Preparation and Self-Assembly Mechanism of Bovine Serum Albumin

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Preparation and self-assembly mechanism of bovine serum albumin-citrus peel pectin conjugated hydrogel: a potential delivery system for vitamin C Hailong Peng, Sha Chen, Mei Luo, Fangjian Ning, Xue-Mei Zhu, and Hua Xiong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02966 • Publication Date (Web): 13 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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Journal of Agricultural and Food Chemistry

1

Preparation and

self-assembly

mechanism of

bovine

serum

2

albumin-citrus peel pectin conjugated hydrogel: a potential delivery

3

system for vitamin C

4 5

Hailong Penga, bǁ, Sha Chenaǁ, Mei Luoa, b, Fangjian Ninga, Xuemei Zhua, Hua Xionga*

6 7

a

8

Nanchang 330047, Jiangxi, China

9

b

10

State Key Laboratory of Food Science and Technology, Nanchang University,

Department of Chemical and Pharmaceutical Engineering, Nanchang University,

Nanchang 330031, Jiangxi, China

11 12

*Corresponding author: Tel: +86-791-86634810; Fax: +86-791-86634810

13

E-mail address: [email protected] (H, Xiong)

14

ǁ

Equally contributed to this work and should be regarded as co-first authors

15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment


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23

ABSTRACT

24

In this study, a novel hydrogel (BSA-pectin hydrogel, BPH) was prepared via a

25

self-assembly method by using the natural polymers of bovine serum albumin (BSA)

26

and citrus peel pectin (pectin). The rheological properties and gel conformational

27

structures were determined, and showed that electrostatic and covalent interactions

28

between BSA and pectin were the main formation mechanisms of BPH. The

29

morphological characteristics of BPH exhibited a stable and solid three-dimensional

30

network structure with a narrow size distribution (polydispersity index, PDI < 0.06).

31

BPH was used as a delivery system to load the functional agent of vitamin C (Vc).

32

The encapsulation efficiency (EE) and release properties of Vc from BPH was also

33

investigated. These results suggested that the EE of Vc into BPH was approximately

34

65.31%, and the in vitro Vc release from BPH was governed by two distinct stages

35

(i.e., burst release and sustained release) under different pH solutions with release

36

mechanisms of diffusion, swelling, and erosion. Meanwhile, the stability results

37

showed that BPH was a stable system with an enhanced Vc retention (73.95%) after

38

10-weeks storage. Thus, this three-dimensional network system of BPH may be a

39

potential delivery system to improve the stability and bioavailability of functional

40

agent in both food and non-food fields.

41 42

KEYWORDS: bovine serum albumin, citrus peel pectin, vitamin C, self-assembly,

43

hydrogel

44 2


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INTRODUCTION

46

Vitamin C (Vc) is an indispensable nutrient required to retain the physiological

47

processes of humans because it removes harmful free radicals and accelerates

48

collagen synthesis.1,2 Vc also exhibits a wide range of pharmacological properties,

49

including anti-aging, anti-oxidation, anti-hypertension, and anti-cancer.3,4 However,

50

the sustainability of Vc is low and most of its functionality is lost during processing

51

and storage of food and feeds due to the exposure to light, heat, moisture, and

52

oxygen.5,6 The utilization of more stable forms of Vc is therefore a crucial

53

requirement for human nutrition. To solve these drawbacks, encapsulation is a

54

technique that has recently been utilized for shelf-life extension of Vc, such as

55

liposome,7,8 nanoparticle,9 and microencapsulation.10 However, organic solvent is

56

usually used in the liposome preparation processing, synthetic polymer is the main

57

wall material for the nanoparticle, and higher temperature is needed for

58

microencapsulation. Consequently, these encapsulated techniques often suffer from

59

various disadvantages of security risk and instability. Thus, food-grade material-based

60

and environmentally-friendly encapsulated technique should be developed to

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encapsulate Vc for effectively overcoming these problems.

62

Hydrogel is a water-swollen network of hydrophilic polymers that can swell in

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water and hold a large amount of water while maintaining a three-dimensional

64

network structure.11 Recently, hydrogel has been applied extensively in food,

65

pharmaceutical, and biomaterial industries because of its ability to improve the

66

stability, solubility, half-life, and bioavailability of the loaded functional agents.12,13 3



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As we all known, traditional hydrogel is usually prepared by using synthetic materials,

68

and has also been successfully applied as an oral drug delivery system. However,

69

these synthetic material-based hydrogels have inherent limitations for food

70

applications because they contain components that are not generally recognized as

71

safe for regular consumption by healthy individuals.11 Thus, to promote the

72

application of hydrogel in the food industry, food-grade and biodegradable materials

73

should be used in food systems during processing, storage, and consumption.

74

Some food-grade materials, such as natural proteins, polysaccharides, and

75

especially their complexes, have been applied in the food industry.12,13 The

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protein/polysaccharide complexes exhibit some functional properties of hydration,

77

structuration, and surface properties.14 Among these properties, structuration has

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received considerable attention because its ability to self-assemble and form into

79

hydrogel with environmentally-friendly, cost-effective and convenient.

