Influence of interfacial compositions on the microstructure, physiochemical stability, lipid digestion and β-carotene bioaccessibility of Pickering emulsions

Journal Pre-proof Influence of interfacial compositions on the microstructure, physiochemical stability, lipid digestion and β-carotene bioaccessibility of Pickering emulsions Yang Wei, Zhen Tong, Lei Dai, Di Wang, Peifeng Lv, Jinfang Liu, Like Mao, Fang Yuan, Yanxiang Gao PII:

S0268-005X(19)32632-3

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105738

Reference:

FOOHYD 105738

To appear in:

Food Hydrocolloids

Received Date: 7 November 2019 Revised Date:

7 January 2020

Accepted Date: 3 February 2020

Please cite this article as: Wei, Y., Tong, Z., Dai, L., Wang, D., Lv, P., Liu, J., Mao, L., Yuan, F., Gao, Y., Influence of interfacial compositions on the microstructure, physiochemical stability, lipid digestion and βcarotene bioaccessibility of Pickering emulsions, Food Hydrocolloids (2020), doi: https://doi.org/10.1016/ j.foodhyd.2020.105738. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Contributions Statement: Yang Wei: Conceptualization, Methodology, Software, Writing- Original draft preparation. Zhen Tong: Data curation. Lei Dai: Software. Di Wang: Investigation. Peifeng Lv: Visualization. Jinfang Liu: Supervision. Like Mao: Software. Fang Yuan: Validation. Yanxiang Gao: Writing- Reviewing and Editing.

Table of Contents:

Influence of interfacial compositions on the microstructrure, physciochemical stability, lipid digestion and β-carotene bioaccessibility of Pickering emulsions

Yang Wei, Zhen Tong, Lei Dai, Di Wang, Peifeng Lv, Jinfang Liu, Like Mao, Fang Yuan, Yanxiang Gao*

Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100083, P. R. China

*Corresponding author. Tel.: + 86-10-62737034

Fax: + 86-10-62737986

Address: Box 112, No.17

Qinghua East Road, Haidian District, Beijing 100083, China E-mail: [email protected] E-mail address of other co-authors: 1

1

Abstract: Novel Pickering emulsions were co-stabilized by solid particles and two

2

types of emulsifiers (lactoferrin or rhamnolipid) for delivery of β-carotene. The

3

influence of the particle-surfactant or particle-protein mixed interface on novel

4

Pickering emulsions was investigated. The droplet size of the particles and lactoferrin

5

co-stabilized Pickering emulsion was increased from 13.34 ± 0.03 µm to 20.70 ± 0.09

6

µm with the rise in lactoferrin level from 0.10% to 1.25% (w/v), but the droplet size

7

of the particles and rhamnolipid co-stabilized Pickering emulsion was increased

8

initially from 17.61 ± 0.10 µm to 38.24 ± 0.11 µm with the increase in rhamnolipid

9

level from 0.10% to 0.75% (w/v) and then decreased to 15.17 ± 0.25 µm at 1.25% of

10

rhamnolipid. Compared to the emulsion stabilized by particles solely, the stability of

11

Pickering emulsions co-stabilized by particles and emulsifiers, and the β-carotene

12

entrapment were improved under environmental stresses. Presence of lactoferrin or

13

rhamnolipid might induce the competitive displacement, adsorption onto particle

14

surface or multilayer deposition at the interface, which were observed through

15

confocal laser scanning microscope and cryo-scanning electric microscopy.

16

Furthemore, the addition of lactoferrin or rhamnolipid inhibited the release of free

17

fatty acids from in vitro digestion effectively, thereby reducing the bioaccessibility of

18

β-carotene slightly. These findings facilitated to understand the effect of interfacial

19

compositions on the stability and digestion fate of the nutraceutical emulsions

20

co-stabilized by particles and protein or surfactant.

21

Keywords: Pickering emulsion; β-carotene; lactoferrin; rhamnolipid; interfacial

22

structure; lipid digestion 2

23

1. Introduction

24

Traditional emulsions were stabilized by surfactants or biopolymers (such as

25

proteins and polysaccharides).(Wei, Sun, Dai, Mao, et al., 2018) Due to their high

26

mobility, surfactants can rapidly adsorb onto the interface and effectively decrease the

27

interfacial tension. High mobility of surfactants at the interface keep the emulsion

28

stable. Comparably, biopolymers can provide sufficient steric repulsion among

29

droplets attributed to their larger molecular weight. Although the adsorption velocity

30

of biopolymers onto the interface is much smaller than surfactants, the adsorption of

31

biopolymer is nearly irreversible.(Pugnaloni, Dickinson, Ettelaie, Mackie, & Wilde,

