Characterization of bovine serum albumin and (-)-epigallocatechin

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Food and Beverage Chemistry/Biochemistry

Characterization of bovine serum albumin and (-)-epigallocatechin gallate/3,4-O-dicaffeoylquinic acid/tannic acid layer-by-layer assembled microcapsule for protecting immunoglobulin G in stomach digestion and releasing in small intestinal tract Chunxu Chen, Guijie Chen, Peng Wan, Dan Chen, Tao Zhu, Bing Hu, Yi Sun, and Xiaoxiong Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04381 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 2018

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Journal of Agricultural and Food Chemistry

1

Characterization

of

Bovine

Serum

Albumin

and

(-)-Epigallocatechin

2

Gallate/3,4-O-Dicaffeoylquinic Acid/Tannic Acid Layer-by-layer Assembled

3

Microcapsule for Protecting Immunoglobulin G in Stomach Digestion and

4

Releasing in Small Intestinal Tract

5

Chunxu Chen,†,‡ Guijie Chen,† Peng Wan,† Dan Chen,† Tao Zhu,§ Bing Hu,† Yi Sun,†

6

Xiaoxiong Zeng†,*

7

†

8

210095, Jiangsu, China

9

‡

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

College of Food Engineering, Anhui Science and Technology University, Fengyang

10

233100, Anhui, China

11

§

12

Collaborative Innovation Center of Advanced Microstructures, Nanjing University,

13

Nanjing 210093, China

*

National Laboratory of Solid State Microstructures and Department of Physics,

Corresponding author. Fax: +86 25 84396791; E-mail address: [email protected] (X Zeng) 1

ACS Paragon Plus Environment


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14

ABSTRACT

15

The protein-polyphenols layer-by-layer (LbL) assembled polymer composite

16

microcapsule is a considerable delivery system that can be used to improve the

17

bioactive stability and effectiveness of natural compounds in various applications. In

18

the present study, three kinds of polyphenols were loaded in the sequence of

19

(-)-epigallocatechin gallate (EGCG), 3,4-O-dicaffeoylquinic acid (3,4-diCQA) and

20

tannin acid (TA) to prepare BSA-polyphenols LbL membrane. The composition of

21

IgG-(BSA-EGCG/3,4-diCQA/TA)n microcapsule and its stability and releasing ability

22

in gastrointestinal tract were evaluated. In addition, by binding these three kinds of

23

polyphenols to BSA, the thermal denaturation temperature and ordered secondary

24

structure of the BSA-polyphenols microcapsules were increased, and the time of

25

scavenging activity on 2,2'-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid) free

26

radicals

27

(BSA-EGCG/3,4-diCQA/TA)n microcapsule can not only protect IgG in food

28

processing and stomach digestion, but also release it in small intestinal tract for

29

bioactive delivery.

30

Keywords: Layer-by-layer; Bovine serum albumin; (-)-Epigallocatechin gallate;

31

3,4-O-Dicaffeoylquinic acid; Tannin acid; Microcapsules; Immunoglobulin G

was

significantly

prolonged.

These

findings

32

2


suggest

that

the

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33

INTRODUCTION

34

Immunoglobulin G (IgG) is one of the primary bovine colostrum bioactive antibodies1

35

showing antibacterial, antivirus and immunomodulatory activities by binding to

36

pathogens and activating special immunological functions (cell killing, activation of

37

complement, antibody dependent cell-mediated cytotoxicity, etc.).2,3 The action is to

38

remove and/or neutralize potential disease-causing agents. For example, IgG has been

39

proved to improve the body’s ability to resist Escherichia coli4 and Rotavirus.5

40

However, while being processed at high temperature,6 exposed to low pH gastric

41

juice7 and especially in pepsin digestion, the activity of IgG decreased because of its

42

heat, pH and pepsin sensitivity.8 Although the F(ab')2 fragment, which is resulted by

43

pepsin treatment of IgG, still can bind to antigen, it does not mediate the other

44

immunological functions as the complete antibodies.9 Thus, nowadays, how to

45

stabilize bioactivity of IgG becomes a research focus.

46

One considerable strategy for protection the bioactivity of IgG is the use of

47

layer-by-layer (LbL) assembled microcapsules. Due to the capability of tailoring

48

multiple functional materials in one shell, LbL shells can load a great variety of

49

molecules and gradually release them through diffusion or force burst by remote

50

activation of physical (ultrasound, light, magnetic field, temperature, etc.) or chemical

51

(pH, enzyme, etc.) factors.10 The thickness of LbL shell can also be adjusted

52

accurately in the range from a few nanometers to micrometers by controlling the

53

number of layers.11 Hence, there is a need to develop a new kind of LbL microcapsule

54

able to deliver IgG with bioactive stability and effectiveness to promote its health