80

As is well known, citrus is often used for the production of single-strength and

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frozen concentrated orange juice. When processing citrus production, the capacity of

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waste management systems is challenged by the concomitant by-products such as

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pees, skins, and pips. Therefore, citrus-processing industries are commercially

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interested in recovering residual amounts of soluble solids after juice extraction. In

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addition, many researchers have shown a way to utilize citrus peel wastes by

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producing pectin.15 As a significant component in citrus peels, pectin is a food-grade

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polymer of a-galacturonic acid with a variable number of methyl ester groups.16

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Recently, pectin extracted from citrus peel has had wide applications in the food 4


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industry, such as thickener, texturizer, emulsifier, stabilizer, and fat substitute in some

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food products.17 As a type of dietary fiber, researchers have reported that such pectin

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has the property of lowering blood cholesterol levels and lowering density lipoprotein

92

cholesterol fractions without changing high density lipoprotein cholesterol or

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triglycerides, which are good for human health.18 However, until now, the application

94

of citrus peel pectin is still very limited, and it is necessary to develop high citrus peel

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pectin value by-product foods. Meanwhile, to the best of our knowledge, the

96

applications of protein and citrus peel pectin complexes as delivery systems in food

97

fields have rarely been reported.

98

Thus, this research firstly developed hydrogel (BPH) via self-assembly method

99

between bovine serum albumin (BSA) and citrus peel pectin (pectin). The aggregation

100

behavior and interaction mechanisms were investigated through dynamic and static

101

rheological properties, conformational structures, and gel morphology. Such hydrogel

102

was successfully applied as a delivery system to encapsulate the functional food agent

103

(Vc). Meanwhile, the stability, encapsulation capacity, and in vitro release properties

104

of Vc loaded hydrogel were also investigated.

105 106

EXPERIMENTAL PROCEDURES

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Materials. Bovine serum albumin (BSA, purity > 98%) and citrus peel pectin

108

(total impurities < 10% moisture, galacturonic acid > 74%, methoxy groups > 6.7%,

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DE approximately 50% calculated from FT-IR) were provided by Sigma (USA) and

110

used without further purification. All other chemical agents used were of analytical 5



111

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grade.

112

Preparation of BSA-pectin hydrogel (BPH). Pectin solution (10 mM, pH 7.0)

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was titrated into 10 mg·mL-1 BSA aqueous solution under magnetic agitation until the

114

weight ratio (WR) of pectin/BSA mixture was 0.5. The BSA-pectin (B-P mixture)

115

solutions (pH 7.0) were magnetic stirred slowly at room temperature for 6 and then

116

placed overnight. After that, the B-P mixture solutions were pre-lyophilized for 30

117

min at -80 °C, and then lyophilized for 48 h by using FD-1 lyophilizer (Beijing,

118

China).The lyophilized B-P mixture was resolved into deionized water (5 mg·mL-1)

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with different pH value values (7.0, 6.0, 5.5, 5.0, 4.5, 4.2 and 4.0). To obtain hydrogel

120

(BPH), solution samples were heated at 90 °C in a water bath for 20 min and the

121

solution temperature was monitored with a thermometer, and the BPH samples were

122

obtained after cooling to room temperature. As the control, BSA hydrogel (BH)

123

without pectin was prepared as followings:19 10 mg·mL-1 BSA aqueous was adjusted

124

to pH 7.0 with 0.1 N sodium hydrate under agitation, diluted in four times volume of

125

acetone and balanced for 10 min. The solutions were then heated (90 °C and 20 min),

126

dialyzed, and lyophilized.

127

Particle size, polydispersity index, and zeta-potential. The particle size,

128

polydispersity index (PDI), and zeta-potential of the BSA solution, B-P mixture, and

129

BPH were determined by using a nanoparticle size analyzer (NICOMP380/ZLS, PSS,

130

USA) at 25 °C with particle detection angle (12° < θ < 150°, 90°) and zeta-potential

131

low-angle (19°), respectively. The PDI was calculated and processed by fast 32-bit

132

digital autocorrelator new DSP design (4×T. I. C31) and internal analysis computer 6


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fast DSP design (T. I. C31).

134

Rheological properties. Static and dynamic rheological properties of B-P

135

mixture and BPH were carried out on a TA DHR-2 type rheometer at 25 °C with a gap

136

of 1 mm. Static rheological properties were investigated for testing viscosity and

137

stress variation, and static rheological properties were fixed at a frequency of 1Hz

138

from 0.1 s-1 to 600 s-1 (ascend curve) and then from 600 s-1 to 0.1 s-1 (descend curve).

139

Dynamic rheological properties were carried out for measuring the dynamic

140

viscoelastic parameters (the storage modulus G’ and the loss modulus G’’) as

141

functions of the vibrational frequency with frequency scanning ranging from 0.01 to

142

100 rad·s-1.