32

2004; Wilde, Mackie, Husband, Gunning, & Morris, 2004) Pickering emulsions solely

33

stabilized by particles are attracting increasing attention. The particles can adsorb onto

34

the interface of emulsions due to the appropriate wettability. Compared to traditional

35

emulsions, Pickering emulsions are more stable with a solid shell against

36

coalescence.(Xu et al., 2018) Many researchers have investigated the effects of

37

particle size, wettability, particle concentration and oil fraction on the stability and

38

structure of Pickering emulsions.(Dai, Sun, Wei, Mao, & Gao, 2018; L.-J. Wang et al.,

39

2015; J. Xiao, Wang, Perez Gonzalez, & Huang, 2016; Y. Zou, Guo, Yin, Wang, &

40

Yang, 2015)

41

Nevertheless, the real food interface often contain combined emulsifiers and

42

particles with a complex interfacial composition.(Binks, Desforges, & Duff, 2007;

43

Dickinson, 2011) Due to different molecular structures, charge characteristics,

44

addition sequences and mass ratios of emulsifiers and particles, there are many 3

45

possibilities for the microstructure of mixed interfaces. Although researchers have

46

reviewed the digestion fate of Pickering emulsions stabilized by different

47

particles,(Sarkar, Zhang, Holmes, & Ettelaie, 2019) the effect of the complex

48

particle-emulsifer interface on the digestion behavior of the Pickering emulsion has

49

not been reported.

50

In a previous work, the effect of polymer-particle interactions was investigated

51

on the interfacial structure of bilayer emulsions co-stabilized by particles and

52

polysaccharides.(Wei, Sun, et al., 2019) The synergism of biopolymers and particles

53

improved the stability of Pickering emulsions and modulated their rheology and

54

interfacial structure. Some previous studies have also reported the inorganic particles

55

(such as silica nanoparticles) and artificial surfactants to stabilize the Pickering

56

emulsion systems.(Binks & Rodrigues, 2007; Cui, Yang, Cui, & P. Binks, 2009;

57

Müller et al., 2017; S. Zou, Yang, Liu, & Wang, 2013) The present study focused on

58

the O/W emulsions stabilized by a combination of particles and natural surfactants or

59

proteins.

60

β-Carotene is lipophilic with a highly unsaturated structure, which makes it

61

liable to chemical degradation and limits its application in food industry.(Donhowe &

62

Kong, 2014) As one of most important carotenoids, β-carotene has received greater

63

attention due to its high vitamin A precursor activity and other physiological

64

functions.(Mao, Wang, Liu, & Gao, 2018; Yuan, Gao, Zhao, & Mao, 2008) Among

65

different delivery systems, food emulsions may be ideal delivery systems of

66

β-carotene.(McClements, 2010) β-Carotene can be embedded into oil droplets of an 4

67

O/W emulsion, thereby improving its bioavailability when co-ingested with lipids,

68

which facilitate the absorption of β-carotene by epithelium cells.(Patrick Borel, 2005;

69

Van Het Hof, West, Weststrate, & Hautvast, 2000)

70

As a globular glycoprotein of the transferrin family, lactoferrin (LF) has various

71

health benefits and applications, which can be utilized to prepare nanoemulsions

72

attributing to its excellent emulsifying ability.(Liu, Zhang, Li, McClements, & Liu,

73

2018; Lonnerdal & Lyer, 1995; Tokle, Lesmes, & Julian McClements, 2010; B. Wang,

74

Timilsena, Blanch, & Adhikari, 2019) Many researchers have reported that LF-based

75

emulsions effectively improved the chemical stability and bioavailability of

76

β-carotene.(Liu, Wang, Xu, Sun, & Gao, 2016) It is interesting to investigate the

77

effect of addition of LF into the Pickering emulsions on their interfacial structure and

78

stability. Rhamnolipid (Rha) is a microbial surfactant, which is applied to deliver

79

hydrophobic molecules through a fully biodegradable transport system.(Wei, Yu, et al.,

80

2019; Wei, Zhang, et al., 2019) Due to its surface activity, Rha is widely utilized to

81

prepare nanoemulsions with the aid of external forces.(Bai & McClements, 2016; Z.