3



55

benefits.12

56

To overcome the use of highly cytotoxic polycations in LbL process, a variety of

57

means have been reported in literatures for the preparation of microcapsules using

58

natural compounds as materials.13,14 Polyphenols and proteins as common natural

59

products can form complexes by LbL. Polyphenol compounds, as one of the most

60

important secondary metabolites in plants, have strong antioxidant, antibacterial and

61

anticancer effects.15,16 Moreover, studies have shown that polyphenols can be used as

62

cross-linking agents since polyhydric phenol structures of polyphenols have unique

63

physical and chemical capability of binding to proteins (especially those rich in

64

proline or hydrophobic proteins).17-19 In addition, the binding of polyphenols to

65

proteins may strength the antioxidant properties of complexes.20,21 The non-covalent

66

interactions between proteins and polyphenols include hydrophobic, H-bonding

67

contacts and Van Der Waals force, where the protein conjugates with larger

68

polyphenols will be more stable.22 Although the individual non-covalent interaction is

69

weak, the whole interaction forces can be enhanced synergistically under certain

70

conditions.23 Therefore, the ability of bridging and precipitating proteins from

71

solution with polyphenols is an simple and effective way to form LbL encapsulating

72

films. Furthermore, polyphenols can enhance the assemblies’ thermal stability by

73

increasing the ordered structure in the protein such as α-helixes and β-strands.24,25

74

Bovine serum albumin (BSA), a primarily transfer protein for both endogenous

75

and exogenous biological substances in the circulatory system, has been found not

76

only in blood but also in milk.26 BSA is able to bind with many kinds of natural

4


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77

compounds such as fatty acids, steroids and polyphenols.27-29 Also, as a protein that

78

may withstand proteolytic degradation of gastric digestive enzymes, BSA presents

79

another characteristic of being cleaved by intestinal enzymes.30 Most researches have

80

devoted to the interactions of forming simple BSA-single/multiligand complexes and

81

some results showed that the binding ability of polyphenols to BSA could be

82

determined by the amount of galloyl groups in polyphenols.31-33 (-)-Epigallocatechin

83

gallate (EGCG), tannic acid (TA) and caffeoylquinic acid (CQA) derivatives are plant

84

bioactive polyphenols with galloyl groups. The kinetics of adsorption of EGCG, TA

85

and thearubigins on BSA fixed layer has already been studied by using quartz crystal

86

microbalance (QCM).34-36 Furthermore, the interactions between BSA and four CQA

87

derivatives (5-CQA, 3,4-diCQA, 3,5-diCQA and 4,5-diCQA), isolated from the leaves

88

of Ilex kudingcha C.J. Tseng, were investigated in our previous works, and 3,4-diCQA

89

showed the highest binding constant with BSA.37 However, there is no research on the

90

interactions between one kind of protein and multiple polyphenol bridging agents at

91

the same time, in spite of the synergistic effect of different kinds of polyphenols has

92

been reported.38,39 Therefore, the aim of this study is to develop delivery system based

93

on BSA and EGCG/3,4-diCQA/TA LbL multilayers microcapsule for protecting IgG

94

of bovine colostrum in stomach digestion and releasing in small intestinal tract.

95

Moreover, the thermal stability, release characteristic and antioxidant properties were

96

investigated. The present study may open the prospect of research on LbL multilayer

97

microcapsules for delivery of bioactive substances including IgG and utilization in

98

food industry.

5



99

MATERIALS AND METHODS

100

Materials. BSA (>96%, molar weight 67 kDa) and IgG from bovine serum (>97%,

101

molar weight 150 kDa) were purchased from Sigma Chemical Co. (St. Louis, MO,

102

USA). Poly-L-lysine hydrochloride (PLL, molar weight 15,000-30,000), TA, EGCG

103

and 3,4-diCQA were obtained from Shanghai Reagent Co., Ltd. (Shanghai, China).

104

Pancreatin (≥100 U/mg) from porcine pancreas, pepsin (3800 U/mg) from porcine

105

gastric mucosa, bile salts (for microbiology), α-chymotrypsin (98.7 U/mg) from

106

bovine pancreas, trypsin (≥10.000 U/mg) from bovine pancreas were purchased from

107

Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). HPLC grade of formic acid

108

and methanol were purchased from TEDIA Co., Inc. (Fairfield, USA). All other

109

chemical reagents, analytical grade used in this work, were purchased from China

110

Pharmaceutical Chemical Reagent Co., Ltd. (Shanghai, China).

111

Preparation of (BSA-polyphenols)n LbL multilayer film. The preparation of

112

(BSA-polyphenols)n LbL multilayer film was carried out according to the reported

113

method30 with some modifications. Firstly, 0.3 mL PLL solution (2.0 mg/mL) was

114

firstly introduced to a clean Si substrate (0.9 × 0.9 cm) for 15 min following with

115

washing of deionized water to afford the anchoring layer. Then, 0.3 mL BSA (2.0

116

mg/mL) and polyphenols (2.0 mg/mL) solutions were added, respectively, for about 5

117

min in turn with washing of deionized water after each step to wash away excess

118

molecules. The (BSA-polyphenols)n LbL films were produced by repeating the

119

procedures as mentioned above.