143

Characteristic group and secondary structure. The Fourier transform infrared

144

(FT-IR) spectra of BSA, B-P mixture, and BPH were recorded by using the FT-IR

145

spectrophotometer (Nicolet 5700, Thermo Electron Corporation, MA, USA), and the

146

circular dichroism (CD) spectra of BSA, B-P mixture, and BPH were measured by

147

MOS-450/AF CD (Biologic, Claix, France). Data of protein secondary structures of

148

the

149

http://dichroweb.cryst.bbk.ac.uk/html/process.shtml.

150 151

α-helix,

β-sheet,

β-turn,

and

unordered

coil

were

obtained

from

Morphology. The morphology of B-P mixture, BH and BPH were determined by using Scanning Electron Microscopy (SEM, Quanta 200F, FEI, Hillsboro, OR).

152

Encapsulation efficiency (EE). BPH (5 mL) was placed into dialysis bag and

153

then added into a tube with Vc solution (1 mg/ml, 50 mL). After shaking 48 h at 37 °C,

154

the amount of Vc in the tube was measured with UV (TU1810, Beijing Purkinje 7



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155

General Company) at 265 nm after removing the dialysis bag, and EE was calculated

156

according to the following equation.

157

EE(%) = [Wtotal-Wremaining]/Wtotal×100

158

where Wtotal is the total added amount of Vc, and Wremaining is the remaining content of

159

Vc.

160

In vitro release of BPH. The in vitro Vc release from the BPH was evaluated

161

using the dialysis method. BPH was placed into dialysis bags and suspended in 50 mL

162

SGF (simulated gastric fluid, pH 1.2, contained 0.32% (w/v) pepsin), PBS (phosphate

163

buffer saline, pH 7.0), and FF (simulated intestinal fluid, pH 7.4, containing 1% (w/v)

164

pancreatin) as the release media at 37 ± 0.1 °C. At specified time intervals, 0.5 mL

165

media were withdrawn, diluted and replaced with an equal volume of the

166

corresponding fresh media to maintain a constant volume. The concentrations of Vc in

167

different samples were estimated via the UV spectrophotometer.

168

For further investigation of the Vc release mechanism from BPH, the Zero-order,

169

First-order, Higuchi, and Peppas models were used to fit the release data of Vc from

170

BPH according the following equations.

171

Zero-order model: Mt/M∞ = kt

172

First-order model: ln (1 - Mt/M∞) = -kt

173

Higuchi model: Mt/M∞ = kt1/2

174

Peppas model: ln Mt/M∞= n ln t + ln k

175

where Mt/M∞ is the fractional active agent released at time t, k is a constant

176

incorporating the properties, and n gives an indication of the release mechanism. The 8


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177

correlation coefficient (R2) is the linear relationship between Vc release and time.

178

Stability of BH, B-P mixture, and BPH. The Vc loaded BH, B-P mixture, and

179

BPH, and blank Vc solutions were all stored at 25 °C for 10 weeks. The diameter, PDI,

180

zeta-potential, and Vc retention were measured for investigation of the samples

181

stability.

182

Statistical analysis. All measurements were performed on three samples and

183

reported as the means. Their standard deviations (SD) were calculated by Excel

184

(Microsoft, Redmond, WA, USA) and pictures were manipulated by OriginPro 8.6

185

software (OriginLab Corporation, Northampton, USA).

186 187

RESULTS AND DISCUSSION

188

Influence of pH on BSA-pectin hydrogel (BPH). The visual observation of B-P

189

mixture and BPH in different pH solutions are shown in Figure. 1A. The results

190

indicated that both B-P mixture and BPH possessed four states of transparent solution

191

(pH 7.0-5.5) (Figure. 1A, a and e), transparent gels (pH 5.0-4.5) (Figure. 1A, b and f),

192

translucent gel (pH 4.5-4.2) (Figure 1A, c and g), and opaque gel (pH 4.2-4.0) (Figure.

193

1A, d and f). As a well-known protein, BSA can aggregate and precipitate at the

194

isoelectric point (pH 4.6-4.9), which was due to BSA displays tendency self-assembly

195

in large macromolecular and reversible conformational isomerization at a function of

196

pH.20 However, B-P mixture and BPH are more stable without any precipitation at pH

197

5.5-4.0, which demonstrated that pectin can hinder the isoelectric point precipitation

198

and thermal-induced aggregation of protein. 9



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199

The formation of BPH in this study is an electrostatically driven process, and so

200

it is important to measure the electrical characteristics of these samples under

201

different pH conditions (Figure. 1A). The zeta-potential of BSA solution changed

202

from negative (-13.25 mv) to positive (+0.79 mv) when the pH decreased from 7.0 to

203

4.5, which may be due to the increasing of H+ concentration and creating a positive

204

charge on BSA. From Figure 1A, it can be observed that pure pectin solutions showed

205

the characteristic behavior of an anionic polyelectrolyte, and the zeta-potential was

206

unaffected by pH change and stable independently under different pH condition.

