Emulsifying Properties of Polysaccharide Conjugates Prepared from

Subscriber access provided by - Access paid by the | UCSF Library

Food and Beverage Chemistry/Biochemistry

Emulsifying Properties of Polysaccharide Conjugates Prepared from Chin-brick Tea Xiaoqiang Chen, Yuntian Zhang, Yu Han, Qian Li, Li Wu, Jia Zhang, Xiaoling Zhong, Jianchun Xie, Shengrong Shao, Yinjun Zhang, and Zhengqi Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03161 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Journal of Agricultural and Food Chemistry

1

Emulsifying Properties of Polysaccharide Conjugates Prepared from

2

Chin-brick Tea

3

Xiaoqiang Chen*1,2,Yuntian Zhang§1, Yu Han§1, Qian Li1, Li Wu3, Jia Zhang1,

4

Xiaoling Zhong1, Jianchun Xie2, Shengrong Shao1, Yinjun Zhang4, Zhengqi Wu1

5 6 7 8 9 10 11 12 13 14

1National

"111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China 2Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU)，Beijing 100048,China 3Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan 430060, China 4College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China

15 16 17 18 19 20 21

§Equal

contribution * Corresponding author Tel: (+86) 27-87950483 Fax: (+86) 27-87950483 E-mail address: [email protected]

22 23 24 25 26 27 28 29 30

ACS Paragon Plus Environment


31

ABSTRACT: Chin-brick tea polysaccharide conjugates (TPC-C) were prepared to

32

study their emulsion capabilities. The interfacial tension and the effects of some

33

factors, such as storage time, metal ion concentrations (Na+, Ca2+), pH (2.0-8.0), and

34

heat treatment (70-100 °C) on the emulsions stabilized by TPC-C were studied. The

35

interfacial tension of TPC-C (10.88 mN/m) was lower than that of gum arabic (15.18

36

mN/m) at the concentration of 0.08%. As TPC-C concentration increased from 0.1

37

wt% to 3.0 wt%, the mean particle diameter (MPD) (d32) of emulsions stabilized by

38

TPC-C decreased from 1.88 μm to 0.16 μm. Furthermore, at a concentration of 0.5

39

wt% or higher, the MPD (d32) of emulsions stabilized by TPC-C at 25 °C and 60 °C

40

for 10 days were between 0.20 μm and 0.50 μm. In the tested pH conditions from 2.0

41

to 8.0, the MPD (d32) of emulsions stabilized by 2.0 wt% TPC-C was less than 0.20

42

μm. At Na+ concentration conditions between 0.10 mol/L to 0.50 mol/L, the MPD (d32)

43

of emulsions were between 0.19 μm to 0.20 μm, and the zeta potential values varied

44

from -34.10 mV to -32.60 mV. However, with increasing Ca2+ concentrations from

45

0.01 mol/L to 0.05 mol/L, the MPD (d32) of emulsions were between 0.20 μm to

46

21.65 μm, and the zeta potential raised sharply from -34.10 mV to -28.46 mV. The

47

emulsions stabilized by TPC-C have decent storage stability after high temperature

48

heat treatment. Overall, tea polysaccharide conjugates (TPC) strongly stabilized the

49

emulsions, which support their new application as natural emulsifiers.

50 51

KEYWORDS: tea polysaccharide conjugates; Chin-brick tea ; emulsifier; stability

52


Page 2 of 40

Page 3 of 40


53

Introduction

54

Oil-in-water emulsions are widely utilized in many food products, including milk,

55

yogurt, dressings, desserts, ice cream, sauces, and beverages. Because they are

56

thermodynamically unstable systems, they require the addition of emulsifiers acting

57

on the interface to provide kinetic stability by reducing the interfacial tension and

58

generating repulsive interactions (steric or electrostatic).1 At present, emulsifiers used

59

in food, cosmetics, and pesticide products are primarily synthetic, including fatty acid

60

monoglycerides, sucrose esters, Tween-80, Span, and polysorbates.2,3 With the

61

growing desire to consume healthy foods, the development of natural emulsifiers has

62

received much attention.4 To date, protein-based emulsifiers have

63

utilized in the food industry due to their abundance and good emulsifying ability.

64

However, the stability of emulsion stabilized by protein-based emulsifiers is easily

65

influenced by high ionic strength, elevated temperature, and pH values close to their

66

isoelectric point.5,6

been widely

67

Polysaccharide-based emulsifiers are also commonly used in the food industry as

68

natural emulsifiers. Tea produced from the buds and leaves of Camellia sinensis L.

69

has been widely utilized in dietary and medicinal applications for thousands of years.7

70

There are six major categories of teas: green, white, yellow, Oolong, black, and dark

71

teas.8 Chin-brick tea, a dark tea similar to Pu'er tea, is primarily produced in Hubei,

72

China. Chin-brick tea is prepared from sundried green tea by commonly used methods,

73

such as pile-fermentation, sieving, shaping, and drying. The pile-fermentation process

74

lasts for several weeks and has greatly improved the quality and functional



75

components in tea.9 Tea polysaccharide conjugates (TPCs), an important bioactive

76

compounds in six categories of tea, have received increased attention because of their

77

various health-promoting activities, including hypoglycemic, immunomodulatory, and

78

anti-oxidation.10,11,12 TPCs are macromolecules containing a small amount of

79

covalently bound protein. It is thought that the binding protein in TPC is coated by the

80

carbohydrate chain and forms a hydrophobic "core",13-16and this polysaccharide and

81

protein conjugate could induce an emulsification function to TPC. To date, it is

82

difficult to find

reports on the emulsification ability of TPC.