120

Preparation of IgG-(BSA-polyphenols)n LbL multilayer microcapsules. Based

6


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121

on the reported method14 with minor modification, 0.6 mL of 0.4 M Na2CO3 and 0.8

122

mL of 4.0 mg/mL IgG solutions were firstly mixed for 2 min. Then, 0.6 mL of 0.4 M

123

CaCl2 solution was injected into the mixed solution under vigorous agitation for 5 min.

124

The IgG-CaCO3 templates were prepared after being rinsed with deionized water and

125

centrifuged. To synthesize LbL outer film, IgG-CaCO3 particles were added into PLL

126

(2.0 mg/mL) solution under agitation for 15 min for the forming of the anchoring

127

layer. After removing residual PLL with rinsing of deionized water and centrifuging,

128

5.0 mL of 2.0 mg/mL BSA and polyphenols solutions were added, respectively, for 5

129

min in turn with washing steps. The (BSA-polyphenols)n LbL outer layers were

130

produced by repeating the procedures as mentioned above. Finally, 0.2 M of EDTA

131

solution was used to extract CaCO3 from the microcapsules for 15 min.

132

Digestion in vitro of (BSA-polyphenols)n multilayer film and microcapsules.

133

According to the previously reported method40 with modification, the simulated

134

gastric and small intestinal digestion of (BSA-polyphenols)n multilayer film and

135

microcapsules was evaluated. The simulated gastric fluid (SGF) was composed of

136

2.48 g NaCl, 0.2 g CaCl2, 0.88 g KCl, 0.48 g NaHCO3, 23.6 mg gastric pepsin and

137

25.0 mg gastric lipase in 1.0 L deionized water. The pH of solution was adjusted to

138

2.0 with 1.0 M HCl solution. The simulated intestinal fluid (SIF) was prepared with

139

5.4 g/L NaCl, 330.0 g/L CaCl2, 0.65 g/L KCl, 10.0 g/L bile salt, 0.105 g/L

140

α-chymotrypsin, 0.1 g/L trypsin, 35.0 g/L pancreatin and deionized water. The pH of

141

solution was adjusted to 7.5 with 0.1 M of NaOH solution. The film and

142

microcapsules were treated with SGF and SIF at 37 ℃ for 2 h and 1 h, respectively,

7



143

for morphology observation.

144

QCM with Dissipation (QCM-D) Measurement. QCM-D of a Q-Sense D300

145

electronic unit (Q-Sense AB, Sweden) was used to investigate the interactions

146

between polyphenols and BSA. PLL solution (2.0 mg/mL) was firstly introduced to

147

prepare the first anchor layer on quartz crystal surface. After getting a stable baseline

148

by washing with deionized water, 2.0 mg/mL BSA and polyphenols solutions were

149

added, respectively, in the QCM-D chamber at a flow rate of 5.0 µL/min with

150

washing after each step. The adsorption was monitored by recording the shifts of

151

frequency (∆f).

152

Circular Dichroism (CD) Measurement. According to previous report,41 CD

153

spectra of BSA, BSA-EGCG, BSA-3,4-diCQA, BSA-TA, BSA-EGCG-3,4-diCQA

154

and BSA-EGCG-3,4-diCQA-TA dissolved in 20 mM phosphate buffer (pH 6.9) were

155

recorded with a Jasco-810 CD spectrometer (JASCO Corp., Tokyo, Japan) at room

156

temperature, respectively. The concentrations of BSA, EGCG, 3,4-diCQA and TA in

157

the system were set at 0.5 mg/mL. Ellipticity was recorded at a speed of 100 nm/min

158

and 1.0 nm bandwidth from 190 to 250 nm. In addition, three scans were accumulated

159

for each spectrum. The CD data were analyzed by the SELCON3 method in CDPro

160

software.

161

Differential Scanning Calorimetry (DSC) Measurement. With Seiko 120 DSC

162

analyzer (Seiko Instruments Inc., Chiba, Japan), 5.0 mg sample (IgG-BSA,

163

IgG-BSA-EGCG or IgG-BSA-EGCG-TA) was placed inside an aluminum pan with a

164

standard procedure and heated from 40 ℃ to 160 ℃ at a constant rate of 10 ℃/min.

8


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165

During the analysis, dry nitrogen was added at a rate of 30.0 mL/min. An empty

166

aluminum pan was used as a control.

167

Morphology Characterization. The morphology of microcapsules after SGF and

168

SIF treatments was observed by scanning electron microscopy (SEM, JEOL

169

JSM-6700F). The morphology of LbL multilayer film after SGF and SIF treatments

170

was investigated by atomic force microscopy (AFM, Q-ScopeTM 250).

