Foam and conformational changes of egg white as affected by ultrasonic pretreatment and phenolic binding at neutral pH

Journal Pre-proof Foam and conformational changes of egg white as affected by ultrasonic pretreatment and phenolic binding at neutral pH Yinxia Chen, Meihu Ma PII:

S0268-005X(19)31710-2

DOI:

https://doi.org/10.1016/j.foodhyd.2019.105568

Reference:

FOOHYD 105568

To appear in:

Food Hydrocolloids

Received Date: 28 July 2019 Revised Date:

2 December 2019

Accepted Date: 3 December 2019

Please cite this article as: Chen, Y., Ma, M., Foam and conformational changes of egg white as affected by ultrasonic pretreatment and phenolic binding at neutral pH, Food Hydrocolloids (2020), doi: https:// doi.org/10.1016/j.foodhyd.2019.105568. 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. © 2019 Published by Elsevier Ltd.

Graphical abstract

1

Foam and conformational changes of egg white as

2

affected by ultrasonic pretreatment and phenolic

3

binding at neutral pH

4 Yinxia Chen, Meihu Ma*

5 6 7

National Research and Development Center for Egg Processing, College of Food

8

Science and Technology, Huazhong Agricultural University, Wuhan, Hubei

9

420070, PR China.

10 11

*: Corresponding author: Meihu Ma

12

College of Food Science and Technology of Huazhong Agricultural University,

13

Wuhan, Hubei 420070, PR China

14

Tel: +86 27 87283177

15

Fax: +86 27 87283177

16

E-mail address: [email protected]

17

1

Abstract

18 19

This study investigated the impact of ultrasound pretreatment and phenolic

20

binding on the structure characteristic and foaming properties of egg white (EW). EW

21

treated without ultrasound (NEW) and with ultrasound (UEW) (power for 28% and

22

time for 25 min: on-time 3 s and off-time 2 s) were incubated separately at 25 oC for 2

23

h with gallic acid (GA) and epigallocatechin gallate (EGCG) at pH 7.0. Phenolic

24

treatment caused a significant loss of sulfhydryl content, with a more remarkable

25

effect observed in UEW, especially at 120 µmol/g concentration. Both phenolics

26

significantly decreased the surface hydrophobicity and slightly increased the

27

disordered secondary structure of protein. Additionally, the microenvironment polarity

28

of protein molecules was increased by phenolic treatment corroborated by UV

29

absorption blue shift, especially for 240 µmol/g GA-UEW. After ultrasound

30

pretreatment, low EGCG concentration (20 µmol/g) significantly increased the

31

foaming ability. The 240 µmol/g EGCG treatment notably increased the reduced

32

foaming stability by ultrasound from 81.00 to 95.10% (p < 0.05), which was due to

33

high absolute ξ-potential value. This study has shed light on the mechanisms

34

underlying the influence of unfolding structure by ultrasound treatment on phenolic

35

binding.

36

Keywords: Egg white; Ultrasound; Phenolic binding; Foaming properties; Structural

37

characteristic.

38

2

39

1. Introduction

40

Egg white displays multiple functional properties, such as foaming, emulsifying

41

and gelling (Singh & Ramaswamy, 2015). The foaming property is an important

42

parameter in aerated food products, and a food product with an excellent foaming

43

property means a desirable structure and unique texture with higher product volume.

44

The foaming properties of egg white (EW) can be affected by factors, such as shell

45

egg storage, pH, temperature and ionic strength (Sheng et al., 2018b). Although cold

46

storage could inhibit harmful microorganisms and restrict quality deterioration, the

47

foaming ability of egg white decreased during storage duration (Sheng et al., 2018a;

48

Chen, Sheng, Gouda, & Ma, 2019).

49

Foams are thermodynamically unstable due to the effects of drainage,

50

coalescence, and disproportionation. Foam stability is an important indicator for the

51

accessibility of a protein as surface-active agent, and the poor foam stability induced

52

by the low concentration of protein cannot meet the needs of food industry. During

53

the process of whipping, the amphiphilic protein molecules undergo unfolding at the

54

air-water interface, and the process is largely affected by different protein structures

55

(Li, Sun, Ma, Jin, & Sheng, 2018; Sheng et al., 2019). Among the main proteins

56

present in egg white, ovalbumin is the only protein that has free -SH groups,

57

containing a single disulfide bond between Cys74 and Cys121. Ovotransferrin and

58

lysozyme possess 15 and 4 disulfide bonds, respectively. Ovomucoid molecule

59

contains three distinct domains crosslinked only by intradomain disulfide bonds. 3

60

Ovomucin is a biopolymer cross-linked by disulfide bonds (Mine, 2014). These

61

sulfhydryl group and disulfide bond are important for protein structure stability.

62

The application of ultrasound to improve the functional properties of protein is

63

increasingly studied. Sheng et al. (2018b) reported that the highest foaming capacity

64

(4.9-fold versus the control group) of EW was obtained after 360 W ultrasound

65

treatment. Additionally, the foam capacity and stability of ultrasound-treated wheat

66

gluten protein gradually increased with the increase of treatment power (Zhang,

67

Claver, Zhu, & Zhou, 2011). Another study showed that emulsion stability was

68

improved by ultrasonic irradiation, due to an improvement in the interfacial layer

69

(O’Sullivan, Beevers, Park, Greenwood, & Norton, 2015). The obtained gels by

70

high-energy ultrasonic (20 kHz) had much higher water-holding capacity (WHC) than

71

untreated gels (Zisu et al., 2011). The ultrasound process is mainly related to

72

cavitation, dynamic agitation, shear stress and turbulence (O’Donnell, Tiwari, Bourke,

73

&

74

microenvironmental changes around tryptophan residues as indicated by the increased

75

intrinsic fluorescence (Xiong et al., 2016) and the decreased α-helix content (Zou et

76

al., 2018).

Cullen,

2010).

Furthermore,

ultrasound

treatment

could

result

in

77

Phenolic compounds are commonly present in fruits and vegetables, with

78

different modulatory activities beneficial to human health (Cao & Xiong, 2017a),

79

especially the antioxidant activity. Meanwhile, phenolic treatment could enhance the

80

functional properties of protein. Previous studies showed that the foaming properties 4

81

of WPI could be significantly improved by GA or EGCG treatment, and more

82

phenolic binding sites could be caused by preheating treatment due to the exposure of

83

more groups from the heat-unfolded structure (Cao, Xiong, Cao, & True, 2018).

84

Combined with high hydrostatic pressure, tea polyphenols increased the protein

85

solubility and emulsifying activity (Chen, Wang, Feng, Jiang, & Miao, 2019).

