dlnC

(3)

229 230

where C is the concentration, R is the gas constant, and T is the absolute temperature.

231

The mean area occupied per molecules of the adsorbed surfactant, A, was calculated

232

according to A=

233

1 NΓ

(4)

234

where N is Avogadro’s constant. The calculation was calculation by DCATSoftware.

235

concentration was adjusted to 2% for all measurements by Biuret method.

236

2.8. Interfacial rheology measurement

237

Measurements of the interfacial rheology of proteins (1%) at the air/water interface

238

were obtained using a HAAKE MARS ℃ rheometer (Thermo Scientific, Germany)

239

equipped with a Du Noüy ring (platinum ring,diameter 19.450 mm). The thickness of

240

the wire was 0.379 mm. 20 mL of the sample was placed in a beaker (50 mm in

241

diameter) and the ring was lowered to contact the surface. To increase repeatability

242

and avoid destruction of the platinum ring, the gap was zeroed with cone geometry

243

and then the gap was kept constant at the position of 48.723 mm. The inertia

244

determination and MSC (Micro Stress Control) calibration were performed for every

245

measurement. Dynamic time sweeps were performed within the linear region at a

246

strain amplitude of γ =1% and an angular frequency of ω =1 rad s-1 for 1 h. Dynamic

247

strain sweeps were carried out with an angular frequency of ω = 1 rad s-1 in the range

248

of = 0.1-100%, with frequency of ω =1 rad s-1 at 25 °C. 8|P a g e

249

2.9. Foaming properties

250

Foaming properties were evaluated according to the method given by Adebowale,

251

Schwarzenbolz, & Henle (2011). Protein solution (100 mL, 1% w/v) was placed in a

252

500 mL graduated cylinder and homogenized with a disperser homogenizer (T 18

253

basic ULTRA-TURRAX R, IKA Corp., Staufen, Germany) at 17,500 rpm for 2 min.

254

The foam volume was recorded at time zero (V0) and after 30 min (V30) of

255

homogenization.

256

Foaming capacity (FC) and foaming stability (FS) were calculated using Equation (5)

257

and (6), respectively and 100 is the initial volume of solutions (mL). FC (%) =

V0 - 100 100

258

× 100%

(5)

259 260

FS (%) =

V30 - 100 V0 - 100

× 100%

(6)

261

2.10. Statistical Analysis

262

All experiments and associated measurements were performed at least in triplicate.

263

Statistical analysis was performed using a two-way ANOVA (P< 0.05) by Statistix 9.0

264

(Statistix, Tallahassee, FL, USA).

265 266

3.0. Results and discussion 3.1 Degree of hydrolysis and soluble protein percentage

267

The effect of heating temperatures on the degree of hydrolysis and soluble protein

268

content of soy protein hydrolysates were examined. SPH95 had the highest degree of

269

hydrolysis while the control had the lowest degree of hydrolysis amongst all the

270

samples. As shown in Fig.1, increasing pre-treatment temperature before hydrolysis

271

resulted in higher degree of hydrolysis of SPI because the unfolding of protein

272

structure caused by thermal treatment favored enzymatic hydrolysis by making more

273

accessible sites available to enzymes (Lam & Nickerson, 2013). Pepsin has a

274

preference to cleave the peptides containing linkages with aromatic or carboxylic

275

L-amino acids (Worthington, 1993). Thermal treatment caused soy protein molecules 9|P a g e

276

to unfold and expose the sulphydryl and hydrophobic groups (Keerati-u-rai &

277

Corredig, 2009). Two distinct thermal transition peaks, ranging from 68-75°C and

278

85-93°C, have been identified for denaturation of β-conglycinin and glycinin

279

respectively (Scilingo & Añón, 1996; Renkema, Lakemond, Jongh, Gruppen, & Vliet,

280

2000). According to Achouri, Zhang, & Shiying (1998), pre-heat treatment of intact

281

soy protein isolate at 80°C for 10 and 30 min prompted unfolding of some fractions

282

and a gradual molecular dissociation, which facilitated enzymatic hydrolysis. The

283

subunits in β-conglycinin would dissociate when it was subjected to thermal treatment

284

(above 70°C), causing structural changes (Iwabuchi, Watanabe, & Yamauchi, 1991).

285

Protein solubility is crucial for protein functional properties such as emulsifying,

286

rheological and surface-active properties. In theory, controlled enzymatic hydrolysis

287

of soy protein would improve the solubility of protein due to decreasing molar mass

288

of protein and increasing number of charge groups (Panyam & Kilara, 1996).