83

Therefore, the objectives of this investigation were to quantify the emulsifying

84

properties and emulsion stability of polysaccharide conjugates from Chin-brick tea

85

(TPC-C). To achieve this objective, emulsions stabilized by different levels of TPC-C

86

were generated by high speed blender and microfluidizer. Droplet sizes, zeta potential,

87

microstructure, and interfacial properties of the oil-in-water emulsions were measured.

88

This work could provide valuable information about the potential utilization of TPC

89

as a natural emulsifier.

90

MATERIALS AND METHODS

91

Materials. Chin-brick tea was purchased from Yichang, Hubei province, China.

92

The monosaccharide standards (D-glucuronic acid, D-galacturonic acid, L-fucose,

93

D-arabinose, D-mannose, D-xylose, D-fructose, L-rhamnose, D-galactose, D-ribose,

94

and D-glucose), gum arabic (GA), and nile red were purchased from Sigma-Aldrich

95

(St. Louis, MO, USA). All other reagents used were of analytical grade and were

96

obtained from either Sigma-Aldrich (St. Louis, MO, USA) or Aladdin (Shanghai,


Page 4 of 40

Page 5 of 40


97

China). Double distilled water was used to prepare all solutions and emulsions.

98

Polysacchride conjugates prepared from Chin-brick tea. Chin-brick tea (1.0 kg)

99

was ground using a blender. The processed material was extracted by hot water (1:16

100

m/v) under 95 °C for 3.3 h with continuous stirring. After centrifugation at 5000 rpm

101

for 15 min, the supernatant was collected and concentrated via a rotary evaporator at

102

50 °C under reduced pressure. Three volumes of ethanol were added to the

103

concentrated solution to precipitate the polysaccharide overnight at 4 °C. The

104

obtained polysaccharide was re-dissolved in double distilled water, was concentrated

105

again to remove the residual ethanol, and then lyophilized to give TPC-C.10

106

Chemical properties of TPC-C. The monosaccharide composition, amino acid

107

composition, and relative molecular weight of tea polysaccharide conjugates were

108

determined according to the reported methods. The sample was derivatized with

109

acetic anhydride, and the monosaccharide fraction was determined by gas

110

chromatography. Gel Permeation Chromatography-Multi angle light scattering (GPC-

111

MALS) was used to analyze the molecular weight distribution of the TPC-C. After the

112

sample was hydrolyzed by HCl, the amino acid composition was determined by the

113

external standard method of Hitachi L-8900 Amino Acid Auto Analyzer.17 The pH of

114

0.1-3.0 wt% TPC aqueous solution was determined by pH meter (Mettler

115

Toledo,Switzerland).

116

Interfacial tension measurements. The interfacial tension of TPC-C solutions

117

with different concentrations was measured at MCT oil-water interfaces using a Drop

118

Tensiometer (Teclis, France).15 The interfacial tension was measured by performing



119

an online automatic analysis of the oil drop profile using the Laplace equation:

p 

120

2

r

121

Where γ is the specific surface free energy, p is the additional pressure and r is the

122

radius of curvature.

123

Ten microliters of oil droplet was delivered using a micro syringe (Exmire, Japan)

124

through a U-shaped stainless steel needle (internal diameter 0.56 mm) into an optical

125

glass cuvette containing 5 mL TPC-C solutions maintained at 25 °C. The

126

measurement was continued at 25 °C for 12000 s. The concentrations of TPC-C

127

ranged from 0.02 wt% to 0.10 wt% at an interval of 0.02, because the polysaccharide

128

conjugate solution was not transparent enough at concentrations above 0.10 wt%, and

129

the measurement results were not reliable. The GA solution with a concentration of

130

0.08 wt% was used as a control. During the detection process, the entire system

131

should be balanced to avoid external vibration interference measurements.

132

Emulsion stabilized by different concentration of TPC-C.

133

i) Emulsion preparation. Emulsions were produced by homogenizing 8.0 wt%

134

MCT with 92.0 wt% phosphate buffer solution (PBS, pH 7.0, 0.01M) containing 0.1 -

135

3.0 wt% TPC-C using a high-speed shearer (25,000 rpm) for 3 min (T18, IKA,

136

Germany). Next, the solution was passed through a high-pressure homogenizer

137

(Microfluidics M-110L, USA) at 75 MPa for three cycles. Sodium azide (0.02 % w/v)

138

was added to final emulsions to prevent microorganism growth. The concentration

139

gradient of TPC-C was set to 0.10 wt%, 0.25 wt%, 0.50 wt%, 1.00 wt%, 2.00 wt%, and

140

3.00 wt%.


Page 6 of 40

Page 7 of 40


141

ii) Particle size (d32) and zeta potential measurements. The particle size

142

distribution of emulsions was determined using static light scattering (Mastersizer

143

2000, Malvern Instruments Ltd., UK). Emulsions were dispersed in phosphate buffer

144

solutions at the same pH until an obscuration rate of 10-20% was obtained. The mean

145

particle size was reported as the surface-weighted mean diameter (d32). The zeta

146

potential of emulsions was measured using Zetasizer Nano-ZS (Malvern Instruments,

147

UK). Prior to analysis, samples were diluted with 10 mM phosphate buffer at the

148

same pH to avoid multiple scattering effects. All tests were carried out at 25 °C and

149

repeated in triplicate. It is worth noting that the sample was vortexed for 5 s before

150

each measurement.

