protein interaction by methyl-Î²

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Inhibition of Epigallocatechin-3-gallate/protein interaction by methyl-#cyclodextrin in myofibrillar protein emulsion gels under oxidative stress Yumeng Zhang, Yuanqi Lv, Lin Chen, Haizhou Wu, Yingyang Zhang, Zhiyao Suo, Shuxin Wang, Yuxin Liang, Xing-lian Xu, Guanghong Zhou, and Xianchao Feng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00275 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Journal of Agricultural and Food Chemistry

1

Inhibition

2

methyl-β-cyclodextrin in myofibrillar protein emulsion gels under oxidative

3

stress

4

Yumeng Zhang,¶, Yuanqi Lv,¶, Lin Chen, Haizhou Wu§, Yingyang Zhangǁ, Zhiyao

5

Suo, Shuxin Wang, Yuxin Liang, Xinglian Xu‡, Guanghong Zhou‡, Xianchao Feng

6

,

of

Epigallocatechin-3-gallate/protein

interaction

by

*

7

8

Road, Yangling, Shaanxi 712100, China

9

§

College of Food Science and Engineering, Northwest A&F University, No. 22 Xinong

Department of Animal Sciences, Meat Science and Muscle Biology Laboratory,

10

University of Wisconsin-Madison, Madison, WI 53706, United States

11

ǁ

12

213164, China

13

‡

14

Technology, Synergetic Innovation Center of Food Safety and Nutrition, Nanjing

15

Agricultural University, Nanjing, Jiangsu 210095, China

16

¶

17

*Corresponding author-Xianchao Feng

18

Associate professor, College of Food Science and Engineering, Northwest A&F

19

University, No. 22 Xinong Road, Yangling, Shaanxi, China 712100. Email address:

20

[email protected]. Tel/Fax: 86029-87092486.

School of food science and technology, Changzhou University, Changzhou, Jiangsu

Lab of Meat Processing and Quality Control of EDU, College of Food Science and

Co-first author

21

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ACS Paragon Plus Environment


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22

ABSTRACT: Nowadays, natural antioxidants abundant in polyphenols have been

23

widely used to substitute synthetic antioxidants in meat products. Generally, high

24

doses of natural antioxidants are required in order to provide comparative antioxidant

25

effects as synthetic antioxidants. Noticeably, the qualities of meat products can be

26

jeopardized due to interactions between polyphenols and myofibrillar proteins (MPs).

27

In this study, methyl-β-cyclodextrin was used to increase the polyphenol loading

28

amount by preventing interactions between polyphenols and proteins. Solubility,

29

electrophoresis, fluorescence spectroscopy and surface hydrophobicity analyses

30

indicated

31

epigallocatechin-3-gallate-induced attacks on MPs under oxidative stress. Gel

32

strength, cooking loss, confocal laser scanning microscopy (CLSM), dynamic

33

rheological testing, and raman spectrum during gelation were further analyzed to

34

investigate

35

epigallocatechin-3-gallate treated emulsion gel. Methyl-β-cyclodextrin addition

36

prevented

37

epigallocatechin-3-gallate. In consequence, the gel and emulsifying properties of MPs

38

were significantly improved. Moreover, β-cyclodextrins could partly inhibit oxidative

39

attacks on MPs, thus increase their solubility. These results indicated that

40

methyl-β-cyclodextrin addition effectively enhanced epigallocatechin-3-gallate

41

loading capacity in meat products.

42

KEYWORDS: EGCG; gel strength; M-β-CD; meat products; natural antioxidant;

43

quinone–protein association

that

methyl-β-cyclodextrin

effects

modification

of

could


of

the

secondary

dose-dependently

on

structure

2 / 40


the

of

inhibit

qualities

MPs

caused

of

by

Page 3 of 40


44

INTRODUCTION

45

Myofibrillar proteins (MPs), a major functional component (50-60%) of muscle

46

food, are prone to be damaged by reactive oxygen species (ROS) during processing

47

and storage, which are reported to be aggravated in presence of transition metals as

48

well as lipid free radicals produced by lipid peroxide.1-3 Generally, the functional,

49

nutritional, and sensorial properties of MPs can be jeopardized due to modification

50

induced by oxidation, resulting in deterioration of the quality in meat products.4-7

51

Numerous antioxidant strategies have been applied to inhibit the oxidation of MPs

52

by the use of natural extracts such as tea polyphenols due to their health-beneficial

53

effect, at the same time avoiding the toxicity problems arising from the use of

54

synthetic antioxidants.8-10 Therefore, natural extracts consisting chiefly of polyphenols

55

have been widely used to restrain oxidation and extend shelf lives of meat products.11

56

Generally, high doses of natural antioxidants are required to provide comparative

57

antioxidant effects as synthetic antioxidants during primary production, processing,

58

distribution and sale of meat products. Unfortunately, antioxidant strategy

59

incorporating polyphenols can not always effectively inhibit proteins oxidation

60

compared with lipid oxidation.

61

Nowadays, interactions between proteins and polyphenols have attracted attentions

62

of researchers, which can lead to the formation of undesirable protein precipitates.11

63

Rosmarinic acid addition (1.25mM) greatly decreased gel strength but increased

64

cooking loss of MP gels under oxidative stress.4 Both 150 µmol/g chlorogenic acid

65

and gallic acid decreased the level of thiol and amine groups, jeopardized the 3 / 40



66

secondary and tertiary structure of MPs, and hence, reduced the strength and

67

water-holding capacity of MP gel under oxidative stress.7, 12 Addition of catechin

68

(50-200µmol/g) greatly decreased the thiol content of MPs, strength and

69

water-holding capacity of MP gel.5 For meat products, addition of 4-methylcatechol

70

(500 ppm) decreased thiol groups of MPs in beef patties.13 Dog rose extract addition

71

(300ppm) greatly jeopardized the water-holding capacity of pork patties during

72

cooking and storage.14 Green tea extract addition (1000 ppm) led to deterioration of

73

water-holding capacity and strength of meat emulsion gel.9 White grape extract

74

addition (500 ppm) significantly decreased thiol content of beef patties.8

75

The aforementioned phenomena could be explained by that polyphenols can

76

non-covalently and covalently interact with MPs, and hence, led to unexpected

77

deterioration to gelation of MPs due to modification of the characteristics of the MPs.4,

78

11, 15-16

79

groups of MPs through addition reaction.4,

80

disulfide linkage and schiff base between MPs could be blocked during heating,

81

resulting in a poor quality of MP gel. Furthermore, polyphenols could non-covalently

82

bind to MPs, change the secondary structure and the unfolding of MPs during gelation

83

of MPs, which were also significantly correlated to texture properties of meat

84

products.4-5, 7

Previous studies found that polyphenols can modify the thiol and free amine 6-7

In consequence, the formation of

85

Therefore, effective methods should be required to disrupt interactions between

86

polyphenols and proteins when natural antioxidants abundant in polyphenols are used

87

as substitutes for synthetic antioxidants in meat products. Glycation can prevent 4 / 40


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88

protein aggregation caused by polyphenols. Researchers reported that glycation of

89

serum albumin via the Maillard reaction inhibited protein aggregation caused by

90

epigallocatechin-3-gallate.17 Maillard reaction does not require a chemical catalyst,

91

making it superior to other catalyst-dependent systems using to modify proteins.18

92

Unfortunately, there are difficulties in controlling the glycation levels, undesirable

93

color changes, and formation of antinutritional compounds during the Maillard

94

reaction, due to the requirements of dry heating and relatively long reaction time.19

95

For these reasons, application of glycation via Maillard reaction is limited in the food

96

industry.