289

However, there was a decrease of soluble protein as the temperature of pre-heat

290

treatment increased in this study. The lowest soluble protein percentage was observed

291

in SPH95. This was explained as follows. Thermal pre-treatment induced unfolding of

292

protein molecules in partial or total form prior to hydrolysis and provided accessible

293

sites to pepsin. Pepsin hydrolysis promoted the exposure of hydrophobic and

294

hydrophilic groups, which led to formation of aggregates and thus greater loss of

295

solubility of hydrolysates (Chen, Chen, Ren, & Zhao, 2011). The formation of more

296

insoluble aggregates was caused by the hydrophobic interaction among the subunits

297

of soy protein isolate (Utsumi, Damodaran & Kinsella, 1984). The samples with high

298

temperature have more hydrophobic groups. However, the complexes formed by

299

β-subunit from β-conglycinin and basic subunit from glycinin were soluble when the

300

temperatures were above 90°C (Scilingo, & Añón, 1996; Renkema, Lakemond, Jongh,

301

Gruppen, & Vliet, 2000).

302

3.2. Composition of SPI hydrolysates

303

The polypeptide profile of SPI hydrolysates after pre-heating at different temperatures

304

was analyzed by SDS-PAGE for both non-reducing and reducing conditions and 10 | P a g e

305

untreated soy protein isolate was included as the reference (Fig.2A and Fig.2B). As

306

shown in Fig. 2A and 2B, all subunits from β-conglycinin (α, α' and β) and glycinin

307

(acidic and basic subunit) were present in all samples. Compared to the reference, the

308

complex of acidic and basic subunit (AB) was missing due to enzymatic hydrolysis.

309

This finding agreed with the reported by Chen et al. (2019). Under non-reducing

310

condition, large protein aggregates were identified on the top of the stacking gel

311

(Fig.2A). The bands of these protein aggregates disappeared under reducing condition

312

(Fig.2B), which indicated the interactions among the polymer molecules were

313

disulfide (S-S) linkage. The densitometric analysis of the main bands in SDS-PAGE

314

was performed to understand the impact of pre-heat treatment on soy protein

315

composition of SPI hydrolysates (Table 1). As shown in Fig. 2A and Table 1, the

316

bands corresponding to β-conglycinin (α', α, β) became faint when the pre-heat

317

temperature was above 65°C. Nearly 50% of α' and α subunit were lost at 65°C and

318

around 75% were lost when the temperature reached 85°C. The reduction rate of

319

β-subunit was lower than α' and α. According to Yamauchi, Yamagishi, & Iwabuchi

320

(1991), thermal treatment induced the dissociation of β-conglycinin into its

321

constituent’s subunits. When the heat treatment temperature was higher than the

322

denaturation temperature of β-conglycinin (68-75°C), ɑ and α' subunits formed

323

soluble aggregates (Petruccelli & Anon, 1995). Moreover, pepsin may decompose

324

ɑ-subunit from β-conglycinin selectively (Cui, Zhao, Yuan, Zhang, & Ren, 2013). In

325

synergy, both procedures led to the loss of ɑ and α' subunits once the thermal

326

temperature was above 65°C. The slow reduction rate of β-subunit was due to the

327

presence of α' and α subunits that prevented thermal aggregation of β-subunit (He, et,

328

al., 2015). Pepsin had little effect on β-subunit at pre-heat treatment temperature of

329

55°C (Table 1 and Fig. 2). However, when the pre-heat treatment temperature was

330

elevated above 65°C, more hydrophobic regions in β-subunit were exposed, which

331

increased the chances of association with other hydrophobic regions. This association

332

was irreversible-intermolecular aggregation that formed the aggregates of different

333

molecular sizes (Yamauchi, Yamagishi, & Iwabuchi, 1991). For glycinin (11S), the 11 | P a g e

334

reduction of acid subunit was faster than basic subunit, because the basic subunits

335

with higher hydrophobicity were buried and shielded by hydrophilic acid subunits in

336

aqueous solution (Kuipers & Gruppen, 2008). During hydrolysis, acidic polypeptides

337

were decomposed into peptides easily because endoproteases such as pepsin has a

338

preference to attack acidic amino acids (Jung, Roussel-Philippe, Briggs, Murphy, &

339

Johnson, 2004). When pre-heat temperature reached 95°C and above, there was

340

formation of soluble complexes between β-subunit from β-conglycinin and basic

341

subunit from glycinin (Lakemond, de Jongh, Hessing, Gruppen, & Voragen, 2000), as

342

shown by the reduction in the amount of β-subunit and basic subunit in Table 1.

343

3.3. Molar mass distribution of SPI hydrolysates

344

The molar mass of SPI hydrolysates after pre-heat treatment at various temperatures

345

can be categorized into four subgroups: group A (larger than 669 kDa), group B (100

346

-669 kDa), group C (10-100 kDa) and group D (less than 10 kDa) (Fig.3a and Fig.3b).