171

Assay of Scavenging Activity on 2,2'-Azinobis-(3-ethyl-benzothiazolin-6-sulfonic

172

acid) (ABTS) Free Radicals. According to the reference,42 the ABTS free radicals

173

were prepared by oxidation ABTS (7.00 mM) with 4.95 mM potassium persulfate

174

(K2S2O8) in the dark at room temperature for 12 h, and the working solution was then

175

generated by diluting the prepared ABTS solution with 0.2 M phosphate buffer saline

176

(PBS, pH 7.4). The absorbance (Abs) of mixture of 20 µL sample and 200 µL

177

working solution at 734 nm was measured after reaction at room temperature for 6

178

min.

179

ABTS free radical scavenging activity (%) = [1-(Abs1-Abs2)/Abs0] × 100

180

where Abs0 is the Abs of the control (water instead of sample), Abs1 is the Abs of the

181

sample, and Abs2 is the Abs of a standard prepared as that for Abs1 (methanol instead

182

of PBS)

183

HPLC Analysis. The contents of EGCG, 3,4-diCQA, TA, BSA and IgG were

184

determined by HPLC (Agilent 1100). For EGCG and TA, separation of samples was

185

completed on TSK-gel ODS-100Z column (4.6 × 150 mm, 5 µm, Tosoh Corp., Tokyo,

186

Japan) with a gradient mobile phase consisted of 1.0% (v/v) formic acid (A) and

9



187

methanol (B) at a flow rate of 1.0 mL/min. The linear gradient of elution was

188

performed as follows: A was reduced from 82% to 40%. The temperature of column

189

oven was set at 40 ℃ and the injection volume was 20 µL. EGCG and TA were

190

detected at 280 nm. For 3,4-diCQA, the analysis was completed on TSKgel ODS-80

191

TsQA column (4.6 × 250 mm, 5 µm, Tosoh Corp.) with an isocratic mobile phase

192

consisted of water (A, 35%), 20% (v/v) methanol (B, 45%) and 1.0% formic acid (C,

193

20%) at a flow rate of 0.5 mL/min. The temperature of column oven was set at 30 ℃

194

and the injection volume was 20 µL. 3,4-diCQA was detected at 280 nm. For BSA

195

and IgG, the separation was completed on TSK-gel G4000 PWXL column (7.8 × 300

196

mm, Tosoh Corp.) with an isocratic mobile phase of 20 mmol/L PBS containing 0.3

197

mol/L NaCl at a flow rate of 0.6 mL/min. The temperature of column oven was set at

198

30 ℃ and the injection volume was 20 µL. BSA and IgG were detected at 280 nm.

199

The amount of sample in microcapsules (mol) was calculated as following:

200

Amount (mol) = (C0-C1) × V/M

201

where C0 is the initial concentration before LbL process, C1 is the remaining

202

concentration after LbL process, M is the molar mass and V is the volume of the

203

reaction solution.

204

Enzyme-linked Immunosorbent Assay (ELISA). The contents of IgG in

205

microcapsules with different treatments were evaluated by a commercial ELISA test

206

kit following the instruction of manufacturer (Bethyl Laboratories, USA). The free

207

IgG and microcapsules were treated with SGF and SIF at 37 ℃ for 2 and 1 h,

208

respectively.

10


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209

Statistical Analysis. All of the data were expressed as the mean ± standard deviation

210

(SD) of triplicates. One-way analysis of variance (ANOVA) was used to analyze

211

significant difference through multiple comparisons by SPSS 22 software (IBM, New

212

York, USA). The difference was considered to be significant with p value < 0.05.

213

RESULTS AND DISCUSSION

214

Effects of Loading Sequence on BSA-polyphenols Interactions. The previous

215

reports showed that the galloyl group of polyphenols was the responsible functional

216

group for the cross-link between proteins and polyphenols,43,44 and the interaction

217

strength was depended on the molecular size, hydrophobicity and number of galloyl

218

group of polyphenols.45,46 Thus, EGCG, TA and 3,4-diCQA with the galloyl groups as

219

bridges can lead to the aggregation of BSA. Also, the sequence of addition of different

220

ligands could affect the production of protein-ligand complexes.47,48 Hence, in this

221

study, EGCG, TA and 3,4-diCQA were loaded in different sequence to evaluate the

222

impact on polyphenol-BSA LbL membranes by QCM-D.

223

As shown in Figure 1A, PLL solution was firstly introduced as the anchoring

224

layer. Right after the injection of BSA, there was a rapid decrease in ∆f (73 Hz),

225

followed with rinsing water until the steady state was reached. The increase of ∆f at

226

around 700 s might be caused by desorption of excessive BSA on the PLL surface.