97

Cyclodextrins are a family of natural cyclic oligosaccharides consisting of α-(1,4)

98

linked glucopyranose subunits. With a hydrophilic outer surface and a lipophilic

99

central cavity, cyclodextrins can be ready to chelate organic molecules to form

100

complexes through non-covalent interactions.20 Studies found that cyclodextrins had

101

high affinity to polyphenols.21-22 Modifications of cyclodextrins at the hydroxyl

102

groups can enhance the interactions between derivatives of cyclodextrins and

103

polyphenols, resulting in the enhanced binding ability of cyclodextrins to polyphenols.

104

Methyl-β-cyclodextrin has a higher affinity to rutin compared with β-cyclodextrin,

105

which could be explained by that methylation at the hydroxyl groups led to a deeper

106

cavity and the increasing hydrophobicity.23 Zheng et al. (2005) reported that the

107

substituents of hydroxyl groups on cyclodextrin might improve the stability of the

108

cyclodextrin-epigallocatechin-3-gallate complex.24 Therefore, methyl-β-cyclodextrin

109

is supposed to effectively prevent interactions between MPs and polyphenols, 5 / 40



110

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meanwhile methyl-β-cyclodextrin is biocompatible, nontoxic, and rather inexpensive.

111

The objective of this study was to investigate the hypothesis that addition of

112

methyl-β-cyclodextrin improved epigallocatechin-3-gallate loading amount through

113

preventing undesirable interactions between epigallocatechin-3-gallate and proteins.

114

The

115

methyl-β-cyclodextrin treatment on quality of the epigallocatechin-3-gallate-mediated

116

MP emulsion gel. Solubility, electrophoresis, fluorescence spectroscopy, surface

117

hydrophobicity, CLSM, dynamic rheological testing, and raman spectrum were

118

investigated in order to understand the mechanism for improvement of

119


120

epigallocatechin-3-gallate under oxidative stress.

121

cooking

loss

and

on

strength

the

were

quality

measured

of

to

emulsion

access

gel

the

treated

effects

with

MATERIALS AND METHODS

122

Materials. Longissimus muscle from pig carcasses within 24 h post mortem was

123

obtained from Haoyouduo supermarket (Yangling, China). Methyl-β-cyclodextrin and

124

epigallocatechin-3-gallate were acquired from Aladdin Industrial Co. (Fengxian,

125

Shanghai, China) and Sigma Chemical Co. (St. Louis, MO, USA), respectively. All

126

other chemicals were at least of analytical grade.

127

Extraction of myofibrillar proteins. MPs were extracted from muscle according

128

to previous methods, with some modifications.6, 25 After chopping into small pieces,

129

pork samples were blended with 20 mM phosphate buffer (containing 4 mM EDTA, 3

130

mM MgCl2, 25 mM KCl, and 150 mM NaCl, pH 7.0). At the last washing step, MP

131

was suspended in 20 mM phosphate buffer (containing 0.1M NaCl, pH ~6.2) before 6 / 40


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132

centrifugation (2000g for 15 min at 4 °C). The protein concentration of the final MP

133

pellets was measured according to the Biuret method.26

134

Treatment of MPs. Firstly, the quality of MP emulsion gels as affected by

135

epigallocatechin-3-gallate was investigated under oxidative stress. MP suspensions

136

containing different concentrations of epigallocatechin-3-gallate (0, 8, and 80 µM/g

137

protein,

138

epigallocatechin-3-gallate/methyl-β-cyclodextrin (mol/mol = 1/0.5, 1/1, and 1/2) were

139

preparedthrough vortexing 12 h under the protection of nitrogen in the darkness.

140

Dependent upon the addition of methyl-β-cyclodextrin (0, 40, 80, and 160µM/g

141

protein, final) and epigallocatechin-3-gallate (80 µM/g protein, final), four different

142

MP

143

epigallocatechin-3-gallate were prepared. In the absence of epigallocatechin-3-gallate,

144

MP suspensions containing methyl-β-cyclodextrin (0, 40, 80, and 160µM/g protein)

145

were as well prepared to analyze the influences of methyl-β-cyclodextrin. All the MP

146

suspensions were incubated at 4 °C for 24 h under oxidative stress induced by a

147

hydroxyl radical-generating system (20 µM FeCl3, 100 µM ascorbic acid, and 5 mM

148

H2O2). The non-oxidized MP suspension with neither methyl-β-cyclodextrin nor

149

epigallocatechin-3-gallate was prepared as the control.

final)

suspensions

were

(40

prepared

mg/mL)

in

containing

five

replicates.

both

Mixtures


of

and

150

Solubility. The original MP suspensions (1 mg/mL) were prepared due to diluting

151

the treated MP suspensions in 20 mM phosphate buffer containing 0.6M sodium

152

chloride (~pH 6.2). After centrifuged at 5000g for 15 min at 4 °C, the supernatants

153

were collected. Quick Start Bradford Protein Assay Kit (Bio-Rad Laboratories, Inc., 7 / 40



154

Hercules, CA, USA) was used to measure the protein concentrations in the collected

155

supernatants. The solubility (%) was expressed as the radio of protein concentrations

156

in the supernatants and original suspensions multiplied by100.

157

Electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

158

(SDS−PAGE) was performed to analyze the protein patterns according to methods

159

used in the previous studies.25, 27 The diluted MP samples (2 mg/mL) were mixed with

160

4-fold SDS-PAGE sample buffer with or without 10% β-mercaptoethanol. After

161

boiled for 5 min, 10 µL of each sample was loaded into the well on the gel.

162

Fluorescence spectroscopy. Intrinsic fluorescence intensity of tryptophan and

163

tyrosine residues in MP suspensions (0.25 mg/mL) was determined with a

164

spectrofluorometer (PerkinElmer LS-55, Waltham, MA, USA). The emission spectra

165

were recorded from 300 to 450 nm under excitation at 280 nm.