86

Different varieties of instant green tea were all shown to increase the foaming and

87

gelling properties of egg white (Wu, Clifford, & Howell, 2007). Furthermore, GA and

88

EGCG were reported to cause significant structural changes of WPI, with the binding

89

of WPI with EGCG being stronger than that of GA at pH 7.0 (Cao & Xiong, 2017b).

90

High foaming ability but low foaming stability of protein were shown by

91

ultrasound treatment, meanwhile phenolic was reported to have a positive effect on

92

stability. Generally, controlled ultrasound treatment could cause proteins to unfold or

93

partially

94

hydrophobic-hydrophilic balance, and thus forming a modified interfacial property

95

(Chen, Sheng, et al., 2019). On the other hand, phenolic compounds can interact with

96

proteins in food systems to induce protein structural change at neutral pH (Zhang &

97

Zhong, 2012). Since hydrophobic interaction is one of the primary force involved in

98

protein-phenolic binding (Ozdal, Capanoglu, & Altay, 2013), ultrasound treatment of

99

proteins is expected to affect their subsequent interaction with phenolic compounds.

100

Moreover, the protein structural changes induced by phenolic addition have great

101

influence on the functional properties and biological activity (Wu et al., 2015; Wu et

unfold,

aggregate,

variation

5

in

molecular

flexibility

and

102

al., 2019). Despite extensive research on improving the foaming properties of proteins

103

by ultrasound or phenolic binding alone, little information is available on the

104

interaction between phenolic compound and egg white under the ultrasound condition.

105

The aim of this study was to evaluate the impact of ultrasound pretreatment (power:

106

28%, time: 25 min, on-time 3 s and off-time 2 s) combined with phenolic binding (GA

107

or EGCG treatment) at pH 7.0 on the foaming and structural characteristic of egg

108

white (EW). Results of this research will provide useful information for improving the

109

foaming properties of phenolic-treated egg white by ultrasound and broadening the

110

application of egg white in the food processing.

111

2. Material and methods

112

2.1. Materials

113

Fresh Hy-Line Brown chicken eggs were purchased from Jiufeng Xinyue

114

Chicken Farm (Wuhan, China). Eggs stored (4 ± 1 oC) for 4 months were used in this

115

study for improving the foaming properties of egg white from stored shell egg. Gallic

116

acid (GA, purity 99.9%) (PubChem CID: 370) and epigallocatechin gallate (EGCG,

117

purity 98.3%) (PubChem CID: 65064) were purchased from Shanghai Yuanye

118

Biotechnology Co., Ltd (Shanghai, China). 8-Anilino-1-naphthalenesulfonic acid

119

(ANS) (PubChem CID: 1369) was purchased from Aladdin Chemical Reagent Co.

120

(Shanghai, China), and 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) (PubChem CID:

121

6254) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The analytical

122

grade sodium dodecyl sulfate (SDS) (PubChem CID: 3423265), urea (PubChem CID: 6

123

1176), glycine (PubChem CID: 750), ethylenediaminetetraacetic acid (EDTA)

124

(PubChem CID: 6049), (hydroxymethyl) aminomethane (Tris) (PubChem CID: 6503)

125

and potassium bromide (KBr) (PubChem CID: 253877) were purchased from

126

Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Other reagents used in this

127

study were all of analytical grade.

128

2.2. Ultrasonic pretreatment

129

Eggs were washed cleanly and broken manually to separate egg white from egg

130

yolk. Next, the egg white was stirred gently at 4 oC for 1 h using a magnetic stirrer

131

(IKA, IKA Works Inc., Wilmington, NC, USA). After adjustment to pH 7.0 with 1

132

mol/L HCl, the egg white was subjected to ultrasonic pretreatment as described in our

133

previous research (Chen, Sheng, et al., 2019) considering the energy conservation and

134

improving effect. Briefly, an ultrasound processor (JY 92-IIN, Scientz, Zhejiang,

135

China) equipped with a 6 mm titanium probe was inserted into 80 mL of egg white in

136

100 mL breaker and operated at power of 28% and time of 25 min, pulse duration of

137

on-time 3 s and off-time 2 s. The whole ultrasonic process was performed in an ice

138

water bath to prevent protein denaturation. The egg white treated with and without

139

ultrasound was named as UEW and NEW, respectively.

140

2.3. Phenolic treatment

141

Egg white, GA and EGCG stock solutions were prepared using phosphate buffer

142

(10 mmol/L, pH 7.0) and stored at 4 oC. Briefly, fresh GA and EGCG solutions (10

143

mmol/L) were prepared and diluted to specific concentrations with phosphate buffer 7

144

(10 mmol/L, pH 7.0) according to the requirements of different tests. Next, NEW or

145

UEW (10 mg/mL, final concentration) was mixed with phenolic solution (GA or

146

EGCG) at different concentrations (20, 120 and 240 µmol/g, protein basis, final

147

concentration). Finally, the mixtures of protein-phenolic solutions were incubated in a

148

25 oC for 2 h (Cao et al., 2018).

149

2.4. Sulfhydryl group analysis

150

The sulfhydryl (SH) content was determined as reported by Chen, Wang, Ma, et

151

al. (2019) with some modifications. The sulfhydryl groups were detected by the

152

reaction of protein with Ellman’s reagent (DTNB).

153

Surface free sulfhydryl content was measured as follows. Briefly, 0.5 mL of the

154

protein-phenolic reaction solution was mixed with 4.5 mL of standard buffer (0.5%

155

(m/v) sodium dodecyl sulfate, 0.086 mol/L Tris, 0.092 mol/L Glycine and 0.004

156

mol/L EDTA, pH 8.0). Then, the reaction was initiated by adding the mixtures to 0.05

157

mL of Ellman’s reagent (4 mg/mL DTNB in Tris-Glycine buffer, pH 8.0). Meanwhile,

158

the total SH content was measured using the same procedure as described above but

159

with a denaturing buffer containing the standard buffer plus 8 mmol/L urea. After

160

incubation in dark at room temperature for 15 min, the absorbance of the mixture was

161

measured at 412 nm on a UV/VIS spectrophotometer (Nanodrop-2000C, Thermo

162

Scientific, USA). The recorded absorbance was used to calculate the SH content

163

according to the following equation:

164

SH ( µmol/g protein) = 73 .53 × A412 × D/C 8

(1)

165

where A412 is the absorbance of the sample at 412 nm, D is the dilution factor, and C

166

is the concentration of the sample.

167

2. 5. Surface hydrophobicity (H0)

168

Change in the H0 of NEW or UEW sample after GA or EGCG treatment was

169

measured using 8-anilino-1-naphthalenesulfonic acid (ANS) as a fluorescence probe.