151

Influencing factors of emulsion stability

152

i) Effect of storage time on TPC-C emulsion stability. The emulsions stabilized

153

by 2.0 wt% TPC-C (pH 7.0) were prepared and distributed into different beakers as

154

test samples. These samples were stored at 25 °C and 60 °C, while their zeta potential

155

and MPD (d32) were determined daily during the storage of 10 days as the methods

156

described above. Their emulsion appearance was observed daily until the 21st day.

157

ii)

Effect of metal ions on the stability of TPC-C emulsions. The emulsions

158

stabilized by 2.0 wt% TPC-C containing 0.10, 0.20, 0.30, 0.40, and 0.50 mol/L NaCl

159

and 0.01, 0.02, 0.03, 0.04, and 0.05 mol/L CaCl2 (pH 7.0) were prepared. The

160

emulsion samples were placed in glass test tubes and then incubated at 25 °C for 10

161

days. The stability of the emulsions was characterized by observing their appearance,

162

measuring the particle size distribution, mean particle diameter (d32), and zeta



163

Page 8 of 40

potential. The measurement methods were performed as described above.

164

The microstructure of TPC-C emulsions treated by metal ions was determined by

165

laser scanning confocal microscope (LSCM) with a SIM scanner for simultaneous

166

laser stimulation and observed with a 100 × oil immersion objective (Olympus Corp.,

167

Tokyo, Japan). The oil phase of emulsions was dyed by nile red solution (1 mg/mL

168

ethanol). The excitation and emission wavelength for nile red were 488 nm and 520

169

nm, respectively. All the microstructure images were acquired and analyzed using

170

Olympus viewer (Olympus Fluoview Ver.3.1 Viewer).

171

iii) Effect of pH on the stability of TPC-C emulsion. The emulsions stabilized by

172

2.0 wt% TPC-C (pH 7.0) were distributed into different beakers, and then each

173

sample was adjusted to a different pH value ranging from 2.0 to 8.0 at an interval of

174

1.0 using HCl or NaOH solutions. The emulsion samples were placed in glass test

175

tubes and then incubated at 25 °C for 10 days. The stability of the emulsions was

176

characterized by observing their appearance, measuring their particle size distribution,

177

mean particle diameter (d32), zeta potential, and the microstructure. These

178

measurement methods were performed as described above.

179

iv) Effect of thermal treatment on TPC-C emulsion stability.

The emulsions

180

stabilized by 2.0 wt% TPC-C (pH 7.0) were prepared and distributed into different

181

beakers, and then each sample was bathed at 70 °C, 80 °C, 90 °C, and 100 °C for 30

182

min. After heat treatment, these TPC-C emulsions were cooled to room temperature

183

and placed at 25 °C for 24 h. The particle size distribution and zeta potential were

184

measured. Their mean particle diameter (d32) and appearance were investigated during


Page 9 of 40


185

the storage of 10 days at 25 °C. The measurement methods were as described above.

186

Statistical analysis. The statistical analysis was carried out using the statistical

187

software Statistical Package for Social Sciences 21.0 (SPSS Inc., 2012). The

188

comparisons between groups were analyzed by Dunnett’s two-tailed t-test after

189

one-way ANOVA.

190

RESULTS

191

Chemical properties of TPC-C. GPC-MALLS results indicated that the average

192

molecular weight of TPC-C was 31580 Da. The monosaccharide and uronic acid

193

compositions of polysaccharides from Chin-brick tea were analyzed by complete acid

194

hydrolysis. The results indicated that the monosaccharide compositions were found to

195

be arabinose, rhamnose, galactose, glucose, xylose, mannose, and galacturonic acid in

196

the molar ratio of 1.00, 1.30, 2.65, 0.85, 0.39, 1.40, and 2.15, respectively. The free

197

amino acids (Cf.a.a) were not detected in TPC-C. The amino acid composition of

198

TPC-C protein moiety were Asp, Glu, Ser, Gly, Thr, Arg, Ile, Phe, Val, Tyr, Pro, Ala,

199

Leu, and Lys with the mass content (mg/g) of 7.12, 9.00, 5.72, 5.12, 4.34, 3.77, 2.19,

200

2.88, 3.72, 2.72, 2.96, 4.17, 3.36, and 1.29, respectively. The total amount of these

201

amino acids (Ca.a.) were 5.84%. The content of the protein moiety of TPC-C was

202

calculated using the formula: (Ca.a. − Cf.a.a) × 110/128 with 5.02% of TPC-C. 13

203

Interfacial tension of TPC-C. An effective emulsifier must be surface-active and

204

have the capacity to reduce the oil-water interfacial tension. Thus, the interfacial

205

tension of emulsifiers is closely related to the formation and stabilization of

206

emulsions.18,19 Therefore, in order to investigate the potential emulsifying activity of



207

TPC-C, we measured the dynamic interfacial tension of TPC-C at different

208

concentrations (0.02 - 0.10 wt%) and 0.08 wt% GA in solution with an MCT oil

209

droplet (Figure 1). As shown in Figure 1A, the interfacial tension decreased rapidly at

210

the beginning of adsorption, indicating that TPC-C can quickly adsorb onto the oil-

211

water interfaces. In addition, the interfacial tension decreased as the TPC-C

212

concentrations increased, with the highest concentration of TPC-C having the lowest

213

interfacial tension. As shown in Figure 1B, at the concentration of 0.08%, the

214

interfacial tension of TPC-C (10.88 mN/m) was lower than that of GA (15.18 mN/m),

215

which indicated that TPC-C can more quickly adsorb onto the oil-water interface and

216

more effectively decrease the interfacial tension. Reports have suggested that the

217

formation of emulsion is dependent on the ability of emulsifiers to absorb onto the

218

oil-water interface and to undergo a conformational rearrangement to form a

219

viscoelastic film surrounding the oil droplets.20 Therefore, TPC-C has the potential to

220

form emulsions as a natural emulsifier.