166

Surface hydrophobicity. Bromophenol blue (BPB) was used to determine surface

167

hydrophobicity of MPs.28 One milliliter of each diluted MP suspension (1 mg/mL)

168

was mixed with 20 µL of BPB solution (1mg/mL). Then, the MP solution was shaken

169

at room temperature for 10 min in the darkness and centrifuged at 5000 × g for 10 min

170

at 4°C. The free BPB in the supernatant was measured at 595 nm against the reagent

171

blank. Then, the percent of bound BPB (µg) by MP was calculated as an index of

172

hydrophobicity. Control was prepared with PBS buffer without MPs. The amount of

173

BPB bound was given by the following formula,

174

BPB bound (µg) = (Acontrol – Asample)/Acontrol × 100%

175

Preparation of MP emulsion gel. MP-stabilized emulsions were prepared by 8 / 40


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176

mixing 20 % (v/v) soybean oil and 80% (v/v) 40mg/mL MP suspension. Then, the

177

mixtures were homogenized at 11000 rpm for 1 min using an Ultra-Turrax

178

homogenizer (T18-Digital, IKA). MP emulsions (~5 g each) were transferred into 25

179

mm (inside diameter) × 50 mm (height) glass vials. All samples were heated from

180

room temperature to 74 °C at about 1°C/min increment in a water bath. Then, vials

181

stayed at 74 °C for 10 min to form emulsion gel. After heating, the formed emulsion

182

gels were immediately chilled.

183 184

Cooking loss. Before and after heating, the weights of the emulsion samples were weighted. Cooking loss was calculated using the following formula: ୛బ ି୛భ

185

Cooking loss (%) =

186

where W0 and W1 are the weight of emulsion and the weight of gel, respectively.

187

Gel strength. Emulsion gels were penetrated using a flat-surface cylinder probe

188

(P/0.5) with a TA-XT plus texture analyzer (Stable Micro Systems, Surry, UK) at a

189

speed of 1.0 mm/min. Gel strength is defined as the initial force (N) required to

190

rupture the gels.

୛బ

× 100%

191

Dynamic Rheological Measurement. The rheological behavior of MP emulsion

192

was measured with a classical rheometer (TA Instruments AR 1000). Emulsions (5g)

193

were placed between two parallel plates. Gelation behaviors of samples were induced

194

by heating at a rate of 2 °C/min from 30 to 80 °C. During heating, a fixed frequency

195

of 0.1 Hz and a strain of 0.02 were applied to investigate the viscoelastic properties in

196

terms of storage modulus (G′) and tan δ values (G″/G′, where G″ is loss modulus).29

197

Confocal laser scanning microscopy (CLSM). The distribution of oil droplets in 9 / 40



198

emulsion gels was investigated by CLSM (A1R, Nikon Inc., Tokyo, Japan). The oil

199

droplets and proteins were stained with a fluorescence dye mixture of Nile Red and

200

Fast Green (eah at 0.038%, w/v).

201

Raman spectrum analysis. Raman spectra of all gels were analyzed by

202

LabramHR800 spectrometer (Horiba/Jobin. Yvon, Longjumeau, France).4 Samples

203

(approximately 0.5 g) were spread on a glass slide. Spectra were recorded in the range

204

of 400–3600 cm−1, then spectra were smoothed, baselines corrected and normalized

205

according to the intensity of phenylalanine band at 1003 cm−1. Secondary structures

206

of MPs were defined as α-helix, β-sheet, β-turn, and random conformations according

207

to Chen and Han.30 The relative content of secondary structures was calculated by

208

areas of the corresponding fitted bands from amide I spectra (1600–1700 cm−1)

209

according to a previous study.31 PeakFit Version 4.12 software (SPSS Inc., Chicago,

210

IL) was used for curve fitting, and area calculation.

211

Statistical analysis. Data were collected from five independent trials and subjected

212

to the statistical analysis using one-way analysis of variance (ANOVA) (SPSS 20.0,

213

Chicago, IL, USA). The Duncan test was used for multiple comparison of mean

214

values between different treatments (P < 0.05). Data were expressed as means ±

215

standard deviations (SD).

216

RERULTS AND DISCUSSION

217

Dose effects of EGCG on the cooking loss and strength of emulsion gels. As

218

shown in figure 1, the cooking loss significantly increased due to oxidation.

219

Compared to oxidized gel, addition of 8µM/g epigallocatechin-3-gallate slightly 10 / 40


Page 10 of 40

Page 11 of 40


220

reduced the cooking loss to the similar level as the control (non-oxidized MPs)

221

(Figure 1). Nevertheless, further addition of epigallocatechin-3-gallate (80µM/g)

222

sharply increased the cooking loss (Figure 1). Changes of strength caused by

223

epigallocatechin-3-gallate were similar as that of cooking loss (Figure 1). The gel

224

strength significantly increased due to addition of 80µM/g epigallocatechin-3-gallate

225

(Figure 1). Generally, high doses of natural antioxidants are required to provide

226

comparative antioxidant effects as synthetic antioxidants for meat products. In the

227

following

228

epigallocatechin-3-gallate loading amount through preventing interactions between

229

MPs and polyphenols, as methyl-β-cyclodextrin is biocompatible, nontoxic, and rather

230

inexpensive.

study,


was

applied

to

increase

the

231

Carbonyl content of MPs. Carbonyl content is widely measured and is the most

232

frequent method for assessing protein oxidation in meat products.6 As shown in

233

supplementary figure 1, the oxidized MPs had a significantly higher carbonyl level

234

compared to the non-oxidized MPs. The carbonyl levels of the oxidized MPs sharply

235

decreased owing to addition of 80 µM/g epigallocatechin-3-gallate (Supplementary

236

figure 1), indicating that free radicals were scavenged and metal ions were chelated by

237

epigallocatechin-3-gallate addition.5 In the presence of epigallocatechin-3-gallate, the

238

carbonyl levels of the oxidized MPs showed a methyl-β-cyclodextrin-induced

239

increase, but was still significantly lower than that of the oxidized MPs

240

(Supplementary figure 1). These results indicated that the antioxidant capacity of

241

epigallocatechin-3-gallate

was

partly

weakened

11 / 40


due

to

addition

of


Page 12 of 40

242

methyl-β-cyclodextrin, but still maintained at a promising level (Supplementary figure

243

1).

244

Solubility of MPs. Solubility of proteins is closely interrelated with their functions,

245

such as gelation and emulsification behavior.18 Oxidized MPs had significantly lower

246

solubility than control (Figure 2). Addition of 80µM/g epigallocatechin-3-gallate

247

further decreased the solubility of the oxidized MPs, indicating aggregation of MPs

248

induced by interactions between epigallocatechin-3-gallate and the oxidized MPs. In

249

the presence of epigallocatechin-3-gallate, methyl-β-cyclodextrin dose-dependently

250

increased the solubility of the oxidized MPs (Figure 2), which could be explained by

251

binding

252

epigallocatechin-3-gallate,

253

epigallocatechin-3-gallate.23-24 Noticeably, the solubility of MPs increased with

254

increasing

255

epigallocatechin-3-gallate. This result indicated that methyl-β-cyclodextrin might bind

256

to MPs and sterically hinder the protein-protein interactions. In consequence, high

257

concentrations of methyl-β-cyclodextrin (80 and 160 µM/g) significantly improved

258

the solubility of MPs under oxidative stress (Figure 2).

interactions

between then



inhibited

the

modification

concentrations

in

the

and of

MPs

absence

the by

of

259

SDS−PAGE Patterns of MPs. The degree of cross-links in MPs induced by

260

oxidation was examined with SDS−PAGE methods. As shown in figure 3, myosin

261

heavy chain (MHC) of the oxidized MPs had an obviously lower band intensity

262

compared to control, and so did the actin (Figure 3A & Supplementary figure 2 A).