347

The relative areas of these four subgroups were different with different pre-heat

348

treatment conditions. It was observed that higher temperature used in pre-heat

349

treatment (55-95°C) resulted in an increase of the proportion of large molecules

350

(group A) significantly, while a decrease of the proportion of small molecules (group

351

B, C, D) was observed. The MW distribution of SPI subjected to different heat

352

treatment was in agreement with the results of SDS-PAGE analysis (Fig. 3).

353

3.4. SPI hydrolysate surface properties

354

As shown in Table 2, increasing pre-heat treatment temperature led to a decrease in

355

the surface excess (Γ) and an increase of molecular area (A) at the air-water interface.

356

Pre-heat treatment did not impact on the surface tension and CMC of hydrolysates at

357

the air-water interface as shown in Table 2. The value of CMC and surface tension of

358

the control sample are similar to the results reported by Li et al. (2016). Compared to

359

the control, pre-heating temperature at 55°C resulted in an increased surface excess (Γ)

360

from 0.84 µmol/m2 to 1.22 µmol/m2. However, Table 2 shows surface excess (Γ)

361

progressively decreased from 1.22 µmol/m2 to 0.45 µmol/m2, when pre-heating

362

temperature was increased from 55°C to 95°C. SPH55 achieved a maximum surface 12 | P a g e

363

excess (Γ) and a minimum molecular area (A) amongst all samples, indicating the

364

amount of adsorbed SPH55 molecules was more than other hydrolysates with a

365

smallest occupied molecular area at air-water interface. With 55°C pre-heat treatment

366

and subsequent hydrolysis, the high ratio of hydrophobic subunits such as β-subunits

367

and basic subunits in hydrolysates favored the rapid adsorption at air-water interface,

368

because at the air-water interface, the hydrophobic subunits can be positioned to form

369

more interactions with the air phase to attain a more thermodynamically stable state,

370

thus indicating a good interfacial activity. Additionally, improved flexible molecular

371

structure by hydrolysis and more 7S compositions in the hydrolysates would

372

contribute to better interfacial activities. According to Shao & Tang (2014), higher

373

extent of protein denaturation and/or aggregation exhibited a higher surface excess (Γ)

374

compared to intact SPI. However, pre-heat treatment above 65°C increased the

375

amount of larger molecular aggregates at the expense of hydrophobic subunits, which

376

reduced the amount of absorbed protein at air-water interface with a lower trend of

377

surface excess (Γ).

378

3.5. Interfacial rheology measurement

379

The formation, structural reorganization and mechanical properties of protein layer at

380

the interface can be monitored by interfacial shear rheology (Qiao, Wang, Shao, Sun,

381

& Miller, 2015). The adsorption kinetics and characteristics of the film formed by soy

382

protein hydrolysates at air-water interface were obtained by interfacial rheology. Fig.4

383

records the change of storage modulus G' and loss modulus G'' during time sweep as

384

the function of pre-heat treatment temperature. The rates of film formation were

385

expressed by the slope (K) by building the linear model of G' as the function of time

386

(0 to 10 min). By comparing K values of all samples, SPH55 K value (0.34) in Fig.4B

387

is the highest amongst all samples. K value demonstrates a descending trend with

388

increasing pre-heat treatment temperature. SPH55 molecules migrated and formed a

389

gel-like film at the fastest rate compared to the other samples. SPH55 molecules can

390

also unfold, change structure conformation, interact with neighboring molecules and

391

form a two-dimensional viscoelastic gel, as was indicated by storage modulus G' 13 | P a g e

392

being higher than loss modulus G'' during the whole time sweep. With time going, the

393

increasing gap between G' and G'' demonstrated the strength of adsorbed SPH55 film

394

and more “gel-like” properties. The interfacial storage modulus G' was nearly one

395

order magnitude higher than loss modulus G'' and this trend did not change

396

remarkably in the whole measurement range, which proved that the higher ratio of

397

β-subunits and basic subunits in hydrolysates favored the formation of gel structure

398

by the association between molecules (Patino, Ortiz, Sánchez, Niño, & Añón, 2003).

399

Moreover, the highest storage modulus G' of SPH55 observed in all samples showed

400

that its film had the best mechanical properties against draining, coalescence and

401

rupturing. All samples except SPH95 demonstrated “solid-like” properties for

402

adsorbed protein layers at air-water interface (G' > G'') and no interfacial gel transition

403

(no cross-over between G' and G'') during the time sweep. The adsorbed protein layer

404

of SPH95 exhibited “fluid-like” properties at the beginning of the sweep and it took

405

some time to get cross-over between G' and G'' at air-water interface, indicating the

406

SPH95 protein molecules needed time to form a layer with some “solid-like”

407

characteristics. As discussed in Section 3.2, the loss of β-subunit and basic subunit

408

during the treatment can explain this difference.