207

However, the zeta-potential of B-P mixture (from -25.95 to -15.70 mv) and BPH

208

(from -21.90 to -16.85 mv) had a similar changing trend in the pH range of 7.0 to 4.5

209

after mixed with pectin. The zeta-potential of B-P mixture and BPH remained

210

negative for all values of pH, which might be due to the anionic polyelectrolyte and

211

the magnitude of the negative charge on the pectin molecule. The zeta-potential of

212

BPH (from -21.90 to -19.30) was slightly smaller than that of the B-P mixture (from

213

-25.95 to -20.30 mv) from pH 7.0 to 5.5, whereas the zeta-potential of BPH was

214

higher (from -20.35 to -16.85 mv) than that of the B-P mixture (from -18.4 to -15.7

215

mv) in a pH range of 5.0-4.5. Higher zeta-potential induced more electrostatic

216

repulsion, which can hinder the hydogel interaction and thus improve BPH stability in

217

the pH range of 5.5-4.0.

218

The means particle diameter and PDI of B-P mixture and BPH, and

219

corresponding SEM from pH 7.0 to 4.0 are shown in Figure 1B. The diameter of B-P

220

mixture decreased greatly (from micro to nano) as the pH decreased in the range of 10


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7.0-4.5, but no significant change occurred in the pH range of 4.5-4.0. From Figure

222

1B, it is noted that the diameter of BPH was generally smaller than that of the B-P

223

mixture at the same pH. The PDI of B-P mixture was between 0.7 and 0.08 in the pH

224

range of 7.0-5.0, and was close to 0.08 at pH 4.5, which indicated that B-P mixture

225

changed from moderate-dispersion to mono-dispersion with pH decreasing. However,

226

the PDI of BPH decreased greatly compared with that of the B-P mixture at pH 4.5

227

(0.084-0.012), which suggested that BPH is a more homogeneous monodisperse

228

system.

229

The stability of BPH can be attributed to a stronger electrostatic interaction

230

between negative charge of pectin and positive charged of BSA in the pH range of

231

4.5-4.0, and the negative charge of pectin and positive change chains of BSA in the

232

pH range of 5.5-4.5. Another reason is that thermal treatment may induce the

233

re-arrangement of pectin molecules on the protein surface by forming a more compact

234

and denser network. Meanwhile, intermolecular hydrophobic interactions and

235

disulfide bonds would happen after heating for BSA. These reasons thus offered better

236

stability against precipitation and smaller PDI values.21 Therefore, pH 4.5 was

237

considered the optimum experimental condition for developing a stable hydrogel with

238

thermal treatments (90 °C) between two biopolymers of BSA and pectin.

239

Rheological properties of B-P mixture and BPH. The static rheological

240

properties (viscosity-shear and stress-shear rate) of B-P mixture and BPH were

241

determined (Figure. 2A and B). The results showed that B-P mixture and BPH have

242

the character of pseudoplastic Bingham fluid with a yield stress τ0 (τ-τ0 = η (dvx/dy) = 11



Page 12 of 32

243

ηγ, η < 1) and a thixotropic lag, which suggested that a gel structure was formed for

244

B-P mixture and BPH. B-P mixture avoided vibrational damage through a weak

245

network structure based on noncovalent bonds (such as hydrogen bonds and van der

246

Waals force) to generate the thixotropic lag. Meanwhile, B-P mixture in pH 5.0

247

solution has a smaller hysteresis loop and τ0 than that in pH 4.5 solution, which

248

indicated that the interaction force was stronger at pH 4.5 and that more external force

249

and energy was needed to break the network structure. Compared with B-P mixture,

250

BPH had lower viscosity, and a smaller hysteresis loop and τ0 at the same pH, which

251

may be caused by the reduction of hydrogen bonds after the thermal treatment. The

252

results (Figure. 2B) also suggested that BPH at pH 4.5 had a larger hysteresis loop

253

and a higher τ0 value than that at pH 5.0, which indicated that BPH is more stable

254

system at pH 4.5 after heating.

255

Dynamic rheological properties are to be an extremely effective method for

256

research of sol-gel systems and their transitions.22 As shown in Figure. 2C, the elastic

257

and viscous modulus of BPH was not sensitive to changes in the low-frequency

258

region, which shows that BPH was a typical chemical cross-linking system at pH 4.5.

259

The elastic modulus of BPH is greater than the viscous modulus, and no intersection

260

between the elastic modulus and viscous modulus occurred. This result indicated that

261

the gel exhibits an elastic response and a strong recovery from deformation. These

262

results further confirmed that BPH has formed a gel network structure. However,

263

BPH system at pH 5.0 shows a strong frequency dependence (Figure. 2C), G’ and G’’

264

increased with increasing in the vibrational frequency, which proved that BPH was a 12


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265

tangling network system supported by a non-covalent interaction and a typically weak

266

network system. The results of dynamic rheological properties were in accordance

267

with the remarkable difference in PDI at pH 4.5 and pH 5.0. These results showed that

268

the electrostatic interaction at pH 4.5 is much stronger than that at pH 5.0 and reveals

269

that the pH is a critical factor in formation of hydrogels.