227

The subsequent loading of 3,4-diCQA solution resulted in a significant decrease of the

228

frequency which implied the formation of complex. However, the value of ∆f kept

229

almost the same after EGCG loading. This phenomenon indicated that the activation

230

did not happen and the mass and the conformation of the molecules on the sensor 11



231

surface did not change. Figure 1B shows the changes in ∆f with loading BSA, TA and

232

EGCG in sequence. Similarly, the combination of PLL and BSA occurred at the

233

beginning. The addition of TA solution resulted in significant change in frequency.

234

Subsequently, the ∆f value not only did not decrease but also increased after loading

235

EGCG, suggesting that the excessive TA molecules were loosely adsorbed on the BSA

236

surface.35,36 From Figure 1A and 1B, it could be concluded that EGCG should be

237

loaded before TA and 3,4-diCQA.

238

As shown in Figure 1C, although 3,4-diCQA could combine with BSA after TA

239

loading process, the decrease in ∆f (20Hz) was less than that in Figure 1A (35Hz),

240

which implied that the binding of TA could reduce the binding amount of 3,4-diCQA.

241

In addition, the value of ∆f also increased by rinsing away loosely excessive

242

3,4-diCQA molecules during loading EGCG on the BSA surface. From Figure 1A

243

and 1C, it could be concluded that 3,4-diCQA should be loaded before TA. Therefore,

244

in order to bind the three polyphenols to BSA better, EGCG, 3,4-diCQA and TA were

245

added in turn. Figure 1D shows that polyphenols could combined with BSA in the

246

order of EGCG, 3,4-diCQA and TA. Furthermore, BSA could still be attached to the

247

combination layer after loading the three polyphenols, indicating that single

248

BSA-polyphenols shell could be combined to assembly LbL to form a

249

(BSA-EGCG-3,4-diCQA-TA)n multilayer shells.

250

Composition of IgG-(BSA-polyphenols)n

Multilayer Microcapsules. The

251

constructed IgG-(BSA-polyphenols)n microcapsules not only provided a quantitative

252

description of the binding capacity of polyphenols, but also could suggest

12


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253

implications for the mechanism of assembly during the binding process. To avoid

254

effects of other proteins during HPLC analysis, the CaCO3-(BSA-polyphenols)3

255

particles were firstly synthesized without IgG. The contents of EGCG, 3,4-diCQA, TA

256

and BSA in samples before (initial concentration) and after (remaining concentration)

257

LbL process were investigated by HPLC, respectively. And the HPLC chromatograms

258

of IgG, BSA, 3,4-di-CQA, TA and EGCG are shown in Figure S1. Table 1 presents

259

the binding amounts including the linear equation of every component and coefficient

260

of determination (R2) in CaCO3-(BSA-polyphenols)3. It can be seen that the molar

261

ratio calculated based on HPLC analysis was nearly constant (BSA: EGCG:

262

3,4-diCQA: TA = 1: 4: 13: 87) when they were investigated in three separate layers of

263

a CaCO3-(BSA-polyphenols)3. This was supported by Siebert’s model,49 in which

264

protein-polyphenols could produce the largest network if the number of polyphenol

265

ends equals the number of protein binding sites. Furthermore, to determine the

266

loading content of IgG in the microcapsules, the IgG-CaCO3 particles without BSA

267

and polyphenols were synthesized and the results showed that the encapsulation

268

extent of IgG in IgG-CaCO3 particles was about 3.27 × 10-8 mol. Since the same

269

amount of Na2CO3 and CaCl2 were used to synthesize CaCO3-(BSA-polyphenols)3

270

and IgG-CaCO3 particles, it could be concluded that after EDTA treatment the molar

271

ratio of each component in IgG-(BSA-EGCG/3,4-diCQA/TA)n microcapsule was

272

nearly IgG: BSA: EGCG: 3,4-diCQA: TA = 3: (1: 4: 13: 87)n.

273 274

Stability and Release of Microcapsules in Simulated SGF and SIF. In order to investigate

the

gastric

stability

and

intestinal

13


release

characteristics

of


275

(BSA-EGCG-3,4-diCQA-TA)n microcapsules, EDTA, SGF and SIF were sequentially

276

introduced into the (BSA-EGCG-3,4-diCQA-TA)2 LbL membrane. As shown in

277

Figure 2, right after the injection of EDTA solution, there was a slowly and slightly

278

increase in ∆f until the steady state reached, which might be caused by rinsing away

279

loosely excessive TA molecules on the (BSA-EGCG-3,4-diCQA-TA)2 surface by

280

EDTA solution. The result indicated that EDTA had limited effect on the structure of

281

(BSA-EGCG-3,4-diCQA-TA)2 membrane. When pepsin was added, the ∆f of

282

(BSA-EGCG-3,4-diCQA-TA)2 film decreased slightly at first. It was probably due to

283

that pepsin as a digestive protein in SGF was adsorbed at the moment of injection by

284

the tannins on (BSA-EGCG-3,4-diCQA-TA)2 surface under the gastric condition.30

285

After that, the ∆f returned back and maintained a stable state for about 2 h with SGF

286

treatment, suggesting that the pepsin was desorbed before binding more to TA. The

287

pepsin resistance of BSA resulted in that the membrane structure was not affected.50 It

288

can be seen that the ∆f value was higher (30 Hz) than that of the first BSA layer after

289

PLL binding, suggesting that SIF could digest BSA into smaller polypeptides on the

290

PLL layer. In a word, the BSA-polyphenols assembled microcapsules could resist

291

gastric digestion and release in intestinal tract.