409

The fracture mechanism and structure strength of the films formed by SPI

410

hydrolysates were investigated by strain sweeps (Fig.5). All the samples exhibited

411

similar trend whereby a plateau in a regime of linear viscoelasticity at the low strain

412

was observed followed by a rapid drop at the high strain due to some fractures in the

413

absorbed protein layer structure. The drop point of the dynamic storage modulus G' is

414

the point where the destroyed and newly formed bonds were balanced. The interfacial

415

layers formed by the control were relatively fragile compared to other sample. The

416

reason is that the control molecules without pre-heat treatment possessed a more rigid

417

structure and the protein was not unfolded, so the film was weak against external

418

strain sweeps. The unfolded protein molecules induced by pre-heat treatment

419

contributed to structural strength of the films.

420

3.6. Foaming capacity and foaming stability

14 | P a g e

421

In soy protein hydrolysates dispersions, when air was incorporated into protein liquid

422

dispersion, the formation of a cohesive air-water interfacial film in rapid rate was a

423

prerequisite for desirable foaming capacity. Fig.6 showed that SPH55 displayed the

424

best foaming capacity and stability amongst all the samples. There was a significant

425

drop in the foaming capacity and stability when the pre-heat temperature was above

426

75°C. SPH55 molecules rapidly adsorbed at air-water interface with more exposed

427

hydrophobic subunits such as β-subunit and basic subunit (Table 1) and higher

428

proportion of small molecule peptides (Fig.2). The adsorption of SPH55 molecules

429

may not undergo a molecular unfolding step. As a result, the formation of adsorbed

430

protein film at air-water interface was accelerated (Davis & Foegeding, 2007), which

431

agreed with the results shown as the highest value of slope (K) in Section 3.5. As

432

discussed in Section 3.2, 55°C pre-heat treatment altered the ratio of 7S/11S in soy

433

protein molecules (Table 1) because pre-heat treatment promoted the hydrolysis of

434

acidic subunit and the increase of ratio of hydrophobic residues such as β-subunits

435

and basic subunit in the hydrolysates, which facilitated the initial anchoring of SPH55

436

molecules to the air phase and formed a high viscoelastic film. Moreover,

437

pepsin-driven hydrolysis produced a certain amount of small molar mass peptides,

438

which also contributed to foaming capacity because of its flexible molecular structure,

439

small molar mass and amphiphilic properties. According to Damodaran (2005), an

440

effective foaming agent requires protein molecules to be adsorbed at the air-water

441

interface rapidly. As indicated in Fig 3, the proportion of small molar mass peptides

442

(less than 10 kDa) was the second largest among all samples. Although higher

443

pre-treatment temperature (above 65°C) would accelerate the exposure of more

444

enzyme accessible sites compared to SPH55, the hydrophobic subunits formed larger

445

molecular aggregates with increasing DH and hydrophobicity (Kuipers & Gruppen,

446

2008). Intact soy protein structure or large molecular aggregates under high

447

temperature treatment (above 65°C) diffused slowly to the air-water interface,

448

indicating as decreasing foaming capacity.

449

Foam destabilization is caused by liquid drainage, disproportionation with growing of 15 | P a g e

450

larger bubbles and coalescence (Talansier et al., 2009). Good viscoelastic and

451

mechanical properties of film with favorable intermolecular interactions can resist a

452

high Laplace pressure in small bubbles against the rupture of foam. SPH55 provided

453

the most stable foams compared with other samples (Fig.6), indicating that pre-heat

454

treatment temperature had a good correlation with the strength of the interfacial film.

455

Combination of 55°C pre-heat treatment with pepsin-driven hydrolysis was desirable

456

for maintaining stable foam (Fig.6). When more hydrophobic subunits were present at

457

air-water interface, they were sufficient to enable the formation of a “gel-like” layer

458

via the formation of intermolecular polymers and prevent the collapse of air bubbles.

459

Additionally, adsorbed protein layers created disjoining pressure to increase the

460

thickness of liquid film and prevented liquid drainage in bubbles (Damodaran, 2005).

461

The disjoining pressure is the development of an osmotic pressure between the bulk

462

phase and the lamella fluid (Damodaran, 2005). When two bubbles approach each

463

other, higher surface excess (Γ) concentration would lead to disjoining pressure.