270

Functional groups and secondary structure of BH, B-P mixture, and BPH.

271

Characterization of functional groups and secondary structure of BSA, B-P mixture,

272

and BPH were carried out by Fourier transform infrared spectroscopy (FT-IR) (Figure.

273

3A) and circular dichroism (CD) spectra (Figure. 3B).

274

In the FT-IR spectra of pure BSA, the absorption bands at 3420.85, 1648.85, and

275

1530.56 cm-1 were attributed to -OH stretching, amide I, and amide II band,

276

respectively. In the FT-IR spectra of pure pectin, a broad absorption peak at 3393.78

277

cm-1 was the -OH stretching vibration, the absorption peaks at 2977.82 and

278

2933.64cm-1 were the symmetric and asymmetric stretching vibrations, respectively.

279

Peaks at 1749.73 cm-1 and 1632.32 cm-1 were considered the stretching vibration of

280

the non-methyl esterified carboxyl group and the methyl esterified carboxyl group,

281

respectively. The FT-IR spectra of B-P mixture was similar to that of pure BSA but

282

with certain blue shifting of -OH stretching peaks from 3420.85 cm−1 to 3446.64 cm−1,

283

and with the peaks of amide I and amide II closer to each other. We can conclude that

284

the amide structure of BSA was affected by the interaction between pectin and the

285

solvent. The characteristic peaks of BSA and pectin were both found in BPH

286

(3420.85-3433.22,

2977.82-2973.47,

1648.85-1646.19,

1530.56-1564.74, 13



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287

1054.24-1055.81, and 998.22-1018.06 cm-1). However, the peaks of 1749.73 and

288

1632.32 cm-1 disappeared in BPH, whereas the peaks of amide I and II (1646.19 and

289

1564.74 cm-1) significantly strengthened with the formation of a new amide bond. On

290

the other hand, the peak of -OH stretching became wider and stronger in the spectra of

291

BPH than that of pure BSA, which was caused by the typical feature of the Milliard

292

reaction between protein and pectin. From these results, it can be concluded that some

293

amino groups of BSA are covalently associated with the carbonyl group of pectin.

294

For further investigation of conformational structure of BPH, CD spectra were

295

used to investigate the secondary structures, and the results are shown in Figure. 3B

296

and Table 1. From the results, no significant difference for the secondary structures

297

was found among B-P mixture, BPH, and BSA in pH 7.0 solutions (Table.1).

298

However, the secondary structure parameters of B-P mixture and BPH changed

299

greatly in acidic solutions (pH 4.5 and 5.0). As shown in Figure. 3B and Table 1, the

300

number of α-helix in the B-P mixture decreased, which resulted from the ε-amino

301

groups decreasing in BSA. Meanwhile, the number of β-sheet, β-turn, and random

302

coils in the B-P mixture increased at pH 4.5 and 5.0. BPH has more remarkable trends

303

that number change from α-helix to β-sheet and β-turn but with a decreasing in

304

random coil. The β-turn structure is considered the product of highly ordered protein

305

structure, and β-sheet structure was available to form three-dimensional hydrogels

306

under proper conditions.23 Thus, BPH has a higher order three-dimensional network

307

structure than B-P mixture. However, BPH at pH 5.0 has a decreasing in β-sheet and

308

β-turn, and an increasing in random coil with no remarkable change of α-helix. These 14


Page 15 of 32


309

results of dynamic rheology further suggested that BPH was a tangling and disordered

310

network system at pH 5.0.

311

Based on the above-mentioned results, a schematic illustration of the

312

charge-charge interaction and forming mechanisms between BSA and pectin at pH 4.5,

313

with a pectin/BSA ration of 0.5:1 upon heating, were proposed in Figure. 4B. The B-P

314

mixture system at pH 7.0 was stable and co-existed with the same negative charge.

315

When the pH of the mixture system became 4.5, two molecules changed with the

316

opposite net and attracted each other to form into a more stable and ordered structure.

317

The aggregates were self-assembled with the formation of interpenetrating polymer

318

networks via hydrophobic interaction and stiffened through the sulfhydryl-disulfide

319

reaction after heating, along with the interaction between an amino group of protein

320

and a carbonyl group of pectin.

321

Morphology of BH, B-P mixture, and BPH. Figure 4 A displayed SEM images

322

of BH, B-P mixture (pH 7.0 and 4.5), and BPH. As shown in Figure 4A (a and b), a

323

loose net structure with pores on the surface is present in BH. With the mixing of

324

pectin into BSA at pH 7.0 (Figure 4A, c and d), the texture of the system changed into

325

smooth silk. When the pH was adjusted to 4.5, the B-P mixture became a system of

326

tufted ordered structure (Figure 4A, e and f), perhaps because of the formation of

327

β-sheet-rich fibrils. Compared with the BH and B-P mixture (pH 7.0 and 4.5),

328

heat-induced BPH at pH 4.5 (Figure 4 A, g and h) showed a solid and ordered

329

three-dimensional network structure, which may be due to heat can denaturate

330

proteins, causing them to lose their compact structure, expose their hydrophobic 15



Page 16 of 32

331

residues to the surface, exchange their disulfide bonds, and finally intermolecular

332

hydrophobic interactions and disulfide bonds.