221

Effect of TPC-C concentration on the emulsion formation. A well- established

222

polysaccharide-based emulsifier, GA (5.0 wt%), was used as a control to compare to

223

the emulsifying capability of TPC-C. An oil-in-water emulsion was generated by

224

passing oil (8.0 wt%) and aqueous phase (92.0 wt%) through a high-pressure

225

homogenizer at 75MPa. The mean particle diameter (MPD) (d32) and particle size

226

distribution of emulsions stabilized by different concentrations of TPC-C and GA

227

were measured.

228

Particle size of emulsions plays an important role in their application.21 As shown


Page 10 of 40

Page 11 of 40


229

in Figure 2A, the emulsion stabilized by 5.0 wt% GA had the widest particle size

230

distribution, followed by 0.1 wt% TPC-C; both stabilized emulsions had bimodal

231

distributions. The particle size distribution of the emulsions stabilized by 0.25-3.0

232

wt% TPC-C exhibited unimodal distribution. The higher the concentration of TPC-C,

233

the smaller the particle size. If there was insufficient emulsifier molecules to cover the

234

oil droplet surface, the emulsions were more susceptible to bridging flocculation.22,23

235

Large droplet surface areas can be covered by increasing concentrations of TPC-C

236

within the homogenizer, leading to the quick formation of a smaller droplet size and

237

the reduction of droplet re-coalescence, indicating that TPC-C possesses excellent

238

emulsifying properties.1

239

As shown in Figure 2B, the MPD (d32) of emulsions stabilized by TPC-C

240

decreased with the increasing concentrations. Specifically, the MPD (d32) value of

241

emulsions decreased from 1.88 to 0.16 µm as the TPC-C concentration increased from

242

0.1 to 3.0 wt%. This phenomenon may occur, because more emulsifiers can cover a

243

larger droplet surface area, leading to smaller droplets sizes within the homogenizer.

244

Additionally, oil droplet surfaces can be covered more quickly, thereby decreasing

245

droplet re-coalescence during homogenization.24,25 The MPD (d32) value of emulsion

246

stabilized by GA was 8.68 µm, which was significantly higher than that of the

247

emulsions stabilized by TPC-C. This suggested that the emulsifying capacity of

248

TPC-C was more effective than that of the commercial polysaccharide-based

249

emulsifiers and that TPC-C can be used as a novel emulsifier.

250

Zeta potential is an indicator of the emulsion stability induced by the electrostatic



251

repulsive interactions.1 As shown in Figure 2B, the surface charges of droplets in

252

TPC-C-stabilized emulsions were above -35.0 mV. In addition, the negative surface

253

charge of emulsion droplets decreased with increasing concentrations of TPC-C.

254

Because TPC is weakly acidic, the pH of the solution decreases slightly as the

255

concentration of TPC increases (Figure 2C), resulting in a slight decrease in the zeta

256

potential. Previous studies have also reported that the zeta potential of emulsion

257

droplets decreases with a reduction of emulsion pH.26,27

258

Emulsion storage stability. Emulsion storage stability is imperative for the shelf

259

life of commercial food and beverage products. In order to further investigate the

260

potential of TPC-C as a natural emulsifier, we measured the storage stability of

261

emulsions stabilized by TPC-C and GA at 25 °C and 60 °C for 10 days.

262

As shown in Figure 3, there was no significant change in the mean particle

263

diameter of a droplet in emulsion stabilized with TPC-C (concentrations of 0.5 to 2.0

264

wt%) at 25 °C for up to 10 days of storage. Particle size distribution analysis showed

265

a unimodal distribution at higher TPC-C concentrations during storage, indicating that

266

TPC-C possess excellent emulsion-stabilizing properties (Shown in Figure 4). In

267

addition, visual observation results showed that TPC-C-stabilized emulsions were still

268

stable, and no visible cream layers were observed on the top of the emulsions when

269

the TPC-C concentration was above 0.5 wt% after 10 days of storage. GA-stabilized

270

emulsions formed visible white-creaming layers on the top after 24 h of storage. The

271

relatively high stability of emulsions stabilized by TPC-C at certain concentrations

272

maybe due to their small droplet size; the creaming rate is proportional to the square


Page 12 of 40

Page 13 of 40


273

of the droplet diameter, and the rate of gravitational separation decreases with a

274

reduction in droplet size.1

275

No significant difference was observed in the d32 values of emulsions stabilized by

276

TPC-C at 0.5, 1.0 and 2.0 wt% at 60 °C after 10 days of storage (Shown in Figure 3).

277

As shown in Figure 5, there were no significant changes in the microstructures of

278

droplets in emulsions at higher TPC-C concentration during 10 days of storage.

279

Interestingly, the particle size distribution of emulsions stabilized by TPC-C

280

(concentrations from 0.5 to 2.0 wt%) remained unimodal throughout storage.

281

Furthermore, negligible or no creaming was observed in emulsions at higher TPC-C

282

concentrations due to the small droplet size in these systems. Nevertheless,

283

GA-stabilized emulsions significantly separated into two phases, with a white cream

284

layer on top and a relative clear phase at the bottom. The above results indicated that

285

the emulsion stabilized by TPC-C had excellent long-term stability; thus, TPC-C

286

could be used as a natural emulsifier in commercial food and beverage applications.