464

SPH55 has the highest surface excess (Γ) concentration at air-water interface,

465

indicating it had the highest disjoining pressure amongst all the samples. As a result,

466

water molecules would migrate from low-pressure area to high-pressure area, which

467

prevented liquid drainage when two bubbles approach.

468 469

4.0. Conclusions

470

In this study, the composition of soy protein hydrolysates can be manipulated through

471

a combination of pre-heat treatment and enzymatic treatment driven by pepsin to

472

prepare a foaming agent with good foaming properties. The results demonstrated that

473

55°C pre-heat treatment promoted the exposure of more accessible sites for pepsin

474

compared to the control. At the same time, increasing ratio of β-subunits and basic

475

subunits in SPH55 led to more surface excess (Γ) concentration and contributed to the

476

mechanical strength of the adsorbed protein film at the air-water interface. Moreover,

477

high ratio of 7S/11S of SPH55 and the presence of high ratio of small molecule

478

peptides facilitated its molecular flexibility and rapid adsorption at the interface. All

479

of them contributed to foaming capacity and stability. The aggregates in hydrolysates 16 | P a g e

480

induced by high temperature treatment (above 65°C) deteriorated foaming capacity

481

and stability. This finding provides useful insight regarding the relationship of soy

482

protein composition and its foaming properties.

483 484 485 486

Conflict of interest

487 488

The authors state that there are no conflicts of interest regarding publication of this article.

489

Acknowledgement

490 491 492

The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant No. 31471583) and National First-class discipline program of Food Science and Technology (Grant No. JUFSTR20180201).

493 494

Reference

495

Achouri, A., Zhang, W., & Shiying, X. (1998). Enzymatic hydrolysis of soy protein

496

isolate and effect of succinylation on the functional properties of resulting

497

protein hydrolysates. Food Research International, 31(9), 617-623.

498

Adebowale, Y. A., Schwarzenbolz, U., & Henle, T. (2011). Protein isolates from

499

Bambara groundnut (Voandzeia Subterranean L.): Chemical characterization and

500

functional properties. International Journal of Food Properties, 14(4), 758-775.

501

Anderson, J. J., Anthony, M. S., Cline, J. M., Washburn, S. A., & Garner, S. C. (1999).

502

Health potential of soy isoflavones for menopausal women. Public health

503

nutrition, 2(4), 489-504.

504 505 506 507

Campbell, G. M., & Mougeot, E. (1999). Creation and characterisation of aerated food products. Trends in food science & technology, 10(9), 283-296. Chang, S.K.C. (2014). Protein Analysis. In S.Suzanne, Nielsen (Eds), Food Analysis (pp.135-144). New York, Springer.

508

Chen, L., Chen, J., Ren, J., & Zhao, M. (2011). Effects of ultrasound pretreatment on

509

the enzymatic hydrolysis of soy protein isolates and on the emulsifying

510

properties of hydrolysates. Journal of Agricultural and Food Chemistry, 59(6),

511

2600-2609. 17 | P a g e

512

Chen, W., Liang, G., Li, X., He, Z., Zen, M., Gao, D.et al. (2019). Impact of soy

513

proteins, hydrolysates and monoglycerides at the oil/water interface in emulsions

514

on interfacial properties and emulsion stability. Colloids and Surfaces B:

515

Biointerfaces, 177, 550-558.

516

Cui, C., Zhao, M., Yuan, B., Zhang, Y., & Ren, J. (2013). Effect of pH and pepsin

517

limited hydrolysis on the structure and functional properties of soybean protein

518

hydrolysates. Journal of food science, 78(12), C1871-C1877.

519 520

Damodaran, S. (2005). Protein stabilization of emulsions and foams. Journal of Food Science, 70(3), 54-66.

521

Davis, J. P., & Foegeding, E. A. (2007). Comparisons of the foaming and interfacial

522

properties of whey protein isolate and egg white proteins. Colloids and Surfaces

523

B: Biointerfaces, 54(2), 200-210.

524

Dickinson, E. (2003). Interfacial, emulsifying and foaming properties of milk proteins.

525

In Advanced Dairy Chemistry—1 Proteins (pp. 1229-1260). Springer, Boston,

526

MA.

527

Dickinson, E., & Matsumura, Y. (1991). Emulsifying and surface properties of the

528

11S and 7S globulins of soybean. Food polymers, gels and colloids,.498-502.

529

Foegeding, E. A., Luck, P. J., & Davis, J. P. (2006). Factors determining the physical

530 531

properties of protein foams. Food hydrocolloids, 20(2-3), 284-292. Harnedy, P. A., Parthsarathy, V., McLaughlin, C. M., O'Keeffe, M. B., Allsopp, P. J.,

532

McSorley,

E.

M.

et

al.

(2018).