170

H0 was determined as described by Cao et al. (2018). Specifically, the

171

protein-phenolic reaction solutions were diluted to five concentrations (0.05, 0.10,

172

0.15, 0.20 and 0.25 mg/mL) using phosphate buffer (10 mmol/L, pH 7.0). Then, 20

173

µL of 8 mmol/L ANS in the same buffer was added to 4 mL of each diluted sample

174

solution. Finally, the fluorescence intensities of samples with and without ANS were

175

measured at an excitation wavelength of 390 nm and an emission wavelength of 470

176

nm (5 nm slit width) using a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan).

177

H0 is defined as the initial slope of the linear regression curve of the fluorescence

178

intensity as a function of protein concentration.

179

2. 6. UV spectral analysis

180

The protein-phenolic reaction solution was diluted to 0.2 mg/mL using phosphate

181

buffer (10 mmol/L, pH 7.0). The UV absorption spectra of diluted solution were

182

recorded in a 1.0 cm quartz cuvette from 240 to 340 nm at 25 oC with the

183

corresponding phenolic solution as background subtraction using a UV/VIS

184

spectrophotometer (Nanodrop-2000C, Thermo Scientific, USA).

185

2. 7. Circular dichroism (CD) analysis 9

186

The protein-phenolic reaction solution was diluted to 0.1 mg/mL using phosphate

187

buffer (10 mmol/L, pH 7.0). The CD spectra were recorded in a 0.1 cm quartz cuvette

188

from 250 to 190 nm at 100 nm/min by subtracting data of corresponding phenolic

189

solution from phenolic-protein sample using a Jasco J-1500 Circular Dichroism

190

Spectrometer (JASCO, Tokyo, Japan) purged with N2. Each spectrum was obtained as

191

an average of three scans to reduce the noise before protein structure analysis and the

192

proportions of α-structure and β-structure were gained by Yang’s equation.

193

2. 8. FTIR analysis

194

The protein-phenolic reaction solution was lyophilized by an Alpha 2-4 LD plus

195

freeze dryer (CHRIST, Germany). The lyophilized samples (2 mg) were mixed with

196

KBr at the ratio of 1: 200 and ground to powder in a mortar with a pestle. The spectra

197

were obtained in the range of 4000-400 cm-1 with an average of 32 scans at a

198

resolution of 4 cm-1. The spectra of free GA and EGCG were also obtained.

199

Background noise was corrected with air data.

200

2. 9. Intrinsic fluorescence spectroscopy measurement

201

The intrinsic fluorescence change induced by phenolic addition was measured to

202

evaluate the binding affinity of phenolic compounds to proteins. The fluorescence

203

quenching analysis was according to Cao and Xiong (2017a). Briefly, diluted protein

204

(0.2 mg/mL in 10 mmol/L phosphate buffer, pH 7.0) was mixed with different

205

concentrations of GA (0, 13.3, 26.6, 53.2, 79.8, 106.4 and 133 µmol/L, final

206

concentration) or EGCG (0, 5.5, 11, 22, 33, 44 and 55 µmol/L, final concentration). 10

207

Next, the mixtures of protein-phenolic solutions were incubated in 25, 30 and 38 oC

208

for 2 h, respectively. Finally, the fluorescence spectra were recorded using a

209

spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). The excitation wavelength

210

was 280 nm and the emission spectra were from 300 to 450 nm with the

211

corresponding phenolic solution as background subtraction (3 nm slit width). The

212

obtained spectra were further analyzed using the Stern-Volmer equation:

213

F 0 / F = 1 + Kqτ0[Q ] = 1 + Ksv[Q ]

(2)

214

where F0 and F are the maximal fluorescence intensity without and with phenolic

215

addition, respectively. Kq is the biomolecular quenching-rate constant (M-1 S-1) and τ0

216

is the lifetime of the fluorophore without a quencher. [Q] is the phenolic concentration

217

(mol/L) and Ksv is the quenching constant (M-1). Finally, Ksv was calculated by linear

218

regression of a plot of F0/F against [Q].

219

2. 10. Fluorescence lifetime measurement

220

To further clarify the quenching mechanism of static and dynamic, the

221

fluorescence lifetime was measured in this study. Diluted protein solution (0.2 mg/mL,

222

final concentration) was mixed with GA (133 µmol/L, final concentration) or EGCG

223

(55 µmol/L, final concentration). Then, the mixture was incubated in 25 oC and the

224

fluorescence intensity decay was performed with Ex: 280 nm and Em: 350 nm using

225

the FLS 980 fluorescence spectrophotometer (FLS 980, Edinburgh Instruments, UK).

226

The fluorescence lifetime was calculated from the single exponential fitting using

227

Origin software (Version 9.0.0). 11

228

2. 11. Solubility measurement

229

The solubility of NEW and UEW before and after incubation with different

230

concentrations of phenolic compounds were measured as described by Jiang, Zhang,

231

Zhao and Liu (2018). Briefly, the protein-phenolic reaction solution was centrifuged

232

at 10,000 r/min for 20 min at 4 oC using a high-speed refrigerated centrifuge (CR 22N,

233

Hitachi, Japan). After centrifugation, the protein concentration in the supernatant and

234

in the original solution were determined using the Biuret method (Itzhaki & Gill,

235

1964). Finally, the solubility is defined as the percentage of protein content over that

236

of total protein content.

237

2. 12. Foaming properties

238

Changes in the foaming properties of NEW or UEW induced by phenolic

239

addition were determined as reported in our previous research (Chen, Sheng, et al.,

240

2019). Specifically, an aliquot volume of protein-phenolic solution (20 mL) was

241

placed in a glass cylinder (internal diameter of 22 mm, height of 150 mm), followed

242

by producing the foam at 8000 r/min for 1 min using a high-speed homogenizer

243

(XHF-DY, Scientz, Zhejiang, China) equipped with a probe (internal diameter of 7.5

244

mm, height of 145 mm). Meanwhile, the foam volumes at 2 and 30 min were recorded.

245

The foaming ability (FA) and foaming stability (FS) were defined according to the

246

following equations:

247

FA(% ) = V 2 / 20 × 100

(3)

248

FS(% ) = V 30 / V 2×100

(4)

12

249

where V2 and V30 are the foam volume at 2 and 30 min after whipping,

250

respectively.

251

2. 13. Determination of particle size and ξ-potential

252

The protein-phenolic reaction solution was diluted to 1 mg/mL using phosphate

253

buffer (10 mmol/L, pH 7.0). The average particle size and size distribution of

254

protein-phenolic complexes were determined by dynamic light scattering (DLS) using

255

a ZS Zetasizer Nano (Malvern Instrument, Ltd., UK). Additionally, the ξ-potential of

256

samples was determined using a ZS Zetasizer Nano (Malvern Instrument, Ltd., UK).

257

All measurements were carried out at room temperature and repeated three times.