82

Li, Dai, Wang, Mao, & Gao, 2018; Lovaglio, dos Santos, Jafelicci, & Contiero, 2011)

83

Similar to other natural small molecular emulsifiers, Rha can rapidly adsorb onto the

84

surface of droplets and reduce the interfacial intension.(Bai & McClements, 2016;

85

Wei, Tong, et al., 2019)

86

Herein the β-carotene Pickering emulsion was designed to be co-stabilized by

87

particles and different emulsifiers (Rha or LF). The droplet size and zeta-potential of

88

Pickering emulsions were measured as a fundamental. Interfacial structures of oil 5

89

droplets were observed through confocal laser scanning microscope (CLSM) and

90

cryo-scanning

91

physicochemical

92

comprehensively. Besides, the digestion behavior of the Pickering emulsions was

93

simulated under in vitro gastrointestinal model. The lipid hydrolysis and

94

bioaccessibility of β-carotene in Pickering emulsions using the particle-emulsifier

95

mixed interface were investigated, which was meaningful for the design of complex

96

interfaces to modulate the physicochemical properties and digestion behavior of novel

97

Pickering emulsions.

98

2. Materials and methods

99

2.1. Materials

electric

microscopy (cryo-SEM).

stability

of

β-carotene

Rheological

Pickering

properties

emulsions

were

and tested

100

Zein with a protein content of 91.3% (w/w) was purchased from Sigma-Aldrich

101

(USA). Propylene glycol alginate (PGA) (esterification content: 87.9%) was

102

generously provided by Hanjun Sugar Industry Co. Ltd. (Shanghai, China).

103

Medium-chain triglycerides (MCT, Miglyol 812N) were purchased from Musim Mas

104

(Medan, Indonesia). β-Carotene suspension (30% by mass β-carotene in sunflower

105

oil) was supplied by Xinchang Pharmaceutical Company, Ltd. (Xinchang, Zhejiang,

106

China). LF was obtained from Hilmar Ingredients (Hilmar Ingredients Inc., Hilmar,

107

Calif., USA), the product contained 1.2% moisture, 0.3% ash and 99% protein.

108

Rhamnolipid (purity ≥ 90%) was obtained from Parnell Biological Technology Co.

109

Ltd (Shaanxi, China). Dyes (Nile blue and Nile red) and enzymes used to simulate

110

digestion included pepsin (P7125), pancreatin (P3292), bile salts and lipase (L3126, 6

111

type II) were all purchased from Sigma-Aldrich. Absolute ethanol (99.99%), solid

112

sodium hydroxide and liquid hydrochloric acid (36%, w/w) were obtained from

113

Eshowbokoo Biological Technology Co.,Ltd. (Beijing, China). All other chemical

114

agents were of analytical grade.

115

2.2. Preparation of particles and rhamnolipid or LF solutions

116

Zein-PGA

composite

nanoparticles

(ZPNPs)

were

prepared

by

the

117

solvent-evaporation co-precipitation method.(Wei, Yu, et al., 2019) Briefly, 7.5 g zein

118

and 1.5 g PGA was co-dissolved in 1000 mL 70% (v/v) aqueous ethanol solution and

119

stirred at 600 rpm overnight at 25 ℃ until their complete dissolution. Thereafter, the

120

ethanol in the mixture solution was removed with a rotary evaporator at 45 ℃ for 60

121

min and the remaining volume was set to around 250 mL. The colloidal dispersion

122

was diluted with pH-adjusted water (pH 4.0) to 300 mL. The ZPNP dispersion was

123

centrifuged (Sigma 3k15, Germany) at 3000 rpm for 10 min to separate large particles

124

and aggregates if any. Finally, the supernatant obtained was adjusted to pH 4.0 using

125

0.1 M HCl solution. One part of ZPNP dispersion was placed at 5 ℃ for further

126

analysis and the other part was freeze-dried for 72 h to obtain powder samples. Rha

127

and LF solutions with different concentrations (0.10%, 0.25%, 0.50%, 0.75%, 1.00%

128

and 1.25%, w/v) were prepared and adjusted to pH 4.0 by 1 M HCl.

129

2.3. Preparation of β-carotene Pickering emulsions co-stabilized by ZPNPs and

130

131

different emulsifiers

β-Carotene suspension (25 g) was first dissolved in MCT oil (225 g) at 140 ℃ 7

132

for 10 s to form oil phase (1.5 wt% β-carotene in final emulsions). The primary

133

emulsion was prepared by mixing 7.5 g of ZPNPs (2.0%, w/v) dispersion with 15 g of

134

oil phase at a speed of 18000 rpm using a blender (Ultra Turrax, model T25, IKA

135

Labortechnic, Staufen, Germany). After the complete dispersion of oil phase, the

136

mixture was further homogenized for another 5 min. Secondary emulsions were

137

fabricated by mixing the primary emulsion with 7.5 g of LF or Rha solution

138

(0.10−1.25%, w/v) and homogenized under the same condition. The ZPNPs and LF

139

co-stabilized Pickering emulsions were termed as Z-P/0.10LF, Z-P/0.25LF,

140

Z-P/0.50LF, Z-P/0.75LF, Z-P/1.00LF and Z-P/1.25LF, respectively. The ZPNPs and

141

Rha co-stabilized Pickering emulsions were termed as Z-P/0.10Rha, Z-P/0.25Rha,

142

Z-P/0.50Rha, Z-P/0.75Rha, Z-P/1.00Rha and Z-P/1.25Rha, respectively. As a control,

143

the ZPNPs stabilized Pickering emulsion were prepared by mixing 15 g of ZPNP

144

(2.0%, w/v) dispersion with 15 g of oil phase, which was named as Z-P. The pH of

145

fresh emulsions was adjusted to 4.0 using 0.5 M HCl.