292

To characterize the BSA-polyphenols film thickness and morphology, 50

293

assembly layers fixed on silicon wafers pre-anchored with PLL were prepared. As

294

results, the AFM micrographs of PLL-(BSA-EGCG-3,4-diCQA-TA)50 film showed

295

the expected large differences in microarchitecture between treatments with SGF and

296

SIF digestion (Figure 3), indicating that the BSA-polyphenols membrane could resist

14


Page 14 of 39

Page 15 of 39


297

SGF in stomach and release in the small intestine. There was no difference between

298

control

299

PLL-(BSA-EGCG-3,4-diCQA-TA)50 film was very rough (Figure 3, control), which

300

might be due to the collapse of the surface morphology of the film after being dried.

301

The diameter of the surface bulge was about 400-500 nm. In contrast to the structure

302

of BSA-polyphenols membrane with SGF treatment (Figure 3A), the surface of the

303

membrane after treatment of SIF was smoother and had more fine particles (about 100

304

nm in diameter, Figure 3B), where the roughness decreased from 46.9 nm to 8.24 nm,

305

only with polypeptide resulted from BSA treated by trypsin and α-chymotrypsin on

306

the PLL surface. Figure 3C is a partially enlarged version of Figure 3B (marked with

307

white dotted line).

and

that

treated

with

SGF.

In

particular,

the

surface

of

the

308

To further study the difference between SIF and SGF treated surfaces, sectional

309

drawings were observed by SEM. After SGF treatment, the thickness of the

310

PLL-(BSA-EGCG-3,4-diCQA-TA)50 film was about 500 nm (Figure 4A and 4A'),

311

which is in a good agreement with that of 8-10 nm for a separate BSA layer as

312

previous report.30 While the thickness was found to have a significant change due to

313

the decomposition of BSA-polyphenol film on silicon surface by SIF treatment

314

(Figure 4B and 4B'). From the SEM observation, the microarchitecture of

315

BSA-polyphenols films with trypsin and α-chymotrypsin treatments in SIF was

316

relatively similar to that measured by QCM-D or AFM, illustrating that

317

(BSA-EGCG-3,4-diCQA-TA)n film could be effectively preserved in stomach and

318

released in small intestine.

15



Page 16 of 39

319

The IgG-(BSA-EGCG-3,4-diCQA-TA)50 microcapsules were prepared with

320

developed BSA-EGCG-3,4-diCQA-TA film as templates. The SEM images of

321

IgG-(BSA-EGCG-3,4-diCQA-TA)50 microcapsules with digestion of SGF, SIF, EDTA

322

and without treatment are shown in Figure 5. The microcapsules had spherical shape

323

and size of 8-10 µm with SGF digestion for 2 h. Compared with the control, the

324

microcapsules without CaCO3 core had much rougher surface and the appearance of

325

IgG-(BSA-EGCG-3,4-diCQA-TA)50 microcapsules was not changed significantly by

326

SGF treatment. Intriguingly, the surface of SGF treated microcapsules was much

327

smoother than that of EDTA treatment. It was probably due to that pepsin as a

328

digestive protein in SGF was adsorbed by TA on microcapsules’ surface.30 In contrast,

329

the capsules decomposed immediately after introduction of SIF, demonstrating the

330

releasing property of (BSA-EGCG-3,4-diCQA-TA)50 microcapsules. Although some

331

microcapsules had concave spherical surface due to the removal of the CaCO3 core,

332

the assembly and degradation of shells in SGF and SIF were the same with the shells

333

formed on flat Si substrate.

334

To further verify the integrity of the encapsulated IgG in SGF and SIF, the

335

contents of IgG in microcapsules with different treatments were evaluated by a

336

commercial ELISA test kit. As shown in Table S1, the encapsulated antibodies were

337

intact upon exposure to pH condition in the stomach and SIF-induced capsule

338

disassembly. Moreover, it was found that microcapsules could protect IgG from

339

stomach digestion when the number of layers was more than 5.

340

Thermal

Characteristic.