258

2. 14. SDS-PAGE analysis

259

Polymerization and depolymerization of EWP treated with phenolic addition

260

were investigated by reduced and non-reduced SDS-PAGE with a 5% stacking gel

261

and a 12% separating gel. The protein-phenolic reaction solution was diluted to 5

262

mg/mL using phosphate buffer (10 mmol/L, pH 7.0). Next, 80 µL of the diluted

263

sample solution was mixed with 20 µL of sample buffer (5×) (reduced and

264

non-reduced). Then, 10 µL of the mixture was loaded in the stacking gel. The

265

electrophoresis ran at a constant voltage (80 V) for about 40 min, followed by 120 V

266

for 1 h. After the run, the gel was fixed in solution (ethanol: glacial acetic acid:

267

distilled water, 5: 1: 4, v/v), and then dyed using dyestuff (Coomassie Brilliant Blue

268

R-250). The obtained gel was washed with destainer (250 mL of 95% ethanol mixed

269

with 80 mL glacial acetic acid to 1000 mL) until background decolorization. 13

270

2. 15. Statistical analysis

271

All measurements were carried out in triplicate and all data were presented as

272

mean ± standard deviation of at least three independent tests. The significance of the

273

results was analyzed using one-way variance (ANOVA) (p < 0.05). Data were

274

compared between different treatments by Duncan’t multiple range test using SPSS

275

statistical software (Version 19.0). Graphs were plotted using Origin software

276

(Version 9.0.0).

277

3. Results and discussion

278

3. 1. Sulfhydryl content changes

279

The sulfhydryl group of protein, especially the free sulfhydryl group of cysteine,

280

has high chemical activity coupled with high reactivity and vulnerability to phenolic

281

molecules. As shown in Fig. 1, both the free and total sulfhydryl content of NEW and

282

UEW showed a gradual downward trend with increasing phenolic concentration at pH

283

7.0. For example, the free SH content of NEW exhibited a significant decrease (p <

284

0.05) of up to 46.48% and 75.81% under the incubation of 120 and 240 µmol/g of GA,

285

respectively (p < 0.05) (Fig. 1 A). At neutral pH, the sulfhydryl groups are easy to

286

deprotonate and form mercaptan anion (RS-) in the presence of phenolic compounds

287

(Strauss & Gibson, 2004), causing a loss of SH content. The SH content changes may

288

also due to the hydrolysis (Wu, Dong, Collins, Babalhavaeji, & Woolley, 2016).

289

Comparatively, an extensive diminishment of SH content was observed when EW was

290

incubated with EGCG solution, especially at 120 µmol/g (Fig. 1), due to more 14

291

hydroxyl groups in the EGCG molecule structure and more structural change induced

292

by EGCG, thus increasing the accessibility of phenolic molecules to sulfhydryl

293

residues (Cao et al., 2017a). This was consistent with the report by Cao and Xiong

294

(2017b) about GA- and EGCG-treated whey protein at pH 7.0. Moreover, with the

295

addition of 20 and 120 µmol/g of phenolic, the UEW displayed a more obvious loss (p

296

< 0.05) in the sulfhydryl content, indicating that ultrasonic pretreatment facilitated the

297

binding of protein to phenolic, due to its effect on the unfolding of protein structure

298

and thus the exposure of more sulfhydryl groups. The same tendency was observed in

299

the total SH content (Fig. 1B).

300

3. 2. Protein structural characterization

301

Changes in the protein structure induced by phenolic addition could be

302

characterized using multiple spectra (UV absorption, CD and FT-IR spectra). The side

303

chain groups of tryptophan (Trp) and tyrosine (Tyr) residues can produce ultraviolet

304

absorption with peaks near 280 nm. The maximum absorption peak location (λmax)

305

was not changed significantly (p > 0.05) with the addition of low concentration (20

306

µmol/g) of phenolic in all tested groups, indicating that the distribution of Trp and Tyr

307

residues was not obviously changed. However, the addition of a high concentration

308

(120 and 240 µmol/g) of phenolic compounds induced not only a remarkable increase

309

in the absorption intensity but also an obvious blue shift (p < 0.05) (Fig. 2A).

310

Generally, the increased absorption intensity was ascribed to the exposure of

311

hydrophobic groups from Trp and Tyr, and the blue shift was attributed to the 15

312

increased microenvironment polarity around Trp and Tyr residues. Moreover,

313

compared with EGCG-treated, the blue shift caused by high phenolic concentration

314

was more obvious for GA-treated samples, indicating a more hydrophilic environment.

315

In the GA treatment, an obvious increase (p < 0.05) was observed in the absorption

316

intensity of the UEW sample, indicating that the exposure of aromatic amino acids

317

was more obvious under ultrasound-assisted treatment (Fig. 2A).

318

Fig. 2B shows the percentage of secondary structure (α-helix, β-sheet, β-turn and

319

random coil) from the CD spectra in the tested groups. For NEW, the GA and EGCG

320

treatments induced a slight and gradual increase in random coil content while a

321

reduction in α-helix and β-structures, suggesting a transition to the disordered

322

structure at the expense of structured motifs (α-helix, β-sheet and β-turn). A similar

323

phenomenon occurred in GA- and EGCG-induced WPI at pH 7.0 (Cao et al., 2018) as

324

well as CA- and EGCG-treated lactoferrin at pH 7.0 (Liu, Wang, Sun, & Gao, 2016).

325

An identical result was observed in the UEW groups, except that the EGCG-treated

326

sample showed a reduction in the random coil content at 120 µmol/g addition,

327

probably due to the dual effects of ultrasound treatment and phenolic structure. The

328

protein structure varies significantly with the structure of phenolic compound. Besides,

329

the UEW groups had more random coil structures than the NEW groups, suggesting

330

that more unfolded structure was formed under the cavitation effect of ultrasound

331

(Sheng et al., 2018b).

332

Fig. 3C shows the FT-IR spectra of NEW and UEW treated with different 16

333

concentrations of GA at pH 7.0. In the spectrum of untreated NEW, two strong bands

334

were observed at 1648.9 and 1542.8 cm-1, corresponding to the vibrations of amide I

335

(1700-1600 cm-1, C=O of the peptide bond) and amide II (1600-1500 cm-1, N-H

336

bending and C-N stretching) (Yakimets et al., 2005), respectively. A new broad

337

absorption band appeared at 3305.4 cm-1 or 3311.2 cm-1 in 120 and 240 µmol/g

338

GA-modified NEW or GA-modified UEW spectra, respectively, attributed to the O-H

339

stretching vibration of phenolic groups (Jia et al., 2016). Hydrogen bonding between

340

aliphatic and aromatic O-H groups, respectively, on protein and GA was observed for

341

O-H stretching since this peak shifted from 3414 cm-1 for protein toward 3305 cm-1

342

and became broad for the phenolic/protein complex (Chen et al., 2010). Therefore, the

343

new peak confirmed the binding of phenolic to protein components in EW partially

344

via the hydrogen bonding (Yang, Liu, Xu, Yuan, & Gao, 2014). However, no new

345

band was observed in the EGCG-treated EW sample (data not shown). In the NEW

346

samples treated by GA at high concentrations (120 and 240 µmol/g), the frequencies

347

of amide I and amide II shifted from 1648.9 to 1652.7 cm-1 and from 1542.8 to 1540.9

348

cm-1, respectively. This phenomenon indicated that the secondary structure of NEW

349

was changed after modification, which was in agreement with the result of CD spectra.