333

EE of BPH, in vitro release and mechanism of Vc from BPH. Vc has been

334

used as the guest molecules and loaded into different delivery systems, such as

335

liposomes and nanoparticels. The EE of Vc within liposomes

336

nanoparticles

337

Compared with these previous studies, the EE of Vc within BPH was increased up to

338

65.31 ± 3.42%. The higher EE of BPH was attributed to the ordered

339

three-dimensional and porous network structures, and its good permeability for Vc

340

molecule.

9,10

7,8

and chitosan

were 48.30% and 62.25%, and 15.70% and 28.00%, respectively.

341

The results of Vc release from BPH in SGF, PBS, and SIF solutions are shown in

342

Figure 5 A. These results indicated that BPH had ideal release behaviors with two

343

stages of burst (stage 1) and sustained (stage 2) release. The release amount of Vc

344

from BPH in SGF solutions was very slow with only 25.11% after 6 h and 28.05%

345

after 24 h, respectively. On the contrary, approximately 61.14% and 90.73% of Vc

346

was released in PBS solutions, and 80.17% and 95.81% in the SIF solution after 6 h

347

and 24 h. These results indicated that the release rate of Vc from BPH is sensitive to

348

the pH, and BPH has a rapid release rate in higher pH solutions. The rapid digestion

349

of BPH in the SIF solutions after possible detachment of pectin caused by the weak

350

electrostatic interaction and some swelling resulting from the repulsive force between

351

the -COO- of pectin, lead to a higher release rate in the SIF solutions.

352

The Zero-order, First-order, Higuchi, and Peppas models were used to investigate 16


Page 17 of 32


353

the probable release mechanism of Vc from BPH. As shown in Table 2, the Higuchi

354

model is the most appropriate model to fit the release kinetics of Vc from BPH. The n

355

value of the Peppas model is applied to further investigate the detailed release

356

mechanisms for BPH, such as n < 0.43 for Case I Fickian diffusion, n > 0.85 for Case

357

II transport (swelling and erosion), and 0.43 < n < 0.85 for anomalous behavior or

358

non-Fickian transport.24 The calculated n values of Vc release processes from BPH

359

are listed in Table 2. The n value for stage 1 of BPH in the SIF solutions was higher

360

than 0.85, suggesting that the main mechanisms were swelling and erosion, and the n

361

value was close to zero for stage 2 because the internal and external concentrations of

362

Vc were similar. In the SGF solutions, the n value indicated that the diffusion,

363

swelling, and erosion release mechanisms coexisted for the release of Vc from BPH

364

for stage 1, and a Fickian diffusion for stage 2. This biphasic release profile of BPH in

365

SGF solution was attributed to the protection of BPH from hydrolysis by the pectin,

366

and BSA hydrolysis products with molecular weights of several thousand Daltons can

367

still bind with pectin to restrict the release of Vc under SGF conditions.

368

Stability of BH, B-P mixture, and BPH. The size diameter, PDI, and visual

369

observation of BH, B-P mixture, and BPH are shown in Figure 6A. Yellow precipitate

370

appeared in the blank Vc samples, and no Vc was detected after a two-week storage.

371

The PDI value and the size diameter of BH increased greatly, with the zeta-potential

372

changing from -13.25 to -0.887 mV, and aggregate forming after storage of two weeks.

373

For B-P mixture, the precipitation appeared, and the zeta-potential changed from

374

-15.70 to 9.93 mV after four-week storage. Meanwhile, almost no Vc retention was 17



Page 18 of 32

375

detected for the BH and B-P mixture after storage of 10 weeks (Figure 6B).

376

Fortunately, the size diameter (200-300 nm), PDI (0-1.5), and zeta-potential (-15 mv

377

to -20 mv) for BPH did not significantly change with higher Vc retention (73.95%)

378

after storage of 10 weeks. These results indicated that BPH is a more stable system

379

than BH and B-P mixture. The reasons for the stability of the system might come from

380

the chemical interaction between BSA and pectin, and the heat-induced more stable

381

three-dimensional network structure. Thus, the shelf-life and bioavailability of Vc

382

could be improved after loading into BPH.

383

In conclusion, a novel self-assembled hydrogel system (BPH) with

384

three-dimensional and porous network structures was developed by using food-grade

385

and natural biopolymers of BSA and citrus peel pectin. The electrostatic and the

386

covalent interactions between hydrophobic groups of BAS and amide groups of pectin

387

were the main mechanisms for forming hydrogel after thermal treatment in pH 4.5

388

solutions. The resultant BPH was a stable system that can be used as a potential

389

carrier for functional food agents. Vc was used as the model active agent and loaded

390

into BPH with higher EE. Vc release from BPH possessed sustained release with

391

diffusion, swelling, and erosion as the release mechanisms. Based on the results of

392

this study, BPH, as a potential delivery system, has a great application in food fields

393

for improving stability and bioavailability of functional agents.