146

2.4. Droplet size and zeta-potential

147

The droplet size and size distribution were measured after preparation of

148

emulsions for 12 h with a laser scattering size analyzer (LS230®, Beckman Coulter,

149

USA). The samples were diluted with deionized water at 3000 rpm until an

150

obscuration rate between 8% to 12% was obtained. The optical properties were

151

applied as followed: a refractive indice of 1.52 for MCT and absorption of 0.001, and

152

a refractive indice of 1.33 for the dispersant (deionized water).(Wei, Sun, Dai, Mao, et

153

al., 2018) The volume-area (D4,3) average diameters were calculated using the 8

154

following equation: 4,3 =

155

∑ ∑

The ni is the number of particles with a diameter of di.

156

The zeta-potential of droplets was determined by measuring the direction and

157

velocity of droplet movement in a well-defined electric field using a Zeta sizer

158

NanoZS90 (Malvern Instruments, Worcestershire, UK). Emulsions were diluted to a

159

final oil droplet concentration of 0.005 wt% with pH-adjusted deionized water (pH

160

4.0) to minimize multiple scattering effects. The data were collected from at least 10

161

sequential readings per sample after 120 s of equilibration and calculated by the

162

instrument using the Smoluchowski model.(Wei, Sun, Dai, Mao, et al., 2018) All

163

measurements were performed in triplicate.

164

2.5. Interfacial tension

165

The interfacial tension was measured using a tensiometer K100 (Kruss, Germany)

166

with the Wilhelmy plate method. The Wilhelmy plate is made of platinum, with a

167

length, width and thickness of 19.9 mm, 10 mm and 0.2 mm, respectively. The

168

Wilhelmy plate was immersed in 20 g of aqueous phase to a depth of 3 mm with a

169

surface detection speed of 15 mm min-1. The surface detection is the speed of the

170

vessel drive used for the detection of the liquid surface. Once the surface has been

171

detected by the microbalance in the tensiometer the vessel moves at the chosen

172

surface detection speed to the position specified by the immersion depth (3 mm).

173

Subsequently, an interface between the aqueous phase and oil phase was created by 9

174

carefully pipetting 20 g of the oil phase over the aqueous phase. The temperature was

175

maintained at 20 ℃ throughout the test. The interfacial tension values and the error

176

bars are reported as the average and the standard deviation of triplicates.

177

2.6. Physicochemical stability of β-carotene Pickering emulsions

178

2.6.1. Effect of UV radiation

179

The photostability of β-carotene in Pickering emulsions against UV photolysis

180

was tested following the method reported by Wei et al. (2018a). Briefly, 15 g of fresh

181

samples was placed into transparent glass vial. Then samples were put into a

182

controlled light cabinet (QSun, Q-Lab Corporation, Ohio, USA) for up to 4 h. The

183

retention rate of β-carotene was plotted against treatment time. All experiments were

184

performed in triplicate.

185

2.6.2. Effect of thermal treatment

186

The emulsions after 12 h storage at ambient temperature (25 ℃) were incubated

187

in water bath (85 ℃) for 60 min and then cooled down to 25 ℃. The retention rate of

188

β-carotene was detected after thermal treatment.

189

2.6.3. Effect of pH

190

The effect of pH on the emulsion stability was evaluated according to a previous

191

study.(Wei, Sun, et al., 2019) The designed emulsions after 12 h storage at ambient

192

temperature (25 ℃) were adjusted to pH 2.5, 6.0 and 8.5 using either 0.1 M NaOH or

193

0.1 M HCl.

194

2.6.4. Effect of ionic strength 10

195

The emulsions after 12 h storage at ambient temperature (25 ℃) were mixed with

196

different weight of NaCl powder for 2 h to assure completely dissolution. NaCl

197

concentrations in different emulsions were adjusted to 10, 50 and 100 mM.(Wei, Sun,

198

et al., 2019)

199

2.6.5. Effect of storage time

200

After the preparation of emulsions, the fresh emulsions were stored at 55 ℃ for 4

201

weeks. The droplet size and chemical stability of β-carotene in emulsions were

202

determined at regular storage periods (1, 7, 14, 21 and 28 days). β-Carotene content

203

was measured according to our reported study.(Wei, Sun, Dai, Zhan, & Gao, 2018) In

204

brief, β-carotene in the emulsions was extracted three times with a mixture of 1 mL

205

ethanol and 3 mL of n-hexane. And then the absorbance at 450 nm was measured with

206

a UV-1800 UV–vis spectrophotometer (Shimadzu, Japan).