350

The protein structure was loosened by GA or EGCG treatment, which was consistent

351

with the finding in the WPI sample treated with tea polyphenols (Jia et al., 2016).

352

3. 3. Surface hydrophobicity

353

The surface hydrophobicity (H0) of UEW was 4.5% (p < 0.05), which was 17

354

greater than that of NEW due to ultrasonic unfolding (Fig. 3). The GA and EGCG

355

treatments significantly decreased (p < 0.05) the H0 of both NEW and UEW samples

356

(except for 20 µmol/g GA-treated NEW). The H0 of NEW was decreased by 14.61%

357

and 10.95% (p < 0.05) with the addition of 240 µmol/g of GA and EGCG,

358

respectively. Most notably, the H0 of UEW was attenuated by 15.06% and 19.23% (p

359

< 0.05) under the treatment of 240 µmol/g GA and EGCG, respectively. The greater

360

reduction of H0 in UEW than in NEW with phenolic addition was ascribed to the

361

increased exposure of hydrophobic groups (higher H0) in ultrasound-pretreated

362

proteins, thus promoting the hydrophobic stacking interactions between phenolic

363

benzene rings and protein aromatic side chains. There are two possible explanations

364

for the decrease of H0 induced by phenolic addition. On the one hand, the binding of

365

phenolic molecules to hydrophobic group led to the availability of less hydrophobic

366

amino acid residues for ANS probe. On the other hand, the excitation of the ANS

367

probe was inhibited by the increased hydrophilic environment caused by the

368

introduction of extra hydroxyl groups from phenolic structure (aromatic rings bearing

369

one or more hydroxyl groups) to protein molecules (Haskard & Li-Chan, 1998; Cao et

370

al., 2018).

371

3. 4. Intrinsic fluorescence and fluorescence lifetime

372

The binding of GA or EGCG to NEW or UEW at pH 7.0 was assessed indirectly

373

by fluorescence quenching spectroscopy, and the results are displayed in Fig. 4. In all

374

tested groups, an obvious fluorescence loss was observed with increasing 18

375

concentrations of phenolic compounds. Many small molecular ligands can change the

376

microenvironment around the chromophore, thus leading to changes in the intrinsic

377

fluorescence intensity of protein (Wang et al., 2019) since protein intrinsic

378

fluorophores are reported to be susceptible to changes in their microenvironment

379

polarity (Mach & Middaugh, 1994). Another possible explanation is the occurrence of

380

the interaction between phenolics and the major fluorophores (Trp and Tyr). Generally,

381

an increase in the microenvironment polarity of protein molecules will lead to a

382

decrease in the fluorescence intensity and thus a red shift due to energy loss (Cao &

383

Xiong, 2017b).

384

Typically, the quenching type can be divided into static and dynamic quenching.

385

In order to judge the quenching mechanism of static and dynamic, the Stern-Volmer

386

quenching plots at three different temperatures (25, 30 and 38 oC) were applied

387

(attached side). In the slope of the plot linear regression, the Ksv values presented an

388

upward trend with the increasing of temperature for the four groups. For NEW-GA

389

sample, the Ksv value increased from 3.22×103 to 7.06×103 M-1 and for UEW-GA

390

sample, it increased from 6.60×103 to 8.63×103 M-1 when the temperature from 25 to

391

38 oC. Similarly, for NEW-EGCG sample, the Ksv value slightly increased from

392

1.71×104 to 1.85×104 M-1 and for UEW-EGCG sample, it increased from 1.46×104 to

393

2.44×104 M-1 when the temperature from 25 to 38 oC (Fig. 4). The result revealed that

394

the quenching process may be dominated by dynamic quenching. The increase extent

395

was more pronounced for GA treatment, suggesting the more obvious dynamic 19

396

quenching process. For the GA-treated samples, the UEW group displayed a larger

397

quenching constant, indicating that ultrasound pretreatment was beneficial for more

398

hydroxyl groups from GA binding to protein molecules. However, the result was the

399

opposite in the EGCG-treated samples, probably due to more steric hindrance from

400

EGCG structure. Higher Ksv value was observed for EGCG treatment, which

401

indicated that molecular flexibility and free galloyl groups were more favorable to the

402

binding of EGCG to EW than that of GA (Dobreva et al., 2014; Wang, Zhou, Ning, &

403

Zhao, 2016).

404

To further clarify the quenching type, the fluorescence lifetime was also

405

measured and the result was shown in Fig. 5. After GA treatment, the fluorescence

406

lifetime (τ value ) decreased from 3.54 to 2.45 ns for NEW, and from 3.51 to 2.62 ns

407

for UEW. However, slight reduction was obtained on EGCG treatment. The

408

fluorescence lifetime was reduced after the quencher was added, which further

409

indicated that the dynamic quenching was dominant in this quenching process.

410

Therefore, the variable temperature experiment combined with the fluorescence

411

lifetime test explained the dynamic quenching process.

412

3. 5. Solubility and foaming properties

413

High solubility is essential to good functional properties, such as foaming,

414

emulsifying and rheological properties. Fig. 6A shows the solubility of NEW and

415

UEW induced by GA and EGCG treatments. It can be seen that the GA treatment

416

could slightly improve the solubility of NEW and UEW, and the slight increase in the 20

417

water binding potential was attributed to less hydrophobic residues and elevated

418

charges (Chen, Wang, Feng, et al., 2019). Moreover, both the hydroxyl and carboxyl

419

groups of GA could further improve the hydrophilicity of the protein surface (Fig. 2A).

420

However, the solubility of NEW and UEW showed a significant decrease (p < 0.05) in

421

the EGCG-treated samples with increasing phenolic concentration, which was 12.57%

422

and 14.81% (p < 0.05) at 240 µmol/g, respectively. The hydrophilic amino acids of

423

lysozyme could be blocked after incubation with phenolic compounds, thus reducing

424

the solubility (Rawel, Kroll, & Rohn, 2001). There was no blue shift in the UV

425

spectra and no new peak of hydrogen bonding in the FT-IR for EGCG-treated EW

426

compared with GA-treated, indicating that the reduced solubility may be attributed to

427

less polar microenvironment and the formation of stable insoluble complexes

428

witnessed by increased particle size.