394 395 396

ACKNOWLEDGEMENTS This study was supported by the Planning Subject of “the Twelfth 18


Page 19 of 32


397

Five-Yea-Plan” National Science and Technology for the Rural development of China

398

(2013AA102203-05), the National Natural Science Foundation of China (31660482),

399

and the Natural Science Foundation of Jiangxi Province (20142BAB213003 and

400

20151BAB203029).

401 402

LITERATURE CITED

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(1) Abudu, N.; Miller, J. J.; Attaelmannan, M.; Levinson, S. S. Vitamins in human

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arteriosclerosis with emphasis on vitamin c and vitamin e. Clin Chim Acta. 2004,

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339, 11-25.

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(2) Talaulikar, V. S.; It, M. Vitamin c as an antioxidant supplement in women's health:

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a myth in need of urgent burial. Eur J Obstet Gynecol Reprod Biol. 2011, 157,

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10-13.

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(3) Paz, M. M. Reductive activation of mitomycins A and C by Vitamin C. Bioorg Chem. 2013, 48, 1-7. (4) Verrax, J.; Calderon, P. B. The controversial place of vitamin c in cancer treatment. Biochem Pharm. 2008, 76, 1644-1652. (5) Abbas, S.; Chang, D. W.; Xiaoming, K. H. Z. Ascorbic acid: microencapsulation techniques and trends-a review. Food Rev Int. 2012, 28, 343-374.

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(6) Spínola, V.; Mendes, B.; Camara, J. S.; Castilho, P. C. Effect of time and

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temperature on vitamin c stability in horticultural extracts. uhplc-pda vs

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iodometric titration as analytical methods. LWT-Food Sci Technol. 2013, 50,

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(7) Li, T.; Yang, S. B.; Liu, W.; Liu, C. M.; Liu, W. L.; Zheng, H. J.; Zhou, W.; Tong,

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G. H. Preparation and characterization of nanoscale complex liposomes containing

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medium-chain fatty acids and vitamin c. Int J Food Prop. 2015, 18, 113-124.

422

(8) Zhou, W.; Liu, W.; Zou, L. Q.; Liu, W. L.; Liu, C. M.; Liang, R. H.; Chen, J.

423

Storage stability and skin permeation of vitamin c liposomes improved by pectin

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coating. Colloids Surf B Biointerfaces. 2014, 117, 330-337.

425

(9) Britto, D. D.; Moura, M. R. D.; Aouada, F. A.; Mattoso, L. H. C.; Assis, O. B. G.

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N,n,n-trimethyl chitosan nanoparticles as a vitamin carrier system. Food

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Hydrocolloid. 2012, 27, 487-493.

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(10) Jimnéz-Fernández, E.; Zuasti, E.; Ruyra, A.; Roher, N.; Infante, C.;

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Fernández-Díaz, C. Nanoparticles as a novel delivery system for vitamin c

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administration in aquaculture. Commun Agric Appl Biol Sci. 2013, 78, 202-203.

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(11) Chen, L. Y.; Remondetto, G. E.; Subirade, M. Food protein-based materials as

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nutraceutical delivery systems. Trends Food Sci Tec. 2006, 17 272-283

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(12) Alina, K,; Katharina A. P.; Jochen, W.; Jörg, H. Environmental response of

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pectin-stabilized whey protein aggregates. Food Hydrocolloid. 2014, 35, 332-340.

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(13) Li, Y. H.; Zhang, Y. F.; Xie, J. J.; Zhang, L.; Fang, Y. P. Interaction between

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gelatin and sugar beet pectin. Food Chem. 2014, 35, 29–33.

437

(14) Bryant, C. M.; Mc Clements, D. J. Influence of xanthan gum on physical

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characteristic of heat-denatured whey protein solutions and gels. Food

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Hydrocolloid. 2000, 14, 383-390.

440

(15) Kim, W. C.; Lee, D. Y.; Lee, C. H.; Kim, C. W. Optimization of narirutin 20


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extraction during washing step of the pectin production from citrus peels. J Food

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Eng. 2004, 63, 191-197.

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(16) Mishra, R. K.; Banthia, A. K.; Majeed, A. B. A. Pectin based formulations for biomedical applications: a review. Asian J Pharm Clin Res. 2012, 5, 1-7. (17) Liu, Y.; Shi, J.; Langrish, T. A. G. Water-based extraction of pectin from flavedo and albedo of orange peels. Chem Eng J. 2006, 120, 203-209.

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18 Osamu, K.; Fujiwara, T.; Yamazaki, E. Characterization of the pectin extracted

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from citrus peel in the presence of citric acid. Carbohyd Polym. 2008, 74,

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725-730.