263

Epigallocatechin-3-gallate addition (80 µM/g) led to further reduction of both bands, 12 / 40


Page 13 of 40


264

especially actin, which almost vanished (Figure 3A & Supplementary figure 2 A).

265

This could be explained by that addition of epigallocatechin-3-gallate resulted in more

266

polymerization of MPs. However, methyl-β-cyclodextrin addition increased the both

267

band intensities, especially the high concentrations of methyl-β-cyclodextrin (80 and

268

160 µM/g). After addition of β-mercaptoethanol, some MHC and actin bands of

269

control and oxidized samples were recovered (Figure 3 B & Supplementary figure 2

270

B). However, addition of 80 µM/g epigallocatechin-3-gallate hindered the recovery of

271

MHC and actin bands, indicating formation of non-disulfide bonds, including possible

272

quinone–protein associations. Previous studies reported that the formations of

273

quinone−thiol and quinone−amine adducts were induced by catechin and rosmarinic

274

acid.4, 7 In the presence of 80 µM/g epigallocatechin-3-gallate, the intensities of both

275

bands

276

Methyl-β-cyclodextrin addition might prevent the formation of quinone−thiol and

277

quinone−amine

278


279

epigallocatechin-3-gallate, intensities of both bands increased with addition of

280

methyl-β-cyclodextrin as well. Addition of methyl-β-cyclodextrin could block

281

interactions between the oxidized MPs, which was also supported by the changes of

282

solubility (Figure 2).

obviously

increased

adducts, (80

due

to

especially µM/g

and

addition

for 160

the

of

methyl-β-cyclodextrin.

high

µM/g).

concentrations In

the

absence

of of

283

Fluorescence spectrum of MPs. The intrinsic fluorescence contributed by some

284

amino acid residues (tryptophan, Trp; and tyrosine, Tyr) is considered as an effective

285

tool for investigating the interactions between polyphenols and proteins.17 As shown 13 / 40



286

in figure 4, 80 µM/g epigallocatechin-3-gallate greatly deceased the fluorescence

287

intensity of the oxidized MPs. This indicated that epigallocatechin-3-gallate interacted

288

with myofibrillar proteins, and hence, changed the polarity of microenvironments

289

around amino acid residues, resulting in the decrease of fluorescence intensity. In the

290

presence of 80 µM/g epigallocatechin-3-gallate, the fluorescence intensity increased

291

with increasing methyl-β-cyclodextrin concentrations (40 - 160 µM/g). These

292

indicated that methyl-β-cyclodextrin addition disrupted the interactions between the

293

oxidized MPs and epigallocatechin-3-gallate. Trp, and Tyr residues in the core (a

294

hydrophobic environment) of the proteins could be exposed to solvent (a hydrophilic

295

environment) due to the attack by the generated free radicals (•OH), and hence, the

296

fluorescence intensity of the oxidized MPs significantly decreased compared with

297

control (Figure 4). Methyl-β-cyclodextrin addition also dose-dependently increased

298

the fluorescence intensity in the absence of epigallocatechin-3-gallate. This suggested

299

that methyl-β-cyclodextrin addition prevented oxidative damage of MPs. Several

300

studies found that the addition of β-cyclodextrins could prevent protein aggregation

301

and increase its dispersivity.32-34

302

Surface hydrophobicity changes of MPs. Surface hydrophobicity is wieldy used

303

as an index of the unfolding of proteins, reflecting distribution of hydrophobic amino

304

acid residues on the surface of proteins. As a result, surface hydrophobicity has an

305

important influence on the quality of meat products due to the changes in emulsifying

306

and gelling properties of MPs. As shown in figure 5, oxidation significantly increased

307

the surface hydrophobicity of MPs, indicating the increase level of unfolding. The 14 / 40


Page 14 of 40

Page 15 of 40


308

surface hydrophobicity of the oxidized MP decreased due to addition of

309


310

epigallocatechin-3-gallate addition (80 µM/g) led to aggregation and precipitation of

311

MPs likely due to excessive unfolding of MPs. Consequently, the effect of unfolding

312

was partially shielded. These results were supported by the even lower surface

313

hydrophobicity compared to that of control, which were in good agreement with the

314

reduced solubility (Figure 2), and were consistent with previous studies.6, 35 Addition

315

of methyl-β-cyclodextrin dose-dependently increased the surface hydrophobicity

316

(Figure 5), indicating that methyl-β-cyclodextrin addition inhibited interactions

317

between the oxidized MPs and epigallocatechin-3-gallate. Consequently, aggregation

318

and polymerization of MPs induced by epigallocatechin-3-gallate were partly blocked,

319

leading to the increase of the surface hydrophobicity. In the absence of

320

epigallocatechin-3-gallate, methyl-β-cyclodextrin addition decreased the surface

321

hydrophobicity of the oxidized MP, especially for a high concentration of

322

methyl-β-cyclodextrin (160µM/g). This result indicated that methyl-β-cyclodextrin

323

addition protected MPs from modifications under oxidative stress (Figure 5).

(80

µM/g).

This

could

be

explained

by

that

324

Cooking loss and strength. Cooking loss can reflect the ability of emulsion gel

325

network to retain water and oil. As shown in figure 6 A, the cooking loss increased

326

with addition of epigallocatechin-3-gallate (80 µM/g), indicating the decrease

327

capacity of gel to hold oil and water. Addition of high concentrations of rosmarinic

328

acid and chlorogenic acid greatly reduced the capacity of gel to hold oil and water as

329

well.4, 7 Under oxidative stress, epigallocatechin-3-gallate could covalently modify the 15 / 40



330

MPs, leading to a poor gel network due to disruption of disulfide linkages, supported

331

by the results of SDS-PAGE (Figure 3).6 Moreover, epigallocatechin-3-gallate

332

addition significantly increased the unfolding of the oxidized MPs likely due to

333

non-covalent interactions, supported by results of fluorescence spectra (Figure 4). The

334

strong hydrophobic forces resulted in aggregation of MPs and led to shrinkage of the

335

emulsion gel, and hence, epigallocatechin-3-gallate addition significantly increased

336

the cooking loss of the oxidized MP emulsion gel.17 In the presence of

337

epigallocatechin-3-gallate, addition of methyl-β-cyclodextrin significantly reduced the

338

cooking loss of emulsion gel, especially with relatively high concentrations of

339

methyl-β-cyclodextrin (80 and 160 µM/g) (Figure 6 A). A previous study found that

340

the A-ring of epigallocatechin-3-gallate could be inserted into the hydrophobic cavity

341

of sulfobutyl ether-β-cyclodextrin sodium and result in the formation of

342

epigallocatechin-3-gallate-sulfobutyl ether-β-cyclodextrin inclusion complexes.36 This

343

indicated that methyl-β-cyclodextrin-epigallocatechin-3-gallate interactions could

344

prevent interactions between epigallocatechin-3-gallate and the oxidized MPs. It is the

345

first time to demonstrate that addition of methyl-β-cyclodextrin prevents interactions

346

between epigallocatechin-3-gallate and the oxidized proteins from pork muscles. In

347

meat emulsion model, methyl-β-cyclodextrin addition prevented the increase of

348

cooking loss owing to addition of 80 µM/g epigallocatechin-3-gallate (Supplementary

349

figure 3 A). This result could be attributed to that methyl-β-cyclodextrin addition

350

restricted the modification of MPs by epigallocatechin-3-gallate in meat emulsion

351

model as well, which was supported by the increase fluorescence intensity of isolated 16 / 40


Page 16 of 40

Page 17 of 40


352

MPs (Supplementary figure 3 B). MPs isolated from meat emulsion had the highest

353

Trp and Tyr fluorescence intensity, while MPs isolated from meat emulsion containing

354

80 µM/g epigallocatechin-3-gallate had the lowest Trp and Tyr fluorescence intensity

355

(Supplementary figure 3 B). Addition of methyl-β-cyclodextrin dose-dependently

356

increased the fluorescence intensity of MPs isolated from meat emulsion containing

357

80 µM/g epigallocatechin-3-gallate (Supplementary figure 3 B).