287

Effects of metal ions on the stability of TPC-C emulsion. Na+ has a small impact

288

on the particle size distribution, zeta potential, and MPD (d32) of 2.0 wt% TPC-C

289

stabilized emulsions. As the concentration of Na+ increased from 0 to 0.5 mol/L, the

290

particle size distribution of the emulsions showed single peaks that almost overlapped

291

each other. All MPD (d32) values were lower than 0.30 μm (Shown in Figure 6A). The

292

absolute values of the zeta potential of the emulsions slightly decreased from 34.10 to

293

32.60 mV (Shown in Figure 6B). These emulsions maintained stability during storage

294

for 10 days at 25 °C (As captured in Figure 7). Calcium ion concentrations above 0.03



295

mol/L have a destructive effect on TPC-C stabilized emulsions. Calcium ions cause

296

droplet aggregation and demulsification during the storage for 10 days at 25 °C

297

(Shown in Figure 7). As the concentration of calcium ions increased from 0 to 0.05

298

mol/L, the MPD (d32) of the emulsions also increased from 0.2 to 21.65 μm (Shown

299

in Figure 8A,and Figure 9), and the absolute values of the zeta potentials of the

300

emulsions decreased linearly from 34.10 to 28.46 (Shown in Figure 8B). The linear

301

equation was Y=11.98X-32.26, R2=0.99, where Y is the Zeta potential, and X is the

302

concentration of calcium ions. The increased calcium ion concentration neutralizes the

303

negative charges on the surface of TPC-C and reduces the absolute value of the zeta

304

potential in a concentration- dependent manner.

305

Effects of pH on the stability of TPC-C emulsion. The effect of pH on the stability

306

of TPC-C emulsions is shown in Figure 10 and Figure 11. The size of the emulsion

307

droplets did not change significantly under different acidity and alkalinity (pH2-8),

308

and the emulsion had a single peak distribution with particle sizes of less than 0.20

309

μm (shown in Figure 10A, 11B). The zeta potential values ranged from -2.95 to -35.4

310

when the pH value increased from 2 to 8 (shown in Figure 10B), which suggests that

311

the emulsion will be more stable in a higher pH solution environment. At a pH greater

312

than 4.0, the absolute value of the zeta potential of the TPC-C emulsions was higher

313

than 25 mV. As shown in Figure 11B, the size of the emulsion droplets did not change

314

significantly under different acidity and alkalinity, and was less than 0.20 μm.

315

Thermal stability of TPC-C emulsion. In order to better promote the commercial

316

application of emulsions, the thermal stability of emulsions during production,


Page 14 of 40

Page 15 of 40


317

processing, transportation, and storage should be studied. The effect of heat treatment

318

at 70-100 °C on the stability of TPC-C emulsions was investigated. The emulsion

319

stabilized by 2.0 wt% TPC-C was stored at 60 °C for 10 days to maintain good

320

stability as a control. All the particle size distributions of TPC-C emulsions

321

heat-treated at different temperatures overlap with that of the control, have a single

322

peak distribution, and maintained a small particle size (Figure 12A). The zeta

323

potential values of the heat-treated emulsions were measured on the first, seventh, and

324

tenth days of storage at 25 °C (as shown in Figure 12B). The zeta potential values of

325

the heat-treated emulsions at different temperatures were close and maintained at

326

approximately -32.5 mV. The magnitude of the change in the absolute values of the

327

zeta potential was 0.30-1.96 during storage for 10 days, indicating that the high

328

temperature heat treatment did not destroy the electrostatic repulsion and steric

329

hindrance between the droplets in the emulsions. The average particle size d32 of the

330

TPC-C emulsions treated at different temperatures was not significantly changed with

331

the increase of storage days, and was maintained near 0.2 μm (Figure 12C). The

332

TPC-C emulsions did not appear to be layered at 25 °C by the 10th day after heat

333

treatment of 70-100 °C (Figure 12D), and they were apparently indistinguishable from

334

the unheated control. The TPC-C emulsions maintained storage stability after high

335

temperature heat treatment.

336 337

DISCUSSION

338

The emulsification properties of TPC have been discovered in this work, which will



339

greatly enhance its development and application value. At present, emulsifiers used in

340

food industry, such as fatty acid monoglycerides and sucrose esters, are primarily

341

synthetic.3, 4 However, with the increasing concern about healthy foods, the

342

development of natural emulsifiers has received much attention.2 Many current

343

studies on natural emulsifiers focus on protein or its complex with exogenous

344

polysaccharides.28 A complex in which a polysaccharide is covalently or

345

non-covalently bound to a protein is typically used as an emulsion stabilizer to

346

synergistically exert the emulsification activity of the protein and the stability of the

347

polysaccharide over a wider range of pH and ionic strength.30 TPC is composed of a

348

polysaccharide and a covalently bound protein, which allows it to have a decent

349

emulsification properties without the need to be attached to external proteins. The

350

polysaccharide moiety provided pH stability to the TPC-C stabilized emulsions .

351

Calcium ions, unlike sodium ions, destroyed the emulsion stability. The Ca2+-induced

352

protein aggregation developed because of three effects: 1) electrostatic shielding, 2)

353

crosslinking of adjacent anionic molecules by forming protein-Ca2+-protein bridges,

354

and 3) ion-specific hydrophobic interaction.29,30 In this study, the absolute value of the

355

zeta potential of the emulsion decreased in a calcium ion concentration-dependent

356

manner. The positively charged calcium ions neutralize the negative charge on the

357

surface of the TPC-C, which resulted in cross-linking between the molecules and

358

emulsion aggregation .