207

After each treatment, the average droplet size and zeta-potential of the emulsions

208

were evaluated after 12 h storage (25 ℃) to acquire a stable state.

209

2.7. CLSM

210

CLSM (Zeiss780, Germany) was used to visualize the interfacial structure of

211

emulsion droplets. The emulsions were stained with a mixed fluorescent dye solution

212

consisting of Nile blue (0.1%) and Nile red (0.1%). Then the dyed emulsions were

213

deposited on concave confocal microscope slides and gently covered with a cover

214

slip(Dai, Sun, et al., 2018). The Nile blue was used to stain the ZPNPs and the Nile

215

red was applied to dye the oil phase. The CLSM was operated using two laser

216

excitation sources: an argon/krypton laser at 488nm (Nile red) and a Helium Neon 11

217

laser (He-Ne) at 633 nm (Nile blue).

218

2.8. Cryo-SEM

219

In the Cryo-SEM technique, the sample is vitrified with liquid nitrogen and

220

maintained at a very low temperature, which can preserve the structure of emulsions

221

in a frozen state and allow them to remain stable during the observation.(Sriamornsak,

222

Thirawong, Cheewatanakornkool, Burapapadh, & Sae-Ngow, 2008) The original

223

structures of the Pickering emulsions co-stabilized by ZPCPs and different NSME

224

were observed by Cryo-SEM. The samples were placed on an aluminum platelet, and

225

then transferred to a cryo-preparation system (PP3010T, Quorum Inc., UK) to

226

flash-freeze the samples in liquid nitrogen slush followed by high vacuum

227

sublimation of unbound water. The samples were freeze-fractured in the

228

cryo-preparation chamber, coated with platinum. Then the images were captured

229

using SEM (Helios NanoLab G3 UC, FEI, USA). The analysis was performed at a

230

working distance between 3 and 5 mm with TLD detection at 2 kV.

231

2.9. In vitro digestion analysis, free fatty acid release and bioaccessibility of

232

233 234

β-carotene

This study used an international standardized in vitro gastrointestinal model:(Minekus et al., 2014)

235

Stomach phase: 20 mL of the emulsion was mixed with 20 mL of simulated

236

gastric fluid (SGF) containing 0.0032 g/mL pepsin to mimic gastric digestion. The pH

237

was adjusted to 2.0 and the sample was then swirled at 150 rpm for 1 h.

238

Small intestine phase: 20 mL of gastric digesta was transferred into a 100 mL 12

239

glass beaker and then adjusted to pH 7.0. Thereafter, 20 mL of simulated intestinal

240

fluid (SIF) containing 5 mg/mL bile salt, 0.4 mg/mL pancreatin and 3.2 mg/mL lipase

241

was mixed with digesta in reaction vessel. The pH was adjusted to 7.0 and the

242

samples were held under continuous vibration at 150 rpm for 2 h to mimic small

243

intestine digestion.

244

The degree of lipolysis was measured through the amount of free fatty acids

245

(FFA) released. The amount of 0.25 M NaOH required to neutralize the released FFA

246

through lipid digestion was determined by a pH-stat automatic titration unit (Metrohm,

247

Switzerland, 916 Ti-Touch).(Y. Xiao et al., 2018) Control samples were carried out in

248

ZPCPs-stabilized Pickering emulsions and NSME-stabilized Pickering emulsions.

249

The amount of FFA released was determined as the percentage of FFA (%) released

250

during the digestion time as described by Li and McClements:(Y. Li & Julian

251

McClements, 2010) %

= 100 ×

2

252

where VNaOH and mNaOH represent the volume (L) and concentration (M) of

253

NaOH solution needed to neutralize the FFA, respectively and Wlipid and Mlipid

254

represent the initial mass (g) and molecular mass (g·mol−1) of the triacylglycerol oil,

255

respectively.

256

The bioaccessibility of β-carotene was determined after the intestinal

257

digestion.(Liu, Ma, Zhang, Gao, & Julian McClements, 2017) Part of digesta was

258

processed using an high-speed centrifuge at 15,000 rpm for 60 min at 4 ℃. The

259

micelle phase containing the solubilized β-carotene was collected. The content of 13

260

β-carotene extracted from the initial emulsion and micelle fraction was determined

261

according to the method described in 2.12. The bioaccessibility (%) of β-carotene

262

was calculated by the equation below: !""#$$ % & '( (%) =

,- ./ ,01

/

/-2 3 40

263

where, Cmicelle and Cinitial emulsion are the contents of β-carotene in the micelle fraction

264

and the initial emulsion.