358

Changes in textural properties of samples are shown in figure 6 B. Oxidation

359

obviously enhanced the strength of the emulsion gel compared to control (Figure 6 B),

360

likely due to the improvement of interactions between the unfolded MPs.4

361

Epigallocatechin-3-gallate addition further increased the gel strength, likely due to the

362

shrinkage of the emulsion gel caused by aggregation of MPs. Addition of

363

methyl-β-cyclodextrin dose-dependently lowered the strength of the oxidized

364

emulsion gel in the presence of epigallocatechin-3-gallate, indicating that

365

methyl-β-cyclodextrin addition enhanced the structure of emulsion gel containing 80

366

µM/g epigallocatechin-3-gallate. This could be explained by that covalent and

367

non-covalent interactions between the oxidized MPs and epigallocatechin-3-gallate

368

were

369

epigallocatechin-3-gallate. These results are in good agreement with the

370

physicochemical and structural changes of MPs (Figure 2, 3, 4, and 5). In the absence

371

of

372

methyl-β-cyclodextrin (160 µM/g) significantly decreased the strength enhanced

373

owing to oxidation (Figure 6 B). A high concentration of methyl-β-cyclodextrin could

blocked

by

interactions

epigallocatechin-3-gallate,

between

addition

of


a

17 / 40


high

concentration

and

of


Page 18 of 40

374

protect MPs from modification by oxidation, and disrupt the interactions between the

375

unfolded MPs.

376

Confocal laser scanning microscopy analysis. Emulsifying properties of different

377

MPs were investigated by confocal laser scanning microscopy (CLSM). MP emulsion

378

can form a heat-induced three-dimensional gel network, which has a good water- and

379

oil-holding capacity.37 As shown in figure 7, oxidation made the size of oil droplets

380

became larger and non-uniform compared with control. Epigallocatechin-3-gallate

381

addition (80 µM/g) can covalently and non-covalently modify the oxidized MPs,

382

resulting in a poor gel microstructure rather than a three-dimensional gel network

383

induced by heating. As a result, oil droplets migrated and coalesced into larger ones

384

due

385

epigallocatechin-3-gallate (80 µM/g), the distribution of oil droplets was improved by

386

addition of methyl-β-cyclodextrin, especially for relatively high concentrations of

387

methyl-β-cyclodextrin (80 and 160 µM/g) (Figure 7). The more homogenous

388

distribution of lipid droplets suggested the overall increase in qualities of emulsion gel

389

due to the methyl-β-cyclodextrin addition. This was in agreement with the previous

390

analysis of cooking loss (Figure 6 A). Moreover, methyl-β-cyclodextrin addition

391

might protect MPs from oxidative attacks, and hence, most of oil droplets with

392

relatively smaller sizes were more evenly dispersed. Nevertheless, a high

393

concentration of methyl-β-cyclodextrin (160 µM/g) might disrupt protein-protein

394

interactions in the gel matrix, resulting in relatively uneven distribution and larger oil

395

droplets (Figure 7).

to


addition.

In

18 / 40


the

presence

of

Page 19 of 40


396

Dynamic rheological analysis. In order to better understand influences of

397

methyl-β-cyclodextrin and epigallocatechin-3-gallate on the viscoelastic properties of

398

MP emulsion gels, dynamic oscillatory measurements were used to analyze the G′ and

399

tan δ.6 As shown in figure 8, oxidation significantly enhanced the final G′ compared

400

to that of the control, suggesting that protein-protein interactions were enhanced by

401

oxidation during heating.6 Epigallocatechin-3-gallate addition (80 µM/g) further

402

increased the G′. Epigallocatechin-3-gallate addition might lead to a compact

403

structure rather than a three-dimensional network gel, resulting in high gel strength.

404

This

405

epigallocatechin-3-gallate (80 µM/g), methyl-β-cyclodextrin addition lowered G′ of

406

the

407

methyl-β-cyclodextrin (80 and 160 µM/g) (Figure 8 A). Methyl-β-cyclodextrin can

408

disrupt the interactions between epigallocatechin-3-gallate and MPs and prevent the

409

intermolecular interactions among the oxidized MPs (Figure 8 A). The loss factor

410

(G′′/G′, tan δ) was also calculated to illustrate the rheological changes of emulsion

411

gels. Tan δ indicates the relative importance between elasticity and viscosity (Figure 8

412

B). Generally, the lower value of final tan δ means a better gel matrix. In the presence

413

of epigallocatechin-3-gallate (80 µM/g), methyl-β-cyclodextrin addition reduced the

414

final tan δ values of emulsion gels, indicating the overall increase in the gel structure

415

jeopardized by 80 µM/g epigallocatechin-3-gallate.

was

consistent

oxidized

with

emulsion

our

gel,

study.6

previous

especially

with

In

high

the

presence

concentrations

of

of

416

Raman spectrum of the MP emulsion gels. As shown in figure 9, a typical raman

417

spectrum ranging from 3600 to 400 cm-1 was derived from the control emulsion gel. It 19 / 40



Page 20 of 40

418

displayed similar features compared to the spectrum derived from MPs of pork.4 The

419

amide I band between 1600 and 1700 cm-1 was investigated as the deconvoluted

420

spectrum to gain insights in changes related to the secondary structure. The bands

421

locating at 1605-1620 cm-1 are attributed to vibrations of the amino acid side chain

422

(AA), the bands locating at 1620-1630 cm-1 and 1673 cm-1 are primarily due to

423

β-sheet peaks, the bands locating at 1650-1660 cm-1 are primarily due to α-helix peaks,

424

the bands locating at 1660-1665 cm-1 are due to random coil, and those of β-turn

425

peaks are locating at 1680 and 1688 cm-1. These were similar to those reported

426

previously.30-31, 38

427

The band intensity of amino acid side chain increased due to exposures of amino

428

acid residues with unfolding of MPs under oxidative stress (Table 1).4,

429

Epigallocatechin-3-gallate addition lowered the intensity of the band, indicating a

430

significant decrease in such residues. Under oxidative stress, ε-NH2 groups of amino

431

acid residues could be modified by epigallocatechin-3-gallate due to the formation of

432

quinone−amine adducts.6,

433

intensity of α-helix, but increased the band intensity of β-sheet compared to control

434

(Table 1). Addition of epigallocatechin-3-gallate decreased the band intensity of both

435

α-helix and β-sheet compared to both control and oxidized emulsion gel.