359 360

Funding This research was financially supported by the National Natural Science Foundation


Page 16 of 40

Page 17 of 40


361

of China (grant number 31871813) and the Beijing Advanced Innovation Center for

362

Food Nutrition and Human Health (grant number 20161012).

363

Notes The authors declare no competing financial interest.

364



366

REFERENCE (1) McClements, D. J.; Gumus, C. E. Natural emulsifiers-Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv Colloid Interface Sci. 2016, 234, 3-26. (2) Krog, N.; Sparsø, F.; Friberg, S.; Larsson, K.; Sjöblom, J. Food emulsifiers: their chemical and physical properties. Food Emulsions. 2004. (3) Wang, F.; Marangoni, A. Advances in the application of food emulsifier α-gel phases: saturated monoglycerides, polyglycerol fatty acid esters, and their derivatives. J Colloid Interface Sci, 2016, 483, 394-403. (4) Mcclements, D. J.; Bai, L.; Chung, C. Recent advances in the utilization of natural emulsifiers to form and stabilize emulsions. Annual Review of Food Science and Technology. 2017, 8(1), 205-236. (5) Dickinson, E. Interfacial structure and stability of food emulsions as affected by protein–polysaccharide interactions. Soft Matter. 2008, 4(5), 932. (6) McClements, D. J.; Gumus, C.E. Natural emulsifiers - Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv Colloid Interface Sci. 2016, 234, 3-26. (7) Nie, S.P.; Xie, M.-Y. A review on the isolation and structure of tea polysaccharides and their bioactivities. Food Hydrocolloids. 2011, 25(2), 144-149. (8) Zhu, Y. F.; Chen, J. J.; Ji, X. M.; Hu, X.; Ling, T. J.; Zhang, Z. Z.; Bao, G. H.; Wan, X. C. Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea. Food Chem. 2015, 170, 110-117. (9) Lu, S. F.; Zheng, P. C.; Liu, P. P.; Wang, S. P.; Teng, J.;Feng, L.; Gong, Z.M. Research Progress on Qingzhuan Tea. Acta Tea Sinica. 2018, 59 (3),162 -167. (10) Chen, X. Q.; Fang, Y. P.; Nishinari, K.; We, H.; Sun, C. C.; Li, J. R.; Jiang, Y. W. Physicochemical characteristics of polysaccharide conjugatesprepared from fresh tea leaves and their improving impaired glucose tolerance. Carbohydrate Polymers. 2014, 112, 77-84. (11) Chen, X. Q.; Zhang, Z. F.; Gao, Z. M.; Huang, Y.; Wu, Z. Q. Physicochemical properties and cell-based bioactivity of Pu’erh teapolysaccharide conjugates. International Journal of Biological Macromolecules. 2017, 104, 1294– 1301. (12) Chen, X. Q.; Ye, Y.; Cheng, H.; Jiang, Y. W.; Wu, Y. L. Thermal Effects on the Stability and Antioxidant Activity of an Acid Polysaccharide Conjugate Derived from Green Tea. J. Agric. Food Chem. 2009, 57, 5795–5798. (13) Chen, X. Q.; Du, Y.; Wu, L.; Xie, J. C.; Chen, X. L.; Hu, B. B.; Wu, Z. Q.; Yao, Q. F.; Li, Q. Effects of Tea Polysaccharide Conjugates and Metal Ions on Precipitate Formation by Epigallocatechin Gallate and Caffeine, the Key Components of Green Tea Infusion. J. Agric. Food Chem 2019, 67(13), 3744-3751. (14) Chen, X. Q.; Xie, J. C.; Huang, W.; Shao, S. R.; Wu, Z. Q.; Wu, L.; Li, Q. Comparative analysis of physicochemical characteristics of green tea polysaccharide conjugates and its decolored fraction and their effect on HepG2 cell proliferation. Industrial Crops and