429

Fig. 6B presents the foaming ability of NEW and UEW of GA and EGCG

430

treatments at pH 7.0. The GA treatment was shown to enhance the foaming ability of

431

NEW by 10.26% and 47.03% (p < 0.05) at 120 and 240 µmol/g, respectively.

432

However, in the UEW samples, only 120 µmol/g GA addition showed improved

433

foaming ability (an increase of 36.08% versus the control2). This increment was

434

related to the improved solubility and unfolding protein structure, leading to a more

435

effective transfer of protein molecules to the air-water interface probably due to

436

enhanced molecular flexibility of proteins. The EGCG treatment was different from

437

the GA treatment in their effects on foaming ability. For NEW, 20 and 120 µmol/g 21

438

EGCG declined the foaming ability by 4.58 and 8.82% respectively (p < 0.05),

439

probably due to the decreased solubility and lower molecular flexibility of proteins.

440

However, 240 µmol/g EGCG increased the foaming ability of NEW sharply by 25.11%

441

(p < 0.05) versus the control1, probably due to the promotion of protein cross-linking

442

(Kuan, Bhat, & Karim, 2011). For the UEW samples, 20 µmol/g EGCG significantly

443

increased the foaming ability from 26.33 to 36.67% (p < 0.05), followed by a

444

decrease with increasing EGCG concentration. Several previous studies have reported

445

no obvious dosage effect and inconsistent phenolic concentration effect on the

446

foaming ability of proteins (Sarker, Wilde, & Clark, 1995; Wu et al., 2007; Cao et al.,

447

2018). The protein-phenolic interactions are complicated, and so are the protein

448

interfacial behaviors at the air-water interface.

449

Fig. 6C illustrates the foaming stability of NEW and UEW of GA and EGCG

450

treatments at pH 7.0. For the NEW samples, 120 µmol/g GA addition significantly

451

declined the foaming stability from 89.61 to 80.16% (p < 0.05) compared with the

452

control1 but EGCG treatment showed a slight improvement of the foaming stability,

453

but not in an EGCG concentration-dependent manner. However, the foaming stability

454

of control2 was lower than that control1 due to the reduced viscosity by ultrasound

455

effect, but the EGCG treatment could lift its foaming stability from 81.00 to 95.10%

456

(at 240 µmol/g). Collectively, EGCG treatment exhibited higher foaming stability

457

than GA treatment through effective deceleration of drainage rate (Davis & Foegeding,

458

2006), indicating that EGCG-treated solution was more stable than GA-treated 22

459

solution witnessed by higher absolute ξ-potential value. Therefore, the foaming

460

stability of EW was greatly improved by EGCG modification, probably due to the

461

increased molecular size and cross-linking reaction.

462

3. 6. Particle size and ξ-potential

463

Table 1 demonstrates the changes in the ξ-potential, average particle size and

464

PDI value of NEW and UEW under GA and EGCG treatments. The absolute

465

ξ-potential value showed a downward trend first and then an upward trend for the

466

GA-treated NEW samples, in contrast to an opposite trend for the EGCG-treated

467

NEW samples. The different effect may be due to different phenolic structure. The

468

ξ-potential changes seemed more complex in UEW samples than in NEW samples,

469

with the absolute zeta potential value being lower in the former (-9.19 mV) than in the

470

latter (-12.83 mV) (p < 0.05), probably due to the exposure of more positively

471

charged amino acid residues and neutralization of negatively changed particle under

472

cavitation effect (Chen, Sheng, et al., 2019). The UEW group had higher absolute zeta

473

that NEW group, which may indicate the unfolding structure by ultrasound effect was

474

advantageous to access of phenolic, thus forming electrostatic exclusion between

475

protein molecules.

476

For the NEW samples, the average particle size of GA-treated sample displayed

477

an increase first and then decrease, while the opposite trend was observed for

478

EGCG-treated. The slight increased solubility of GA-treated solution may be due to

479

the decreased particle size and the formation of soluble complexes. Additionally, the 23

480

PDI value followed the same tendency, an increase first from 0.47 to 0.51 (p < 0.05)

481

under 20 µmol/g GA treatment and then a decrease from 0.51 to 0.32 (p < 0.05) under

482

240 µmol/g GA treatments. There were three typical size distribution population for

483

all GA-treated NEW (Fig. 7A). However, the average particle size of EGCG-treated

484

sample showed a decrease first (from 362. 67 to 131.87 nm) and then increase (from

485

131.87 to 1123.33 nm). This increase coupled with a gradual increase in the PDI value

486

from 0.47 to 0.54 suggested the low uniformity of the solution system. The phenolic

487

molecules were bound to the surface of protein, leading to the formation of metastable

488

dispersions of particles (Yang et al., 2014). Fig. 7B displays the size distribution of

489

EGCG-treated NEW samples. Combined with the conversion of multimodal

490

distribution (control1 and 20 µmol/g) to unimodal distribution, a larger particle size

491

was observed in the NEW samples treated with high concentrations of EGCG (120

492

and 240 µmol/g). This result may be ascribed to the reaction of EGCG (higher

493

molecular weight) with protein through hydrophobic interaction.

494

For the UEW samples, either GA or EGCG treatment could increase the average

495

particle size, which was obviously elevated (p < 0.05) from 139.20 to 1592.33 and

496

650.83 nm by the treatment of 240 µmol/g GA or EGCG versus the control,

497

respectively. However, UEW had a lower increase rate than NEW under 120 and 240

498

µmol/g EGCG treatment, probably because the rupture of the protein aggregates by

499

ultrasound is a stronger mechanism than the aggregation effect under phenolic

500

interaction. An opposite result was observed for GA treatment, probably due to a 24

501

different quenching constant between NEW and UEW samples. Therefore, the

502

unfolding structure by ultrasound treatment could promote the binding of phenolic to

503

protein, coupled with a similar increase in the PDI values for UEW samples (Table 1).

504

Additionally, the smaller particles disappeared in 240 µmol/g GA- or EGCG-treated

505

UEW samples. The EGCG-UEW complexes exhibited a larger particle size

506

distribution than GA-UEW (Fig. 7C and 7D), which could be proved by visible

507

aggregates in EGCG treated samples.