436

Epigallocatechin-3-gallate might covalently and non-covalently interact with the

437

oxidized MPs, and disrupt the protein-protein interactions (Table 1). These results

438

were in accordance with a previous study.4 The secondary structure changes are

439

closely

related

to

12

the

28

Oxidative treatment significantly decreased the band

protein

gel

texture.4,

20 / 40


31

In

consequence,

Page 21 of 40


440

epigallocatechin-3-gallate addition resulted in the much rough gel texture and the

441

poor water- and oil-holding capacity (Figure 6 A & B). Some researchers found a

442

positive correlation between β-sheet content and gel texture,39-40 and some others

443

reported that the α-helical content had a positive correlation with textural properties of

444

gel.31 Methyl-β-cyclodextrin addition increased the band intensities of both α-helix

445

and β-sheet in MP emulsion gels treated with epigallocatechin-3-gallate (Table 1).

446

Methyl-β-cyclodextrin addition might disrupt the interactions between the oxidized

447

MPs and epigallocatechin-3-gallate under oxidative stress. In consequence,

448

methyl-β-cyclodextrin addition improved the gel texture jeopardized by 80 µM/g

449

epigallocatechin-3-gallate, which was supported by the decrease of both cooking loss

450

and gel strength (Figure 6 A & B).

451

In conclusion, natural antioxidants abundant in polyphenols have been widely used

452

to substitute synthetic antioxidants in meat products. Generally, high concentrations of

453

natural antioxidants are required in order to provide comparative antioxidant effects as

454

synthetic antioxidants. Noticeably, phenolic compounds can non-covalently and

455

covalently interact with MPs under oxidative stress, especially at a high

456

concentration. Consequently, the functional properties of the MPs could be

457

jeopardized. Therefore, the quality of MP emulsion gel is going to be deteriorated by

458

phenolic compounds. In the present study, methyl-β-cyclodextrin could bind to both

459

epigallocatechin-3-gallate and the oxidized MPs. Therefore, methyl-β-cyclodextrin

460

dose-dependently improved the overall quality of the oxidized MP emulsion gel

461

deteriorated by epigallocatechin-3-gallate. In addition, methyl-β-cyclodextrin addition 21 / 40



462

as well improved the qualities of the meat emulsion gel deteriorated by a high

463

concentration of epigallocatechin-3-gallate. The results of these two different meat

464

model systems (MP and meat emulsion gel) provided a potentially more convenient

465

way to enhance the loading amount of polyphenols compared to some other ways,

466

such as glycation of MPs via the Maillard reaction.

467

SPPORTING INFORMATION

468

The supporting information is available. Supplementary figure 1. Carbonyl levels

469

of MPs as affected by methyl-β-cyclodextrin and epigallocatechin-3-gallate under

470

oxidative stress. Supplementary figure 2. Intensities of MHC bands and actin bands of

471

MPs as affected by methyl-β-cyclodextrin and epigallocatechin-3-gallate under

472

oxidative stress. Supplementary figure 3. Effect of methyl-β-cyclodextrin and

473

epigallocatechin-3-gallate addition on the cooking loss of meat emulsion gel and

474

fluorescence intensity of isolated MPs.

475

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

476 477

(Grant No.: 31771991).

478

REFERENCES

479 480 481 482 483 484 485 486 487 488

1.

Lund, M. N.; Heinonen, M.; Baron, C. P.; Estevez, M., Protein oxidation in muscle foods: A review.

Mol. Nutr. Food Res. 2011, 55 (1), 83-95. 2.

Zhou, F.; Sun, W.; Zhao, M., Controlled Formation of Emulsion Gels Stabilized by Salted

Myofibrillar Protein under Malondialdehyde (MDA)-Induced Oxidative Stress. J. Agr. Food Chem. 2015, 63 (14), 3766–3777. 3.

Vossen, E.; De Smet, S., Protein Oxidation and Protein Nitration Influenced by Sodium Nitrite in

Two Different Meat Model Systems. J. Agr. Food Chem. 2015, 63 (9), 2550-2556. 4.

Tang, C.-b.; Zhang, W.-g.; Zou, Y.-f.; Xing, L.-j.; Zheng, H.-b.; Xu, X.-l.; Zhou, G.-h., Influence of

RosA-protein adducts formation on myofibrillar protein gelation properties under oxidative stress. Food Hydrocolloid. 2017, 67, 197-205. 22 / 40


Page 22 of 40

Page 23 of 40


489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532

5.

Jia, N.; Wang, L.; Shao, J.; Liu, D.; Kong, B., Changes in the structural and gel properties of pork

myofibrillar protein induced by catechin modification. Meat Sci. 2017, 127, 45-50. 6.

Feng, X.; Chen, L.; Lei, N.; Wang, S.; Xu, X.; Zhou, G.; Li, Z., Emulsifying Properties of Oxidatively

Stressed Myofibrillar Protein Emulsion Gels Prepared with (−)-Epigallocatechin-3-gallate and NaCl. J. Agr. Food Chem. 2017, 65 (13), 2816-2826. 7.

Cao, Y.; Xiong, Y. L., Chlorogenic acid-mediated gel formation of oxidatively stressed myofibrillar

protein. Food Chem. 2015, 180, 235-243. 8.

Jongberg, S.; Skov, S. H.; Tørngren, M. A.; Skibsted, L. H.; Lund, M. N., Effect of white grape

extract and modified atmosphere packaging on lipid and protein oxidation in chill stored beef patties. Food Chem. 2011, 128 (2), 276-283. 9.

Jongberg, S.; Terkelsen, L. d. S.; Miklos, R.; Lund, M. N., Green tea extract impairs meat emulsion