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(19) Chen, G. Q.; Lin, W.; Coombes, A. G. A.; Davis, S. S.; Ilium, L. Preparation of

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human serum albumin microspheres by a novel acetone-heat denaturation method.

452

J Microencapsul. 1994, 11, 395-407.

453

(20) Vetri, V.; Librizzi, F.; Leone, M. Thermal aggregation of bovine serum albumin at

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different pH: comparison with human serum albumin. Eur Biophys J. 2007, 36,

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717-725.

456

(21) Sejersen, M. T.; Salomonsen, T.; Ipsen, R.; Clark, R., Rolin, C.; Engelsen, S. B.

457

Zeta potential of pectin-stabilized casein aggregates in acidified milk drinks. Int

458

Dairy J. 2007, 17, 302-307.

459

(22) Mijangos, C.; Lopez, D.; Munoz, M. E.; Santamaria, A. Study of poly(vinyl

460

chloride) gels by means of stereospecific substitution reactions. Micromolecules.

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1993, 26, 5693-5697.

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(23) Gosal, W. S.; Clark, A. H.; Pudney, P. D.; Ross-Murphy, S. B. Novel amyloid 21



463

fibrillar

networks

derived

464

Langmuir. 2002, 18, 7174-7181.

from

a

globular

protein:

Page 22 of 32

β-lactoglobulin.

465

(24) Ritger, P. L.; Peppas, N. A. A simple equation for description of solute release II.

466

Fickian and anomalous release from swellable devices. J Control Release. 1987, 5,

467

37-42.

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 22


Page 23 of 32


485

Figure captions

486

Figure. 1. Zeta-potential of BSA, B-P mixture, and BPH in solution of pH 7.0-4.0, the

487

visual observation of B-P mixture and BPH in pH 7.0-5.5 (a and e), 5.0-4.5 (b and f),

488

4.2 (c and g), and 4.0 (d and h) (A), and diameter and PDI of B-P mixture and BPH

489

(B).

490

Figure. 2. Stress (A) and viscosity (B) of B-P mixture and BPH variation with shear

491

rate at pH 4.5 and pH 5.0, and frequency dependence on storage modules and loss

492

modules (G’ and G’’) of BPH at pH 4.5 and pH 5.0 (C).

493

Figure. 3. FT-IR (A) of BSA, pectin, B-P mixture, and BPH, and CD (B) of BSA,

494

B-P mixture, and BPH.

495

Figure. 4. SEM images (A) of the BH (a and b), B-P mixture (pH 7.0) (c and d), B-P

496

mixture (pH 4.5) (e and f), and BPH (pH 4.5) (g and h), and schematic illustration (B)

497

of charge-charge interaction between BSA and pectin (pH 4.5) after thermal

498

treatment.

499

Figure. 5. Accumulated release (A) and release kinetics (B) of Vc from BPH in SIF,

500

PBS, and SGF solutions, respectively.

501

Figure. 6. The size diameter, PDI and visual observation of BH, B-P mixture, and

502

BPH (A), and Vc retention of BH, B-P mixture, and BPH (B) after 10 weeks storage

503 504 505 506 23



Page 24 of 32

507

Table 1 Secondary structure distribution of BSA, B-P mixture and BPH measured

508

from circular dichroism.

pH 7.0

samples

509

BSA a–Helix (%) 56.32 ß–Sheet (%) 1.98 ß–Turn (%) 19.87 Random coil (%) 21.83

B–P 56.57 4.32 19.28 19.83

pH 5.0 BPH 55.63 3.38 19.37 21.62

B–P 10.18 40.74 20.76 28.32

BPH 11.64 37.95 16.73 33.68

pH4.5 B–P 19.87 31.78 23.73 24.56

BPH 14.71 43.97 23.02 18.00

510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 24


Page 25 of 32


526

Table 2 Correlation coefficients (r2) and release exponent (n) of different models in

527

SIF, PBS, and SGF solutions.

SIF

PBS

SGF

Model

528

Stage 1

Stage 2

Stage 1

Stage 2

Stage 1

Stage 2

Zero–order (r 2)

0.99

0.72

0.96

0.76

0.95

0.74

First–order (r2 )

0.99

0.84

0.98

0.87

0.96

0.74

Higuchi (r2)

1.00

0.87

0.99

0.90

0.99

0.88

Peppas (n)

0.93

0.038

0.79

0.18

0.62

0.077

529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 25



545

Page 26 of 32

Figure 1.

546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 26


Page 27 of 32


563

Figure 2.

564 565 566 567 568 569 570 27



571

Page 28 of 32

Figure 3.

572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 28


Page 29 of 32


588

Figure 4.

589 590 591 592 593 594 595 596 29



597

Page 30 of 32

Figure 5.

598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 30


Page 31 of 32


614

Figure 6.

615

31



254x190mm (96 x 96 DPI)


Page 32 of 32

Preparation and Self-Assembly Mechanism of Bovine Serum Albumin

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