508

3. 7. SDS-PAGE analysis

509

Fig. 8 displays the non-reduced SDS-PAGE profile of NEW and UEW treated

510

with phenolic at pH 7.0. The profile of non-reduced SDS-PAGE had an obvious

511

change under high concentration of phenolic incubation, while the profile of reduced

512

SDS-PAGE remained unchanged (Fig. 1S), indicating the protein cross-linked

513

interaction through disulfide bond. For GA-treated samples (Fig. 8A), high

514

concentrations of GA treatment (120 and 240 µmol/g) induced the formation of

515

cross-linked proteins, as demonstrated by the increasing intensity band (35-55 kDa,

516

100-250 kDa). However, for EGCG-treated samples (Fig. 8B), more bands appeared

517

(35-55 kDa, 100-250 kDa) with increasing concentrations of EGCG when compared

518

with

519

protein-phenolic interaction.

520

4. Conclusion

521

the

control,

indicating

the

potential

formation

of

oligomers

from

This study provided evidence that GA and EGCG can bind to EW at neutral pH 25

522

7.0. Both the phenolic derivatives can significantly modify the protein structure,

523

including the loss of sulfhydryl content, decrease of surface hydrophobicity and

524

increase of disordered secondary structure. Moreover, phenolic binding induced the

525

increased microenvironment polarity of Trp and Tyr residues and a more hydrophilic

526

environment. The fluorescence quenching process was dominated by the dynamic

527

quenching witnessed by the gradual increasing of Ksv value with increasing of

528

temperature and the reduced fluorescence lifetime. Combined with ultrasound

529

pretreatment, EW possessed more binding sites in the presence of phenolic

530

compounds, due to the unfolding structure by ultrasound. The modified foaming

531

properties were greatly dependent on the specific protein-phenolic interaction and the

532

structure of proteins. Further research should focus on the detailed mechanism among

533

the adsorbed protein molecules at air-water interface induced by phenolic compounds

534

combined with ultrasound treatment.

535

Acknowledgments

536

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

537

(grant numbers 31571784).

538

Conflict of interest statement

539

All the authors declare that they have no conflict of interest.

540

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541 542 543 544

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29

Figure Captions Fig. 1. Free sulfhydryl content (A) and total sulfhydryl content (B) of NEW and UEW treated with different concentrations of GA and EGCG (20, 120 and 240 µmol/g, protein basis) at pH 7.0. Control1: egg white treated without ultrasound and without phenolic addition; Control2: egg white treated with ultrasound but without phenolic; NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound. Mean values with different small letters (within a single graph) indicate significant differences (p<0.05). Error bars represent mean values ± standard deviations (n=3). Fig. 2. UV spectra (A), the secondary structure fractions from CD spectra (B) and FT-IR spectra (C) of NEW and UEW treated with different concentrations of GA and EGCG (20, 120 and 240 µmol/g, protein basis) at pH 7.0. Control1: egg white treated without ultrasound and without phenolic addition; Control2: egg white treated with ultrasound but without phenolic; NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound. Fig. 3. Surface hydrophobicity of NEW and UEW treated with different concentrations of GA and ECGC (20, 120 and 240 µmol/g, protein basis) at pH 7.0. Control1: egg white treated without ultrasound and without phenolic addition; Control2: egg white treated with ultrasound but without phenolic; NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound. Mean values with different small letters indicate significant differences (p<0.05). Error bars represent mean values ± standard deviation (n=3). Fig. 4. Fluorescence emission spectra of NEW and UEW (0.2 mg/mL) treated by GA

and EGCG at 25 oC. a → g: NEW (A) and UEW (C) with 0, 13.3, 26.6, 53.2, 79.8, 106.4 and 133 µmol/L of GA or NEW (B) and UEW (D) with 0, 5.5, 11, 22, 33, 44 and 55 µmol/L of EGCG. Attached side: Stern-Volmer plots for the quenching of protein by phenolic at different temperatures (25 oC, 30 oC and 38 oC). NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound. Fig. 5. The fluorescence intensity decay curves of samples (NEW, NEW-GA, NEW-EGCG, UEW, UEW-GA and UEW-EGCG) and the corresponding calculated fluorescence lifetime from single exponential fitting. NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound. Fig. 6. Solubility (A), foaming ability (B) and foaming stability (C) of NEW and UEW treated by different concentrations of GA and EGCG (20, 120 and 240 µmol/g, protein basis) at pH 7.0. Control1: egg white treated without ultrasound and without phenolic addition; Control2: egg white treated with ultrasound but without phenolic; NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound. Mean values with different small letters (within a single graph) indicate significant differences (p<0.05). Error bars represent mean values ± standard deviation (n=3). Fig. 7. Intensity size distribution of of NEW (A and B) and UEW (C and D) treated by different concentrations of GA (A and C) and EGCG (B and D) (20, 120 and 240 µmol/g, protein basis) at pH 7.0. Control1: egg white treated without ultrasound and without phenolic addition; Control2: egg white treated with ultrasound but without phenolic; NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound.

Fig. 8. Non-reduced SDS-PAGE profile of NEW and UEW treated by GA (A) and EGCG (B) treatment at pH 7.0. (1-4: control1, 20, 120 and 240 µmol/g for NEW, 5-8: control2, 20, 120 and 240 µmol/g for UEW). M: Marker protein (10-250 kDa); Control1: egg white treated without ultrasound and without phenolic addition; Control2: egg white treated with ultrasound but without phenolic; NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound.

Table 1 The average droplet size, ξ-potential and polydispersity index (PDI) of NEW or UEW induced by different concentrations of GA or EGCG (20, 120 and 240 µmol/g, protein basis). Control1: egg white treated without ultrasound and without phenolic addition; Control2: egg white treated with ultrasound but without phenolic; NEW: egg white treated without ultrasound; UEW: egg white treated with ultrasound. Sample

Treatment

ξ-potential (mV)

Z-Average (nm)

PDI

NEW

Control1

-12.83±0.67cd

362.67±24.30e

0.47±0.03cde

GA 20

-12.77±0.67cd

372.53±47.94e

0.51±0.06cd

GA 120

-10.48±0.75e

262.57±34.49ef

0.38±0.05efg

GA 240

-12.00±0.46d

185.80±50.37fg

0.32±0.03fgh

EGCG 20

-13.47±1.29c

131.87±38.97f

0.47±0.09cde

EGCG 120

-15.63±1.04b

928.67±27.94c

0.49±0.07cd

EGCG 240

-14.97±0.86b

1123.33±69.24b

0.54±0.12c

Control2

-9.19±0.89e

139.20±5.80fg

0.27±0.01h

GA 20

-10.33±0.29e

175.73±29.23fg

0.35±0.05fgh

GA 120

-9.74±0.23e

601.00±67.61d

0.71±0.03b

GA 240

-12.53±0.21cd

1592.33±192.02a

1.00±0.00a

EGCG 20

-6.79±1.40f

201.20±88.04fg

0.33±0.09fgh

EGCG 120

-17.07±0.25a

631.57±5.44d

0.40±0.04def

EGCG 240

-15.60±0.46b

650.83±24.49d

0.28±0.03gh

UEW

Different small letters in the same column indicate significant differences between different treatments (p<0.05). Values are presented as mean ± standard deviation

(n=3).