properties by disturbing protein disulfide cross-linking. Meat Sci. 2015, 100, 2-9. 10. Vossen, E.; Utrera, M.; De, S. S.; Morcuende, D.; Estévez, M., Dog rose (Rosa canina L.) as a functional ingredient in porcine frankfurters without added sodium ascorbate and sodium nitrite. Meat Sci. 2012, 92 (4), 451-7. 11. Ozdal, T.; Capanoglu, E.; Altay, F., A review on protein–phenolic interactions and associated changes. Food Res. Int. 2013, 51 (2), 954–970. 12. Cao, Y.; True, A. D.; Chen, J.; Xiong, Y. L., Dual Role (Anti- and Pro-oxidant) of Gallic Acid in Mediating Myofibrillar Protein Gelation and Gel in Vitro Digestion. J. Agr. Food Chem. 2016, 15 (64), 3054-3061. 13. Jongberg, S.; Lund, M. N.; Waterhouse, A. L.; Skibsted, L. H., 4-methylcatechol inhibits protein oxidation in meat but not disulfide formation. J. Agr. Food Chem. 2011, 59 (18), 10329-35. 14. Lara, M.; Gutierrez, J.; Timón, M.; Andrés, A., Evaluation of two natural extracts (Rosmarinus officinalis L. and Melissa officinalis L.) as antioxidants in cooked pork patties packed in MAP. Meat Sci. 2011, 88 (3), 481-488. 15. Liang, M.; Liu, R.; Qi, W.; Su, R.; Yu, Y.; Wang, L.; He, Z., Interaction between lysozyme and procyanidin: Multilevel structural nature and effect of carbohydrates. Food Chem. 2013, 138 (2-3), 1596. 16. Liu, Y.; Liu, H.; Hu, Y.; Jiang, J., Density Functional Theory for Adsorption of Gas Mixtures in Metal−Organic Frameworks. J. Phys. Chem. B 2010, 114 (8), 2820-7. 17. Xia, S.; Li, Y.; Xia, Q.; Zhang, X.; Huang, Q., Glycosylation of bovine serum albumin via Maillard reaction prevents epigallocatechin-3-gallate-induced protein aggregation. Food Hydrocolloid. 2015, 43, 228-235. 18. Saeki, H.; Hettiarachchy, N.; Sato, K.; Marshall, M., Protein–saccharide interaction. Food proteins and peptides: Chemistry, functionality, interactions, and commercialization. Crc Press. 2012, 230-253. 19. Zhu, D.; Damodaran, S.; Lucey, J. A., Formation of Whey Protein Isolate (WPI)− dextran conjugates in aqueous solutions. J. Agr. Food Chem. 2008, 56 (16), 7113-7118. 20. Le, B. C.; Renard, C. M., Interactions between polyphenols and macromolecules: quantification methods and mechanisms. Crit. Rev. Food Sci. 2012, 52 (3), 213-248. 21. Bourvellec, C. L.; Renard, C. M. G. C., Interactions between Polyphenols and Macromolecules: Quantification Methods and Mechanisms. Crit. Rev. Food Sci. 2012, 52 (3), 213-48. 22. Folch-Cano, C.; Guerrero, J.; Speisky, H.; Jullian, C.; Olea-Azar, C., NMR and molecular fluorescence

spectroscopic

study

of

the

structure

and

thermodynamic

parameters

of

EGCG/β-cyclodextrin inclusion complexes with potential antioxidant activity. J. Incl. Phenom. Macro. 23 / 40



533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576

2014, 78 (1-4), 287-298. 23. Letellier, S.; Maupas, B.; Gramond, J. P.; Guyon, F.; Gareil, P., Determination of the formation constant for the inclusion complex between rutin and methyl-β-cyclodextrin. Anal. Chim. Acta 1995, 20 (315), 357-363. 24. Zheng, Y.; Haworth, I. S.; Zuo, Z.; Chow, M. S.; Chow, A. H., Physicochemical and structural characterization of quercetin-beta-cyclodextrin complexes. J. Pharm. Sci. 2005, 94 (5), 1079. 25. Feng, X.; Li, C.; Ullah, N.; Hackman, R. M.; Chen, L.; Zhou, G., Potential Biomarker of Myofibrillar Protein Oxidation in Raw and Cooked Ham: 3-Nitrotyrosine Formed by Nitrosation. J. Agr. Food Chem. 2015, 63 (51), 10957-10964. 26. Gornall, A. G.; Bardawill, C. J.; David, M. M., Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 1949, 177 (2), 751-766. 27. Chen, L.; Feng, X.; Zhang, Y.; Liu, X.; Zhang, W.; Li, C.; Ullah, N.; Xu, X.; Zhou, G., Effects of ultrasonic processing on caspase-3, calpain expression and myofibrillar structure of chicken during post-mortem ageing. Food Chem. 2015, 17 (7), 280-287. 28. Feng, X.; Li, C.; Ullah, N.; Cao, J.; Lan, Y.; Ge, W.; Hackman, R. M.; Li, Z.; Chen, L., Susceptibility of whey protein isolate to oxidation and changes in physicochemical, structural, and digestibility characteristics. J. Dairy Sci. 2015, 98, 7602-7613. 29. Martinez-Alvarenga, M.; Martinez-Rodriguez, E.; Garcia-Amezquita, L.; Olivas, G.; Zamudio-Flores, P.; Acosta-Muniz, C.; Sepulveda, D., Effect of Maillard reaction conditions on the degree of glycation and functional properties of whey protein isolate–Maltodextrin conjugates. Food Hydrocolloid. 2014, 38, 110-118. 30. Chen, H.; Han, M., Raman spectroscopic study of the effects of microbial transglutaminase on heat-induced gelation of pork myofibrillar proteins and its relationship with textural characteristics. Food Res. Int. 2011, 44 (5), 1514-1520. 31. Zhou, F.; Zhao, M.; Su, G.; Cui, C.; Sun, W., Gelation of salted myofibrillar protein under malondialdehyde-induced oxidative stress. Food Hydrocolloid. 2014, 40, 153-162. 32. Yi, Z.; Qasim, M.; Qasim, S.; Warrington, T.; Laskowski, M., Ring-Toss: Capping highly exposed tyrosyl or tryptophyl residues in proteins with β-cyclodextrin. BBA-Gen. Subject. 2006, 1760 (3), 372-379. 33. Niccoli, M.; Oliva, R.; Castronuovo, G., Cyclodextrin–protein interaction as inhibiting factor against aggregation. J. Therm. Anal. Calorim. 2017, 127 (2), 1491-1499. 34. Girek, T.; Goszczyński, T.; Girek, B.; Ciesielski, W.; Boratyński, J.; Rychter, P., β-Cyclodextrin/protein conjugates as a innovative drug systems: synthesis and MS investigation. J. Incl. Phenom. Macro. 2013, 75 (3-4), 293-296. 35. Li, C.; Xiong, Y. L.; Chen, J., Oxidation-induced unfolding facilitates myosin cross-linking in myofibrillar protein by microbial transglutaminase. J. Agr. Food Chem. 2012, 60 (32), 8020-8027. 36. Liu, F.; Antoniou, J.; Li, Y.; Majeed, H.; Liang, R.; Ma, Y.; Ma, J.; Zhong, F., Chitosan/sulfobutylether-β-cyclodextrin nanoparticles as a potential approach for tea polyphenol encapsulation. Food Hydrocolloid. 2016, 57, 291-300. 37. Gibis, M.; Schuh, V.; Weiss, J., Effects of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) as fat replacers on the microstructure and sensory characteristics of fried beef patties. Food Hydrocolloid. 2015, 45, 236-246. 38. Xu, X.; Han, M.; Fei, Y.; Zhou, G., Raman spectroscopic study of heat-induced gelation of pork myofibrillar proteins and its relationship with textural characteristic. Meat Sci. 2011, 87 (3), 159-164. 24 / 40


Page 24 of 40

Page 25 of 40


577 578 579 580 581

39. Shao, J.; Zou, Y.; Xu, X.; Wu, J.; Zhou, G., Evaluation of structural changes in raw and heated meat batters prepared with different lipids using Raman spectroscopy. Food Res. Int. 2011, 44 (9), 2955-2961. 40. Herrero, A. M., Raman spectroscopy for monitoring protein structure in muscle food systems. Crit. Rev. Food Sci. Nutr. 2008, 48 (6), 512.