Atlantic

salmon

(Salmo

salar)

533

co-product-derived protein hydrolysates: A source of antidiabetic peptides.

534

Food Research International, 106, 598-606.

535

He, Z., Li, W., Guo, F., Li, W., Zeng, M., & Chen, J. (2015). Foaming characteristics

536

of commercial soy protein isolate as influenced by heat-induced aggregation.

537

International Journal of Food Properties, 18(8), 1817-1828.

538

Iwabuchi, S., Watanabe, H., & Yamauchi, F. (1991). Thermal denaturation of.

539

beta.-conglycinin. Kinetic resolution of reaction mechanism. Journal of

540

agricultural and food chemistry, 39(1), 27-33.

541

Jung, S., Roussel℃Philippe, C., Briggs, J. L., Murphy, P. A., & Johnson, L. A. (2004).

542

Limited hydrolysis of soy proteins with endo℃and exoproteases. Journal of the

543

American Oil Chemists' Society, 81(10), 953.

544

Keerati-u-rai, M., & Corredig, M. (2009). Effect of dynamic high pressure

545

homogenization on the aggregation state of soy protein. Journal of agricultural

546

and food chemistry, 57(9), 3556-3562. 18 | P a g e

547

Kuipers, B. J., & Gruppen, H. (2008). Identification of strong aggregating regions in

548

soy glycinin upon enzymatic hydrolysis. Journal of agricultural and food

549

chemistry, 56(10), 3818-3827.

550

Lakemond, C. M., de Jongh, H. H., Hessing, M., Gruppen, H., & Voragen, A. G.

551

(2000). Soy glycinin: influence of pH and ionic strength on solubility and

552

molecular structure at ambient temperatures. Journal of Agricultural and Food

553

Chemistry, 48(6), 1985-1990.

554

Lam, R. S., & Nickerson, M. T. (2013). Food proteins: a review on their emulsifying

555

properties using a structure–function approach. Food chemistry, 141(2), 975-984.

556

Li, W., Zhao, H., He, Z., Zeng, M., Qin, F., & Chen, J. (2016). Modification of soy

557

protein hydrolysates by Maillard reaction: Effects of carbohydrate chain length

558

on structural and interfacial properties. Colloids and Surfaces B: Biointerfaces,

559

38, 70-77.

560

Liu, G., Xiong, Y. L., & Butterfield, D. A. (2000). Chemical, physical, and gel℃

561

forming properties of oxidized myofibrils and whey℃and soy℃protein isolates.

562

Journal of Food Science, 65(5), 811-818.

563

Morris, V. J., & Gunning, A. P. (2008). Microscopy, microstructure and displacement

564

of proteins from interfaces: implications for food quality and digestion. Soft

565

569

Matter, 4(5), 943-951. Murray, B. S. (1998). Interfacial rheology of mixed food protein and surfactant adsorption layers with respect to emulsion and foam stability. In Studies in Interface Science (Vol. 7, pp. 179-220). Elsevier. Nicorescu, I., Vial, C., Talansier, E., Lechevalier, V., Loisel, C., Della Valle, D. et al.

570

(2011). Comparative effect of thermal treatment on the physicochemical

571

properties of whey and egg white protein foams. Food Hydrocolloids, 25(4),

572

797-808.

566 567 568

573 574

Nielsen, N.S. (1985). Structure of soy proteins. In: New Protein Foods. Altschul AM, Wilcke HL, editors. Vol 5. New York: Academic Press. pp 27-64.

575

Ortiz, S. E. M., & Wagner, J. R. (2002). Hydrolysates of native and modified soy

576

protein isolates: structural characteristics, solubility and foaming properties.

577

Food Research International, 35(6), 511-518.

578

Panyam, D., & Kilara, A. (1996). Enhancing the functionality of food proteins by

579

enzymatic modification. Trends in food science & technology, 7(4), 120-125.

580

Patino, J. M. R., Niño, M. R. R., & Sánchez, C. C. (2003). Protein–emulsifier

581

interactions at the air–water interface. Current Opinion in Colloid & Interface

582

Science,8(4-5), 387-395. 19 | P a g e

583

Patino, J. M. R., Ortiz, S. E. M., Sánchez, C. C., Niño, M. R. R., & Añón, M. C.

584

(2003). Dynamic properties of soy globulin adsorbed films at the air–water

585

interface. Journal of colloid and interface science, 268(1), 50-57.

586 587 588

Petruccelli, S., & Anon, M. C. (1995). Thermal aggregation of soy protein isolates. Journal of Agricultural and Food Chemistry, 43(12), 3035-304 Puppo, C., Chapleau, N., Speroni, F., de Lamballerie-Anton, M., Michel, F., Añón, C.,

589

&

Anton,

M.

et

al.