The

thermal stability

16


of

the BSA-polyphenols

Page 17 of 39


341

microcapsules was investigated by DSC. As shown in Figure 6, the microcapsules

342

showed a low thermal denaturation temperature (72 ℃) when the outer film had only

343

BSA without polyphenols. Upon modification with EGCG, there was a

344

correspondingly decrease to about 70 ℃. In a similar manner, the thermal

345

denaturation temperature dropped to around 68 ℃ with the further combination of

346

3,4-diCQA. Intriguingly, the denaturation temperature raised back to approximately

347

78 ℃ when TA was combined. These results suggested that the increased thermal

348

stability of (BSA-EGCG-3,4-diCQA-TA)n microcapsules was attributed to the

349

conjugation with TA in spite of the heat temperature drop by EGCG and 3,4-diCQA.

350

It is well known that the thermal stability of protein is directly proportional to the

351

amount of ordered structures (α-helix and β-sheets) and inversely proportional to the

352

amount of unordered structures.25,51 To further understand these structures modified

353

by polyphenols, CD spectroscopy was conducted to evaluate the influence of EGCG,

354

3,4-diCQA and TA on the secondary structure of BSA in the far-UV CD range (250 to

355

190 nm).41 As shown in Figure 7, the two mainly negative peaks at 208 and 222 nm

356

can represent spectra of BSA, which are caused by the n–π* transition of the helix

357

structure.52 With the binding of EGCG and 3,4-diCQA simultaneously or separately,

358

the two negative peak signals of BSA at 208 and 222 nm became weak, indicating that

359

the content of ordered structures in the secondary structure was reduced. On the

360

contrary, binding of TA enhanced the signal of the two peaks, which is in a good

361

agreement with the thermal stability results of Figure 6. As shown in Table 2, the

362

secondary structure proportions for free BSA were ordered 83.4% and unordered

17



363

15.6%. With the addition of EGCG, 3,4-diCQA and TA to BSA in sequence, the

364

ordered contents changed to 81.6, 79.3 and 84.1%, while the ordered contents of

365

BSA-TA and BSA-3,4-diCQA were 82.6 and 81.0%, respectively. In addition, it was

366

found that BSA-EGCG-3,4-diCQA-TA had more ordered contents than BSA-TA,

367

meaning the addition of EGCG and 3,4-diCQA could promote the increasing ordered

368

ability of TA for BSA.

369

Long-term Antioxidant Characteristic. The scavenging activity on ABTS free

370

radicals is inhibited by forming protein-polyphenols complexes,53 resulting in a

371

long-term antioxidant activity.54 In this study, the long-term antioxidant activity of

372

(BSA-EGCG-3,4-diCQA-TA)50 microcapsule was investigated by measuring the

373

scavenging activity on ABTS free radicals. Figure 8A shows that the time of

374

completed scavenging on ABTS free radicals was more than 24 h by adding

375

(BSA–EGCG-3,4-diCQA-TA)50, while EGCG-3,4-diCQA-TA mixture solution with

376

the same content of polyphenols almost got the maximum clearance rate at the

377

beginning. To further study the effects of different layers on antioxidation time,

378

microcapsules with 10 to 50 layers were investigated by recording the time of 100%

379

scavenging on ABTS free radicals. As shown in Figure 8B, the antioxidant

380

maintaining time prolonged with the increase of the number of layers. These

381

phenomenon suggested that (BSA-EGCG-3,4-diCQA-TA)n LbL multilayer film could

382

prolong the antioxidation time of microcapsule and effectively protect the antioxidant

383

from being destroyed in shell and core.

384

In conclusion, it was found that the three polyphenols (EGCG, 3,4-diCQA and TA)

18


Page 18 of 39

Page 19 of 39


385

should be loaded in the sequence of EGCG, 3,4-diCQA and TA to form a combination

386

layer

387

IgG-(BSA-EGCG/3,4-diCQA/TA)n microcapsules was determined to be 3: (1: 4: 13:

388

87)n. The (BSA-EGCG/3,4-diCQA/TA)n multilayer films and microcapsules were

389

proved to be stable in SGF and release quickly in SIF by analysis with AFM, SEM

390

and QCM-D. In addition, by binding EGCG, 3,4-diCQA and TA to BSA, the thermal

391

denaturation temperature and ordered secondary structure content of the

392

BSA-polyphenols microcapsules increased accompanying with the prolonged time of

393

scavenging

394

(BSA-EGCG/3,4-diCQA/TA)n microcapsule can not only protect IgG in food

395

processing and stomach digestion, but also release it in intestinal tract for bioactive

396

delivery.

397

ASSOCIATED CONTENT

398

Supporting information

399

The Supporting Information is available free of charge on the ACS Publications

400

website at DOI: 10.1021/acs.jafc.8b04381. Table S1 for contents of IgG in

401

microcapsules with different treatments and Figure S1 for HPLC chromatograms of

402

IgG, BSA, 3,4-di-CQA, TA and EGCG (PDF).