265

2.10.

Statistical analysis

266

All the samples were repeated three times and the data obtained were average

267

values of triplicate determinations, which were subjected to statistical analysis of

268

variance using SPSS 18.0 for Windows (SPSS Inc., Chicago, USA). Statistical

269

differences were determined by one-way analysis of variance (ANOVA) with

270

Duncan’s post hoc test and least significant differences (p < 0.05) were accepted

271

among the treatments.

272

3. Results and discussion

273

3.1. Characteristics of ZPNPs

274

As depicted in Fig. S1A, ZPNPs exhibited a spherical shape with a non-uniform

275

size. A slight aggregation among particles might take place due to the process of

276

freeze-drying. Besides, the mean hydrodynamic size and zeta-potential of ZPNPs

277

were 442.2 ± 12.0 nm and -12.33 ± 0.24 mV. The large size of ZPNPs limited them to

278

adsorb onto the surface of oil droplets rapidly, but endowed them with an excellent

279

stability attached at the interface due to a much higher desorption energy.(Binks, 2002; 14

280

Binks & Rodrigues, 2007; Cui et al., 2009) The isoelectric point (pI) of zein is around

281

pH 6.2 and PGA has a dissociation constant (pKa) around pH 3.5.(Wei, Sun, Dai,

282

Zhan, et al., 2018) At pH 4.0, zein can successfully complex with PGA through the

283

electrostatic attraction. The Pickering emulsion stabilized by ZPNPs showed a

284

long-term stability against coalescence and Ostwald ripening due to a synergistic

285

effect of the steric hinderance and electrostatic repulsion (Dai, Zhan, et al., 2018).

286

The interfacial wettability plays a crucial role for the interfacial adsorption of

287

Pickering stabilizers, and is indicated by the contact angle (θo/w) of particles.(Binks,

288

2002) As shown in Fig. S1B, the θo/w of ZPNPs was around 76.6 ± 1.2 °, indicating a

289

strong hydrophilicity of particles during the solvent-evaporation co-precipitation,

290

which was consistent with our previous work.(Wei, Sun, Dai, Mao, et al., 2018)

291

3.2. Droplet size and zeta-potential.

292

As shown in Fig.1, the Pickering emulsion stabilized by ZPNPs (Z-P) exhibited

293

the smallest droplet size (6.73 ± 0.36 µm). The result demonstrated that ZPNPs could

294

effectively prevent the aggregation and coalescence of droplets. With the addition of

295

LF at a low concentration (0.10%, w/v), the droplet size was significantly increased to

296

13.52 ± 0.15 µm (p<0.05). The pI of LF was ranged from 8.4 to 9.0, and therefore LF

297

molecules showed high positive charge when pH was 4.0.(Farnaud & Evans, 2003)

298

Therefore, ZPNPs and LF would form the electrostatic complex through the attractive

299

force with the opposite charges, which promoted the bridging flocculation among the

300

droplets. With a gradual increase in LF level, the droplet size was continuously

301

elevated from 13.34 ± 0.03 µm to 20.70 ± 0.09 µm. When a higher concentration of 15

302

LF was added, a "simultaneously adsorbed layer" or a "sequentially adsorbed layer"

303

(multilayer) interfacial structure might be formed at the interface of the

304

emulsion.(Dickinson, 2011) On the one hand, the presence of LF might decrease the

305

density of particles adsorbed at the interface and form a more diffuse layer compared

306

to pure particle-stabilized interface.(A. Ganzevles, Kosters, van Vliet, A. Cohen Stuart,

307

& H. J. de Jongh, 2007; A. Ganzevles, Zinoviadou, van Vliet, A. Cohen Stuart, & H. J.

308

de Jongh, 2006; Ganzevles, Fokkink, van Vliet, Cohen Stuart, & de Jongh, 2008) On

309

the other hand, LF could also form a secondary layer outside the particle layer.(Liu et

310

al., 2018) This multilayer interface structure provided sufficient steric hindrance due

311

to the thicker interfacial layer, but charge neutralization reduced the electrostatic

312

repulsion between the droplets. Through the determination of zeta-potential of

313

droplets, a transition of surface charge from negative to positive pattern occurred (Fig.

314

1).