Page 18 of 40

Page 19 of 40


Products. 2019, 131, 243-249. (15) Chen, X. Q.; Shao, S. R.; Xie, J. C.; Yuan, H.; Li, Q.; Wu, L; Wu, Z. Q.; Yuan, H. B.; Jiang, Y. W. Analysis of Protein Moiety of Polysaccharide Conjugates Water-extracted from Low Grade Green Tea. Chem. Res. Chin. Univ. 2018, 3(44), 691-696. (16) Chen, X. Q.; Song, W.; Zhao, J.; Zhang, Z. F.; Zhang, Y. T. Some physical properties of protein moiety of alkali-extracted tea polysaccharide conjugates were shielded by its polysaccharide. Molecules 2017, 22(6), 914 (17) Chen, X. Q.; Lin, Z.; Ye, Y.; Zhang, R.; Yin, J. F.; Jiang, Y. W.; Wan, H. T.; Suppression of diabetes in non-obese diabetic (NOD) mice by oral administration of water-soluble and alkali-soluble polysaccharide conjugates prepared from green tea. Carbohydrate Polymers, 2010, 82, 28–33. (18) Dickinsion, E. Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids. 2003, 17, 25-39 (19) Jiang, J.; Jin, Y.; Liang, X.; Piatko, M.; Campbell, S.; Lo, S. K.; Liu, Y. Synergetic interfacial adsorption of protein and low-molecular-weight emulsifiers in aerated emulsions. Food Hydrocolloids. 2018, 81, 15-22. (20) Uruakpa, F. O.; Arntfield, S.D. Emulsifying characteristics of commercial canola protein– hydrocolloid systems. Food Research International. 2005, 38(6), 659-672. (21) Xu, X.; Zhong, J.; Chen, J.; Liu, C.; Luo, L.; Luo, S.; Wu, L.; McClements, D. J. Effectiveness of partially hydrolyzed rice glutelin as a food emulsifier: Comparison to whey protein. Food Chem. 2016, 213, 700-707. (22) Dickinson, E. Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids. 2009,.23(6), 1473-1482. (23) Mc, C.; David, J. Enhanced delivery of lipophilic bioactives using emulsions: a review of major factors affecting vitamin, nutraceutical, and lipid bioaccessibility. Food & function, 2018, 9, 22-41. (24) Dickinson, E. Strategies to control and inhibit the flocculation of protein-stabilized oil-in-water emulsions. Food Hydrocolloids. 2019, 96, .209-223 (25)Tisserand, C.; Brambilla G.; Meunier G.; Parker A. Predicting the long-term stability of depletion-flocculated emulsions by static multiple light scattering (SMLS). Journal of Dispersion Science and Technology. 2019, 1-8. (26) Ma, F.; Zhang, Y.; Yao, Y.; Wen, Y.; Hu, W.; Zhang, J.; Liu, X.; Bell, A. E.; Tikkanen-Kaukanen, C. Chemical components and emulsification properties of mucilage from Dioscorea opposita Thunb. Food Chem. 2017, 228, 315-322. (27) Nakauma, M.; Funami, T.; Noda, S.; Ishihara, S.; Al-Assaf, S.; Nishinari, K.; Phillips, G. O. Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids. 2008, 22(7), 1254-1267. (28) Krstonosic, V.; Dokic, L.; Nikolic, I.; Milanovic, M. Influence of xanthan gum on oil-in-water emulsion characteristics stabilized by OSA starch. Food Hydrocolloids. 2015, 45, 9-17. (29) Ju, Z. Y.; Kilara,A. Aggregation induced by calcium chloride and subsequent thermal gelation of whey protein isolate, J Dairy Sci ,1998, 81, 925-931.



(30) Wang, C. H.; S. Damodaran. Thermal gelation of globular proteins: influence of protein conformation on gel strength. J. Agric. Food Chem. 1991, 39, 434-442.


Page 20 of 40

Page 21 of 40


Figure Captions Figure 1. Interfacial tension at different concentrations of TPC-C (A) and 0.08 wt% GA (B) at 25 °C . Figure 2. Particle size distribution (A), the mean particle diameter (d32) and zeta potential of emulsions stabilized by different concentrations of TPC-C at 25 °C (B); pH of different concentrations of TPC-C aqueous solution at 25 °C (C). Figure 3. The mean particle diameter (d32) of emulsions stabilized by different concentrations of TPC-C during 10 days of storage at 25 °C (A) and 60° C (B). Figure 4. Particle size distribution of emulsions stabilized by different concentrations of TPC-C during 10 days storage at 25 °C (A) and 60 °C (B). Figure 5. Appearance changes were observed on day 0 and day 10 (A) and day 21 and day 31 (B) for different concentrations of TPC-C emulsions and 5.00% GA emulsion. (The numbers 1, 2, 3, 4, 5, 6, and 7, in pictures represent 0.10% TPC-C emulsion, 0.25% TPC-C emulsion, 0.5% TPC-C emulsion, 2.0% TPC-C emulsion, 3.0% TPC-C emulsion, and 5.00% GA emulsion, respectively. Figure 6. Particle size distribution (A) and zeta potential (B) of emulsions stabilized by 2.0 wt% TPC-C with different concentrations of NaCl. Figure 7. Appearance change of emulsions stabilized by 2.0 wt% TPC-C with different metal ion concentrations over 10 days at 25 °C. The numbers 1-5 in pictures represent the concentration of NaCl from 0.10 to 0.50 mol/L, respectively. The numbers 6-10 in pictures represent the concentration of CaCl2 from 0.01mol/L to 0.05 mol/L, respectively. Figure 8. Particle size distribution (A) and zeta potential (B) of emulsions stabilized by 2.0 wt% TPC-C with different concentrations of CaCl2. Figure 9. Microscopic appearance of 2.0 wt% TPC-C emulsions stained with nile red dye and observed by using LSCM under different metal ion concentrations on the 10th day of storage at 25 °C. The ionic strength of the solution was prepared by NaCl (0.10-0.50 mol/L) and CaCl2 (0.01-0.05 mol/L). Figure 10. Particle size distribution (A) and zeta potential (B) of the emulsions stabilized by 2.0 wt% TPC-C under different pH conditions on the 10th day at 25 °C. Figure 11. Appearance (A) and 10th-day microscopic appearance (B) of emulsions stabilized by 2.0 wt% TPC-C under the conditions of pH 2-8 and 25 °C, stained with nile red dye and observed using LSCM. Figure 12. Particle size distribution (A) and zeta potential (B) of 2.0 wt% TPC-C stabilized emulsion under the thermal treatment at 70-100 °C during 24 h storage at 25 °C, MPD (d32) (C) and appearance (D) during the 10 day storage at 25 °C .The emulsion stabilized by 2.0 wt% TPC-C was stored at 60 °C as an untreated control.



Page 22 of 40

Figure 1.