403

AUTHOR INFORMATION

404

Corresponding Author

405

*Phone: +86-25-84396791. E-mail: [email protected] (X Zeng).

with

binding

on

of

ABTS

BSA.

free

The

molar

radicals.

All

ratio

these

19


of

each

findings

component

suggest

in

that


406

ORCID

407

Xiaoxiong Zeng: 0000-0003-2954-3896

408

Funding

409

The study was supported by the National Key Research and Development Program of

410

China (2017YFD0400600) and a project funded by the Priority Academic Program

411

Development of Jiangsu Higher Education Institutions.

412

Notes

413

The authors have declared no conflict of interest.

414

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415

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416

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tannic acid with bovine serum albumin, egg ovalbumin and bovine beta-lactoglobulin.

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Sci. Technol. 2016, 53, 3455-3464.

573

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574

dichroism calculations. J. Am. Chem. Soc. 1999, 121, 9636-9644.

575

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576

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577

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578

layer-by-layer assembled films and microcapsules. J. Colloid Interf. Sci. 2009, 330,

579

276-283.

27



Page 28 of 39

580

Figure Captions

581

Figure 1. Impact of different loading sequence on polyphenol-BSA LbL films. A,

582

BSA-3,4-diCQA-EGCG; B, BSA-TA-EGCG; C, BSA-TA-3,4-diCQA-EGCG; D,

583

BSA-3,4-diCQA-EGCG-TA-BSA.

584

Figure 2. In vitro digestion of polyphenol-BSA multilayers.

585

Figure 3. AFM image of PLL-(BSA-EGCG-3, 4-diCQA -TA)50 film with SGF (A),

586

SIF (B and C) digestion and without treatment (control) on a flat silicon wafer.

587

Figure 4. SEM images of PLL-(BSA-EGCG-3,4-diCQA-TA)50 film with SGF (A and

588

A’) and SIF (B and B’) digestion on a flat silicon wafer.

589

Figure 5. SEM images of IgG-(BSA-EGCG-3,4-diCQA-TA)50 microcapsules with

590

SGF (A1, A2), SIF (B1, B2) digestion, EDTA treatment (remove CaCO3) and without

591

treatment (control).

592

Figure 6. Differential scanning calorimetry thermographs of CaCO3-PLL-BSA,

593

CaCO3-PLL-BSA-EGCG,

594

CaCO3-PLL-BSA-EGCG-3,4-diCQA-TA microcapsules.

595

Figure 7. Circular dichroism spectra of BSA, BSA-EGCG, BSA-3,4-diCQA, BSA-TA,

596

BSA-EGCG-3,4-diCQA and BSA-EGCG-3,4-diCQA-TA conjugates.

597

Figure 8. ABTS radical scavenging effects of (BSA-EGCG-3,4-diCQA-TA)50

598

microcapsules and EGCG-3,4-diCQA-TA mixture solution with the same polyphenol

599

content (A) and effects of different layers on the prolonged time of ABTS radical

600

scavenging activity (B).

CaCO3-PLL-BSA–EGCG-3,4-diCQA

28


and

Page 29 of 39

601


Table 1. Binding Amount of Polyphenols and BSA in Different Layers BSA (moL) 1st layer 9.81×10-9 2nd layer 9.62×10-9 3rd layer 9.90×10-9 Equation Y=1046.4X-49.20 R2 0.9930

EGCG (moL) 4.14×10-8 4.36×10-8 4.58×10-8 Y=326.7X+134.37 0.9963

3,4-diCQA (moL) 1.24×10-7 1.30×10-7 1.30×10-7 Y=41.03X-285.84 0.9958

TA (moL) 8.46×10-7 8.43×10-7 8.70×10-7 Y=2675.5X-73.48 0.9988

29


Molar ratio

IgG (moL)

1: 4.22: 12.63: 86.3 1: 4.54: 13.51: 87.8 1: 4.63: 13.11: 87.9

3.20×10-8 Y=291.08X-234.47 0.9927


602

Table 2 Secondary Structure Contents of BSA with EGCG, 3,4-diCQA and TA by Circular Dichroism Spectroscopy at Room Temperature BSA BSA-EGCG BSA-EGCG-3,4-diCQA BSA-EGCG-3,4-diCQA -TA BSA-TA BSA-3,4-diCQA

Α-Helix (%) 81.3 79.2 77.0 82.0 79.7 80.1

Β-Sheet (%) 2.1 2.4 2.3 2.1 2.9 0.9

Turn (%) 5.6 5.9 6.2 6.3 5.7 6.2

Ordered (%) 83.4 81.6 79.3 84.1 82.6 81.0

30


Unordered (%) 15.6 16.8 16.2 10.4 15.6 16.1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5 35



Figure 6

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Figure 7

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Figure 8

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Graphic abstract

39


Characterization of bovine serum albumin and (-)-epigallocatechin

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