315

The cooperative stabilization of ZPNPs and LF was proved to be better than that

316

of ZPNPs and Rha at the interface. At a low concentration of Rha, the droplet size of

317

Pickering emulsions was increased significantly (p<0.05). The droplet size reached

318

the maximum (38.24 ± 0.11 µm) in Z-P/0.75rha, interpreting that the addition of Rha

319

promoted the aggregation of droplets. When Rha was added to the particle-stabilized

320

interface, it would interact with ZPNPs and promote the bridging flocculation

321

between the droplets.(Pugnaloni et al., 2004) Furthermore, it was speculated that Rha

322

entered into the gaps between the particles at the interface and changed the interfacial

323

distribution of particles. When the level of Rha was increased, the competitive 16

324

adsorption between particles and Rha molecules occurred at the interface, resulting in

325

a declination of the emulsion stability.(Bai & McClements, 2016; Binks & Rodrigues,

326

2007) An obvious decrease was observed in the droplet size of Pickering emulsions

327

when the concentration of Rha was above 0.75% (w/v). Due to its amphiphilic small

328

molecular structure, Rha might adsorb into the interfacial pores between the particles,

329

reducing the interfacial tension, thereby the large droplets would break up during the

330

emulsification to form smaller droplets.(Wilde et al., 2004) When the concentration

331

of Rha reached 1.25% (w/v), the droplet size was decreased to 15.17 ± 0.25 µm. The

332

presence of Rha altered the interfacial composition of oil droplets and formed a more

333

compact interfacial layer.(Wilde et al., 2004) Similarly, Binks and Rodrigues (2007)

334

found a synergistic behavior between particles and surfactants that displayed in

335

stabilizing the emulsion.(Binks & Rodrigues, 2007) In addition, there was a slight

336

declination in the zeta-potential of droplets with the rise in Rha level, which was

337

attributed to the competitive displacement of ZPNPs by the excessive Rha at the

338

interface.

339

3.3.Interfacial tension.

340

The dynamic interfacial tension of two types of individual emulsifiers at the

341

oil-in-water interface was demonstrated in Fig. 2A. Initially, the interfacial tension (γ0)

342

of oil-water interface was 23.578 ± 0.012 mN/m without any emulsifier.(Zhu et al.,

343

2019) With the rise in Rha level (0.01-0.50%, w/v), the interfacial tension (γ) was

344

decreased from 16.349 ± 0.019 mN/m to 4.564 ± 0.007 mN/m. A slight increase was

345

observed in γ when Rha concentration was further elevated, indicating that the 17

346

interface was saturated by the adsorption of Rha molecules. Similarly, the γ value in

347

the Pickering emulsions co-stabilized by particles and LF was continuously decreased

348

with the rise in LF level. A turning point of γ value was observed at 0.25% (w/v)

349

(7.715 ± 0.008 mN/m), and the γ value was reversely increased at a higher level of LF,

350

which interpreted that LF could cover the interface fully at a lower level compared to

351

Rha. Nevertheless, the Rha molecules could reduce the γ value more effectively than

352

LF due to the small molecular weight and flexible spatial structure of Rha.

353

Interestingly, the adsorption of emulsifiers has a significant influence on the

354

particle-stabilized interface (Fig. 2B). With the addition of different levels of LF or

355

Rha, the γ values of all emulsions were increased. In terms of ZPNPs and Rha

356

co-stabilized Pickering emulsions, the γ value made a jump upward from 4.993 ±

357

0.029 mN/m (the γ of ZPNPs adsorbed interface) to 44.020 ± 0.023 mN/m at 0.01%

358

(w/v) of Rha. As the level of Rha was elevated from 0.05% to 0.50% (w/v), the γ

359

value was significantly (p<0.05) increased from 34.752 ± 0.024 mN/m to 71.692 ±

360

0.024 mN/m. The increased interfacial tension could elevate the surface free energy of

361

Gibbs, making the system less stable, thus decreasing the membrane strength.

362

As we reported, the solid particles could scarcely be replaced by the surfactants

363

at a relatively low level.(Wei, Tong, et al., 2019), which could be combined with

364

particles through van der Waals’ force and hydrophobic attraction, instead of

365

absorbing onto the interfacial gaps between the particles. However, both the particles

366

and Rha molecules carried similar negative charge, thereby facilitating surfactant

367

adsorption onto interface at a low surfactant concentration due to electrostatic 18

368

repulsion (reducing interfacial tension slightly).(Binks et al., 2007; Xu et al., 2018)

369

The surfactants at a level of 0.05% (w/v) and particles could have a synergetic effect

370

in the stabilization of the oil-water interface, which has been reported previously by

371

other researchers.(Binks et al., 2007; Xu et al., 2018) However, the γ value was

372

visibly reduced at a higher concentration of Rha, attributing to the diffusion of Rha

373

molecules into the interfacial pores between the particles.(Mackie & Wilde, 2005;