(2004).

Physicochemical

modifications

of

590

high-pressure-treated soybean protein isolates. Journal of Agricultural and Food

591

Chemistry, 52(6), 1564-1571.

592

Qiao, X., Miller, R., & Sun, K. (2017). Interfacial adsorption, viscoelasticity and

593

recovery of silk fibroin layers at different oil/water interface. Colloids and

594

Surfaces A: Physicochemical and Engineering Aspects, 519, 179-186.

595

Renkema, J. M., Lakemond, C. M., De Jongh, H. H., Gruppen, H., & van Vliet, T.

596

(2000). The effect of pH on heat denaturation and gel forming properties of soy

597

proteins. Journal of Biotechnology, 79(3), 223-230.

598 599

Sadoc, J. F., & Rivier, N. (Eds.). (2013). Foams and emulsion. Springer Science & Business Media, (Chapter XVII).

600

Scilingo, A. A., & Añón, M. C. (1996). Calorimetric study of soybean protein isolates:

601

effect of calcium and thermal treatments. Journal of agricultural and food

602

chemistry, 44(12), 3751-3756.

603

Shao, Y. Y., Lin, K. H., & Kao, Y. J. (2016). Modification of foaming properties of

604

commercial soy protein isolates and concentrates by heat treatments. Journal of

605

food quality, 39(6), 695-706.

606

Shao, Y., & Tang, C. H. (2014). Characteristics and oxidative stability of soy

607

protein-stabilized oil-in-water emulsions: Influence of ionic strength and heat

608

pretreatment.Food Hydrocolloids, 37, 149-158.

609

Spellman, D., McEvoy, E., O’cuinn, G., & FitzGerald, R. J. (2003). Proteinase and

610

exopeptidase hydrolysis of whey protein: Comparison of the TNBS, OPA and pH

611

stat methods for quantification of degree of hydrolysis. International Dairy

612

Journal, 13(6), 447-453.

613

Talansier, E., Loisel, C., Dellavalle, D., Desrumaux, A., Lechevalier, V., & Legrand, J.

614

(2009). Optimization of dry heat treatment of egg white in relation to foam and

615

interfacial properties. LWT-Food Science and Technology, 42(2), 496-503.

616

Tsumura, K., Saito, T., Tsuge, K., Ashida, H., Kugimiya, W., & Inouye, K. (2005).

617

Functional properties of soy protein hydrolysates obtained by selective 20 | P a g e

618

proteolysis. LWT-Food Science and Technology, 38(3), 255-261.

619

Utsumi, S., Damodaran, S., & Kinsella, J. E. (1984). Heat-induced interactions

620

between soybean proteins: preferential association of 11S basic subunits and.

621

beta. subunits of 7S. Journal of Agricultural and Food Chemistry, 32(6),

622

1406-1412.

623

Wagner, J. R., & Gueguen, J. (1999). Surface functional properties of native,

624

acid-treated, and reduced soy glycinin. 2. Emulsifying properties. Journal of

625

Agricultural and Food Chemistry, 47(6), 2181-2187.

626

Were, L., Hettiarachchy, N. S., & Kalapathy, U. (1997). Modified soy proteins with

627

improved foaming and water hydration properties. Journal of Food Science,

628

62(4), 821-824.

629 630

Worthington (1993). Worthington Enzyme Manual: Enzymes, Enzyme Reagents, Related Biochemicals, Freehold. New Jersey: Worthington.

631

Yamauchi, F., Yamagishi, T., & Iwabuchi, S. (1991). Molecular understanding of

632

heat℃induced phenomena of soybean protein. Food Reviews International, 7(3),

633

283-322.

634

Zeng, M., Adhikari, B., He, Z., Qin, F., Huang, X., & Chen, J. (2013). Improving the

635

foaming properties of soy protein isolate through partial enzymatic hydrolysis.

636

Drying technology,31(13-14), 1545-1552.

637 638 639 640

List of Figures

641

Fig.1 Degree of hydrolysis and percentage of soluble protein hydrolysate. Data are

642

means (n=3) ± standard deviation; Values with different superscript letter are

643

different P < 0.05. Abbreviation: DH: degree of hydrolysis, PSP: percentage of

644

soluble protein. 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95: SPH95. A-F is

645

used for DH and a-e is used for PSP.

646 647

Fig.2 SDS-PAGE patterns of hydrolysates subjected to different temperature pre-heat

648

treatment. Samples for SDS-PAGE were without β-mercaptoethanol (A) or with

649

β-mercaptoethanol (B). MW: molar mass marker (Da). β-Conglycinin: α' (86 kDa), ɑ

650

(66 kDa), and β (51kDa); Glycinin: A, acidic subunit (34-43 kDa), and B, basic 21 | P a g e

651

subunit (17-26 kDa). Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85,

652

95: SPH95, C: Control, SPI: Untreated soy protein isolate as reference.