Fig. 1 A a b

40

e f

20

g

gh

a

j

k

10

h i

0

GA EGCG

50

b d

Free SH (µmol/g, protein)

a

b

c

30

B

Total SH (µmol/g, protein)

50

GA EGCG

a

b

c

40

d

30

e 20

f fgh

fg

gh gh h

i

10

0 Control1

20

120 NEW

240 Control2

20

120

240

UEW

Phenolic concentration (µmol/g, protein basis)

Control1

20

120 NEW

240 Control2

20

120

240

UEW

Phenolic concentration (µmol/g, protein basis)

Fig. 2 1.4

1.0 0.8

Control1 NEW-EGCG 20 NEW-EGCG 120 NEW-EGCG 240

1.2 1.0

0.6 0.4

0.8 0.6 0.4

0.2

0.2

0.0

0.0 240

260

280

300

320

340

240

260

Wavelength/nm Contro2 UEW-GA 20 UEW-GA 120 UEW-GA 240

1.0 0.8 0.6 0.4

1.0 0.8 0.6 0.4 0.2 0.0

320

340

240

260

Wavelength/nm

55 50

45

45

40

40

25

4000

3000

2000

1000

0

GA 240

5000 114

76

4000

3000

2000

0 CG

24

12

1000

0

GA 240

76

38

38 3305.4 1652.7

0

3311.2 1650.8

1540.9

1542.8

0

GA 120

114

76

GA 120

76

38

38 3305.4 1652.7

0

1540.9

1542.8 3311.2 1650.8

0

GA 20

114

76

GA 20

76

38

38 3414.0

1648.9

3414.0 1650.8

1540.9

1542.8

0 114

20

(UEW)

(UEW)

(NEW)

114

G

20

(NEW)

EG

EG

Co

CG

nt ro

24

l2

0

0

20

12

G

CG EG

Co

EG C

0

0

24 G A

12

A

GA

G

Co

nt ro l1

10 20

15

10 nt ro l1

15

0

20

G

20

30

EG C

25

35

l2

30

114

340

nt ro

35

Co

Percentage content (%)

50

114

320

α-helix β-sheet β-turn Random coil

A

55

300

EG C

α-helix β-sheet β-turn Random coil

G

B

280

Wavelength/nm

0

300

0

280

24

260

12

240

5000

340

Control2 UEW-EGCG 20 UEW-EGCG 120 UEW-EGCG 240

1.2

0.0

C

320

1.4

0.2

Percentage content (%)

300

GA

Absorbance

1.2

Absorbance

1.4

280

Wavelength/nm

G A

Absorbance

1.4

Control1 NEW-GA 20 NEW-GA 120 NEW-GA 240

1.2

Absorbance

A

0 Control1

114

76

Control2

76

38

38 1542.8

0 114

1648.9

3414.0

1542.8

1648.9 3414.0

0

GA

114

76

76

38 0 5000

GA

38 3282.3

4000

3000

2000

Wave number (cm-1)

1000

0

0 5000

3282.3

4000

3000

2000

Wave number (cm-1)

1000

0

Fig. 3 GA EGCG

800

a

Surface hydrophobicity (H0)

700

b

a c ef

600

def g

f

cd c cde c

f

g

500 400 300 200 100 0 Control1

20

120 NEW

240 Control2

20

120

240

UEW

Phenolic concentration (µmol/g, protein basis)

Fig. 4

Fig. 5 3000

3000 2500

1500

1500

1000

1000

500

500

0

0 0

20

40

UEW τ=3.51 ns R2=0.9952

2000

Counts

2000

Counts

2500

NEW τ=3.54 ns R2=0.9953

60

80

0

100

20

40

3000

100

60

80

100

60

80

100

2500

NEW-GA τ=2.45 ns R2=0.9810

UEW-GA τ=2.62 ns R2=0.9854

2000

Counts

2000

Counts

80

3000

2500

1500

1500

1000

1000

500

500

0

0 0

20

40

60

80

100

0

20

40

Time (ns)

Time (ns)

3000

3000

2500

2500

NEW-EGCG τ=3.49 ns R2=0.9945

1500

1500

1000

1000

500

500

0

0 0

20

40

60

Time (ns)

UEW-EGCG τ=3.44 ns R2=0.9943

2000

Counts

2000

Counts

60

Time (ns)

Time (ns)

80

100

0

20

40

Time (ns)

Fig. 6 GA EGCG

A 100

d

e

ab f

g

bc

f

a

a f

B a

c

a

h i

i

b

Foaming ability (%)

Solubility (%)

80

60

40

GA EGCG

a

40

30

bc cd

bc bc

bc cde

bc

cde

d

de 20

10 20

0

0 Control1

20

120

240 Control2

20

120

240

Control1

UEW

NEW

a

a c

c

bc c

bc

c

80

Foaming stability (%)

a

a

d

60

40

20

0 Control1

20

120 NEW

240 Control2

20

120

240

UEW

Phenolic concentration (µmol/g, protein basis)

240 Control2

20

120

240

UEW

Phenolic concentration (µmol/g, protein basis)

C ab bc a

120 NEW

Phenolic concentration (µmol/g, protein basis)

100

20

GA EGCG

Fig. 7 A

B 50

50

40

Control1 NEWP-GA 20 NEWP-GA 120 NEWP-GA 240

30

Intensity(%)

Intensity(%)

40

20

20

10

0

0 1

10

100

1000

Droplet size (nm)

C

10000

0.1

1

10

100

1000

10000

1000

10000

Droplet size (nm)

D 50

50

40

40

Control2 UEWP-GA 20 UEWP-GA 120 UEWP-GA 240

30

Intensity(%)

Intensity(%)

30

10

0.1

Control1 NEWP-EGCG 20 NEWP-EGCG 120 NEWP-EGCG 240

20

30

20

10

10

0

0 0.1

1

10

100

Droplet size (nm)

1000

10000

Control2 UEWP-EGCG 20 UEWP-EGCG 120 UEWP-EGCG 240

0.1

1

10

100

Droplet size (nm)

Fig. 8

Highlights Phenolic treatment especially EGCG significantly decreased sulfhydryl content. Protein surface hydrophobicity was obviously decreased by phenolic treatment. EGCG treatment increased the foaming stability of egg white. EGCG was stronger than GA in binding affinity with egg white. Ultrasound pretreatment contributed to the binding of phenolic to protein.

Author statement No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication. On behalf of all the authors, I declare that this paper is original and none of the content in the paper has been published or is under consideration for publication elsewhere. All the authors listed have read the manuscript and approved the submission of the paper to your journal.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.