A 0.10% TPC-C 0.08% TPC-C 0.06% TPC-C 0.04% TPC-C 0.02% TPC-C

Interfacial tension (mN/m)

40

30

20

10

0

2000

4000

6000

Time (s)


8000

10000

12000

Page 23 of 40


B

32

0.08% GA 0.08% TPC-C

30

Interfacial tension (mN/m)

28 26 24 22 20 18 16 14 12 10 0

2000

4000

6000

8000

10000

12000

Time(s)

Figure 2. 5.00% GA 0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C 3.00% TPC-C

A 70 60

Volume (%)

50 40 30 20 10 0 0.01

0.1

1

10

100

Particle size m)


1000

10000


B

Page 24 of 40

Mean particle size D[3,2] 2.0

-44

Zeta potential

D[3,2] (m)

1.5

-40 -38

1.0

-36 -34

0.5

-32 0.0

-30

0.10%

0.25%

0.50%

1.00%

2.00%

3.00%

Concentration (%)

C

7.1 7.0 6.9

pH

6.8 6.7 6.6 6.5 6.4 6.3

0.10%

0.25%

0.50%

1.00%

2.00%

Concentration (%)


3.00%

Zeta Potential (mV)

-42

Page 25 of 40


Figure 3. 0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C

A 3.5

D[3,2]-25C

3.0

D[3,2] (m)

2.5 2.0 1.5 1.0 0.5 0.0 0

2

4

6

8

10

Time (Day)

B 7

0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C

D[3,2]-60C

6

D[3,2] (m)

5 4 3 2 1 0 0

2

4

6

8

Time (Day)


10


Page 26 of 40

Figure 4. 40

Day0 Day1 Day3 Day5 Day7 Day10

0.10%-25C

30

30

25

25

20 15

20 15

10

10

5

5

0

0

0.01

0.1

1

10

100

1000


0.25%-25C

35

Volume (%)

Volume (%)

35

40

0.01

10000

0.1

1


0.50%-25C

40

30

30

25

25

20 15

5

5

0

0 10

100

1000

10000

0.01

0.1


2.00%-25C

35

Volume (%)

30 25 20 15 10 5 0 0.01

0.1

1

10

1

10

Partical Size (m)

Partical Size (m) 40


1.00%-25C

15 10

1

10000

20

10

0.1

1000

35

Volume (%)

Volume (%)

35

0.01

100



10

100

1000

10000


A


100

1000

10000

Page 27 of 40


40


0.10%-60C

30

30

25

25

20 15

15 10

5

5 0

0.01

0.1

1

10

100

1000


0.01

10000

0.50%-60C

0.1

1

10

100

1000



35

40

10000 Day0 Day1 Day3 Day5 Day7 Day10

1.00%-60C

35 30

30 25

Volume (%)

Volume (%)

0.25%-60C

20

10

0

20 15

25 20 15 10

10

5

5

0

0 0.01

0.1

1

10

100

1000

0.01

10000

40


2.00%-60C

35 30 25 20 15 10 5 0 0.01

0.1

1

10

0.1

1

10



Volume (%)


35

Volume (%)

Volume (%)

35

40

100

1000

10000


B


100

1000

10000


Figure 5.

A

B


Page 28 of 40

Page 29 of 40


Figure 6.

A Fresh emulsion 0.10 mol/L NaCl 0.20 mol/L NaCl 0.30 mol/L NaCl 0.40 mol/L NaCl 0.50 mol/L NaCl

10

Volume (%)

8

6

4

2

0 0.01

0.1

1

10

100

1000

Particle Size (m)

B

-40

Zeta Potential (mV)

-35

-30

-25

-20

-15 0.00

0.10

0.20

0.30

0.40

Concentration (mol/L)


0.50

10000


Figure 7.


Page 30 of 40

Page 31 of 40


Figure 8.

A

Fresh emulsion 0.01 mol/L CaCl2 0.02 mol/L CaCl2 0.03 mol/L CaCl2 0.04 mol/L CaCl2 0.05 mol/L CaCl2

35 30

Volume (%)

25 20 15 10 5 0 0.01

0.1

1

10



100

1000

10000


B

Page 32 of 40

-40

Zeta Potential (mV)

-35

-30

-25

-20

-15 0.00

0.01

0.02

0.03

0.04

Concentration (mol/L)


0.05

Page 33 of 40


Figure 9.



Page 34 of 40

Figure 10. pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8

A 10

Volume (%)

8

6

4

2

0 0.01

0.1

1

10

100



1000

10000

Page 35 of 40


B

-40 -35

Zeta Potential (mv)

-30 -25 -20 -15 -10 -5 0 2

3

4

5

6

pH


7

8


Figure 11.


Page 36 of 40

Page 37 of 40


Figure 12.

A

12

CK 70C 80C 90C 100C

24h

10

Volume (%)

8

6

4

2

0 0.01

0.1

1

10

100



1000

10000


Page 38 of 40

CK 70℃ 80℃ 90℃ 100℃

B -40

Zeta Potention (mv)

-35

-30

-25

-20

-15

-10 0

2

4

6

8

10

Time (Day)

C

CK 70℃ 80℃ 90℃ 100℃

0.40 0.35

D[3,2] (m)

0.30 0.25 0.20 0.15 0.10 0.05 0.00 0

2

4

6

Time (Day)


8

10

Page 39 of 40




Table of Contents (TOC) Graphic


Page 40 of 40

Emulsifying Properties of Polysaccharide Conjugates Prepared from

Recommend Documents