653 654 655 656 657 658 659

Fig.3 Molar mass distribution of hydrolysates (a) High performance size exclusion chromatographic profiles of hydrolysates (A: > 669 kDa; B: 100– 669 kDa; C: 10– 100 kDa; D: <10 kDa). The equation of the standard curve was y =-0.4708x+6.6162; R2= 0.9856. (b) Relative areas (%) of the four protein fractions (A, B, C, and D) of hydrolysates. Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95: SPH95.

660 661

Fig. 4 The time evolution of the interfacial storage modulus G' and loss modulus G''

662

of layer at the air/water interface obtained from the time sweep. Abbreviation: A)

663

Control; B) SPH55; C) SPH65; D) SPH75; E) SPH85; F) SPH95.

664 665

Fig.5 Strain dependence of interfacial elastic modulus G' of hydrolysates at the

666

air/water interface. Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95:

667

SPH95.

668 669 670 671 672 673 674 675 676

Fig.6 Foaming capacity and stability of SPI hydrolysates. Data are means (n=3) ± standard deviation.Values with different superscript letter are significantly different P<0.05. Abbreviation: 55: SPH55, 65: SPH65, 75: SPH75, 85: SPH85, 95: SPH95. A-E is used for foaming capacity and a-e is used for foaming stability.

22 | P a g e

1/1

CRediT author statement

Guijiang Liang: Conceptualization, Methodology, Software, Formal analysis,Writing original draft Wenpu Chen:Data curation, Writing- Original draft preparation, Investigation Xuejiao Qie: Writing - Review & Editing, Visualization Maomao Zeng: Writing - Review & Editing, Resources Fang Qin: Investigation, Resources Zhiyong He: Supervision Jie Chen: Supervision

Table 1 Relative Compositions (%) of SPI hydrolysates

-βME (Non-reducing condition) 7S

+βME (Reducing condition)

11S

7S/11S

α'

α

β

A

B

Control

6.4

4.7

8.2

6.2

27.3

SPH55

6.5

4.8

7.8

3.1

SPH65

3.1

2.3

5.5

SPH75

1.9

2.0

4.6

SPH85

1.6

2.0

SPH95

1.4

2.3

7S

11S

7S/11S

α'

α

β

A

B

0.58

14.1

11.8

10.7

16.1

10.8

1.36

26.3

0.65

12.3

11.5

10.1

13.9

11.6

1.33

4.6

27.2

0.34

7.2

7.9

7.7

19.5

14.2

0.68

4.9

28.5

0.25

5.8

5.9

6.7

21.8

17.0

0.47

2.9

4.8

27.5

0.20

6.4

6.4

4.7

25.8

19.1

0.39

2.5

5.2

17.3

0.28

6.3

6.2

4.9

29.7

15.8

0.38

*β-Conglycinin: α' (86 kDa), ɑ (66 kDa), and β (51kDa); Glycinin: A, acidic subunit (34-43 kDa), and B, basic subunit (17-26 kDa).

Table 2 Surface properties of hydrolysates Sample

CMC

Surface tension

Control

12.23±2.51

SPH55

13.57±0.96

SPH65

14.66±2.13

SPH75

14.86±1.53

SPH85

14.54±1.34

SPH95

16.68±1.16

2

γ (mN/m)

(mg/ml) a a a a a a

47.89±0.90 46.40±1.41 47.05±1.35 46.60±1.26 46.67±0.76 47.24±2.17

Surface excess Γ (mol/m )

a a a a a a

0.84±0.09

ab

1.22±0.23 0.99±0.27 0.88±0.29 0.62±0.22

a

Molecular area A(nm2) ab

1.97±0.34 a 1.35±0.15

ab

1.67±0.22

ab

ab

1.87±0.13

ab

ab

2.65±0.96

ab

b

3.64±0.23

0.45±0.16

b

* Suface excess (Γ) and molecular area (A), were calculated from the critical micelle concentration (CMC) and suface tension (γ). The experiments were performed in three replicates. The result were expressed as mean values (n=3) ± standard deviation. Values with different superscript letter are significantly different at P<0.05.

Highlight: 

The hydrolysate with pre-heat treatment at 55 °C had the highest ratio of 7S/11S.



β subunit and basic subunits contributed to the desirable foaming properties.



Heat treatment (above 65°C) deteriorated hydrolysates' foaming capacity and stability.

1

Conflict of interest The authors state that there are no conflicts of interest regarding publication of this article.