582

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Page 26 of 40

583

Figure captions

584

Figure 1. Cooking yield and strength of MP emulsion gels as affected by

585

methyl-β-cyclodextrin and epigallocatechin-3-gallate under oxidative stress. Control:

586

non−oxidized;

587

epigallocatechin-3-gallate at 8 and 80 µmol/g protein, respectively. Different letters

588

(cooking loss, a-c; gel strength, x-z) denote a statistical difference between means (p

589

< 0.05).

590

Figure

591

epigallocatechin-3-gallate under oxidative stress. Control: non−oxidized; E, E-M

592

(1:0.5), E-M (1:1), and E-M (1:2): oxidized with 80 µM/g epigallocatechin-3-gallate

593

in the presence of 0, 40, 80, and 160 µM/g methyl-β-cyclodextrin, respectively. M

594

(0.5), M (1), and M (2): oxidized without epigallocatechin-3-gallate in the presence of

595

40, 80, and 160 µM/g methyl-β-cyclodextrin, respectively. Different letters denote a

596

statistical difference between means (p < 0.05).

597

Figure

598

methyl-β-cyclodextrin and epigallocatechin-3-gallate under oxidative stress. (A:

599

without β-mercaptoethanol; B: with β-mercaptoethanol). Control: non−oxidized; E,

600

E-M

601

epigallocatechin-3-gallate in the presence of 0, 40, 80, and 160 µM/g

602

methyl-β-cyclodextrin, respectively. M (0.5), M (1), and M (2): oxidized without

603


604

methyl-β-cyclodextrin, respectively.

2.

3.

E-8µM

and

Solubility

of

MPs

Representative

(1:0.5),

E-M

E-80µM:

as

affected

SDS–PAGE

(1:1),

in

and

the

oxidized

by

patterns

E-M

(1:2):

presence

of

in

the

presence

of


of

MPs

oxidized

40,

26 / 40


80,

as

with

and

and

affected

80

160

by

µM/g

µM/g

Page 27 of 40


605

Figure 4. Fluorescence spectra of MPs as affected by methyl-β-cyclodextrin and

606


607


608


609


610

40, 80, and 160 µM/g methyl-β-cyclodextrin, respectively.

611

Figure 5. Surface hydrophobicity of MPs as affected by methyl-β-cyclodextrin and

612


613


614


615


616

40, 80, and 160 µM/g methyl-β-cyclodextrin, respectively. Different letters denote a

617

statistical difference between means (p < 0.05).

618

Figure 6. Cooking loss (A) and strength (B) of MP emulsion gels as affected by

619


620

non−oxidized; E, E-M (1:0.5), E-M (1:1), and E-M (1:2): oxidized with 80 µM/g

621


622


623


624

methyl-β-cyclodextrin, respectively. Different letters denote a statistical difference

625

between means (p < 0.05).

626

Figure 7. Representative CLSM images of MP emulsion gels as affected by

in

the

presence

of

40,

27 / 40


80,

and

160

µM/g


Page 28 of 40

627


628


629


630


631


632


633

Figure 8. Storage modulus (G′) and loss factor (tan δ) of MPs as affected by

634


635


636


637


638


639


640

Figure 9. Raman spectra in 1600~1700 cm-1 region of MP emulsion gels (Control).

in

in

the

the

presence

presence

of

of

40,

40,

28 / 40


80,

80,

and

and

160

160

µM/g

µM/g

Page 29 of 40


70% Cooking loss

Gel strength

z

0.5 c

50%

0.4

40% 30%

y x

0.2

20% 10%

0.3

y

a

b

ab

0.1

0%

0 Control

Oxidized

E-8µM

Figure 1.

29 / 40


E-80µM

Gel strength (N)

Cooking loss (%)

60%

0.6


Page 30 of 40

0.5 a

Solubility (mg/mL)

0.4 b

b 0.3

c

d e

0.2 f 0.1

0

Figure 2.

30 / 40


d

c

Page 31 of 40


Figure 3.

31 / 40



Figure 4.

32 / 40


Page 32 of 40

Page 33 of 40


7 a

a

ab

b

M (0.5)

M (1)

M (2)

Surface hydrophobicity (BPB bound, µg)

6 c

c 5

d

e f

4 3 2 1 0 Control Oxidized

E

E-M (1:0.5)

E-M (1:1)

E-M (1:2)

Figure 5.

33 / 40



0.7

B

a

b

0.5 b

0.5 0.4

Gel strength (N)

Cooking loss (%)

0.6

0.6

A

a

Page 34 of 40

c de

0.3 f

d e

de

e

0.2

0.4 c

0.3 0.2

d e

0.1

0.1

0

0

Figure 6.

34 / 40


cd

d

cd e

Page 35 of 40


Control

E-M (1:0.5)

Oxidized

E-M (1:1) 35 / 40


E

E-M (1:2)


M (0.5)

M (1)

Figure 7.

36 / 40


Page 36 of 40

M (2)

Page 37 of 40


Figure 8.

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Figure 9.

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Table 1 Raman percentages of secondary structures amino acid side chain vibrations (AA), α-helix, β-sheet, β-turn and random coil of MP gel samples. Samples Control Oxidized E E-M (1:0.5) E-M (1:1) E-M (1:2) M (0.5) M (1) M (2)

AA (%) 5.03±0.12d 5.78±0.15a 5.26±0.09c 5.48±0.07b 5.74±0.10a 5.75±0.09a 5.83±0.15a 5.95±0.11a 5.91±0.12a

α-helix (%) 18.04±0.84a 16.02±0.75bc 10.66±0.66f 13.11±0.90e 14.70±0.58cd 14.47±0.88d 17.24±0.92ab 17.07±0.83ab 17.15±0.64ab

β-sheet (%) 36.10±0.24b 37.02±0.21a 34.01±0.27e 35.20±0.26c 34.83±0.23cd 34.75±0.19d 36.46±0.21b 36.40±0.22b 36.32±0.23b

β-turn (%) 15.59±0.71c 14.81±0.87c 20.71±0.93a 18.97±0.77b 18.11±0.63b 18.46±0.92b 14.70±0.59c 15.15±0.66c 15.13±0.55c

Radom coil (%) 25.24±0.48e 26.37±0.56bcd 29.37±0.51a 27.24±0.76b 26.63±0.88bc 26.57±0.47bcd 25.77±0.83cd 25.43±0.91cd 25.50±0.72cd

Note: Control: non−oxidized; E, E-M (1:0.5), E-M (1:1), and E-M (1:2): oxidized with 80 µM/g epigallocatechin-3-gallate in the presence of 0, 40, 80, and 160 µM/g methyl-β-cyclodextrin, respectively; M (0.5), M (1), and M (2): oxidized without epigallocatechin-3-gallate in the presence of 40, 80, and 160 µM/g methyl-β-cyclodextrin, respectively. Different letters denote a statistical difference between means (p < 0.05).

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protein interaction by methyl-Î²

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