374

Pugnaloni et al., 2004)1 Furthermore, the competitive displacement between particles

375

and surfactants occurred. Similarly, the γ value of ZPNPs and LF co-stabilized

376

Pickering emulsions was gradually increased with the rise of LF level until it reached

377

the maximum value (73.348 ± 0.023 mN/m) at 0.25% (w/v) of LF. Thereafter, an

378

obvious decrease in the γ value was observed when the LF level was over 0.25%

379

(w/v), which revealed that higher concentrations of LF also competitively adsorbed

380

onto the interface with particles.(Dickinson, 2011)

381

3.4. Environmental stability.

382

3.4.1. Physical stability.

383

The influence of LF on physical stability of β-carotene Pickering emulsions was

384

investigated (Fig. S2A). As the concentration of LF was increased, physical stability

385

of Pickering emulsions was decreased continuously until the concentration of LF

386

reached 0.75% (w/v), attributing to the reduced electrostatic repulsion between

387

droplets (1.1 mV). With further a rise in LF level, the emulsion stability was slightly

388

improved, which was ascribed to the secondary layer of LF at the interface with

389

sufficient electrostatic and steric repulsion against the aggregation and coalescence. 19

390

Similarly, the stability of ZPNPs/Rha co-stabilized Pickering emulsion initially

391

decreased and then increased with the rise in Rha level (Fig. S2B). With the addition

392

of Rha at a low concentration, the stability of the Pickering emulsion was

393

progressively reduced, revealing that the adsorption of Rha molecules induced the

394

bridging depletion of particles at the interface. Nevertheless, physical stability of

395

Pickering emulsions was reversely enhanced when the Rha level was elevated above

396

0.75% (w/v). Rha molecules would diffuse into the interfacial gaps between the

397

particles and reduce the interfacial tension, which was beneficial to stabilize

398

Pickering emulsions.(Wei, Tong, et al., 2019)

399

3.4.2. Photo stability.

400

Due to its unsaturated structure, β-carotene was prone to chemical degradation

401

upon the exposure of light, heat or oxygen.(Qian, Decker, Xiao, & McClements, 2012)

402

With the addition of LF ranging from 0.10% to 0.50% (w/v), the chemical stability of

403

β-carotene entrapped in the Pickering emulsions against UV radiation was improved

404

compared to Z-P (Fig. 3A). The highest retention rate of β-carotene in the Pickering

405

emulsions was elevated visibly to 80.84 ± 0.07% (Z-P/0.10LF) upon the exposure of

406

light for 4 h compared to Z-P (69.75 ± 1.19%). The addition of LF filled the

407

interfacial gaps between the particles and then formed a secondary layer to cover the

408

particle-stabilized interface. The better stability of β-carotene in the ZPNPs and LF

409

co-stabilized Pickering emulsion was ascribed to two aspects: firstly, the

410

particle/protein complexes or multilayered structure would form a thicker interface to

411

resist the invasion of oxygen, free radicals and pro-oxidants from bulk phase to inner 20

412

core of lipid droplets; secondly, the antioxidant capacity of LF itself could scavenge

413

free radicals at the interface and inhibit the oxidation and degradation of β-carotene in

414

the emulsion.(Yang et al., 2018) Nonetheless, chemical stability of β-carotene was

415

reduced when the concentration of LF was above 0.75% (w/v). The accelerated

416

degradation of β-carotene in the emulsion was ascribed to the larger droplet size and

417

poor stability at higher concentrations of LF due to the depletion flocculation. The

418

physical breakdown of the emulsions made β-carotene lose the protection of the

419

interfacial layer, resulting in a faster degradation.

420

Similarly, chemical stability of β-carotene entrapped in ZPNPs and Rha

421

co-stabilized Pickering emulsions was improved with the aid of Rha (0.10-0.50% ,w/v)

422

(Fig. 3B). The highest retention rate of β-carotene against UV radiation for 4 h was

423

84.45 ± 0.28% in Z-P/0.50Rha. Comparably, the photo stability of β-carotene in the

424

ZPNPs/Rha co-stabilized Pickering emulsion became better than that in the

425

ZPNPs/LF co-stabilized Pickering emulsion. It was interpreted that Rha molecules

426

would diffuse into the gaps between the particles, effectively prevented light or free

427

radicals entering into the inner core of lipid droplets at the interface.(Mackie & Wilde,

428

2005; S. Zou et al., 2013) Among all the samples, β-carotene in Z-P/0.75Rha

429

degraded most quickly after 4 h exposure of UV radiation (52.40 ± 0.52%), which

430

was consistent with its largest droplet size. As the concentration of Rha was elevated

431

above 0.75% (w/v), the retention rate of β-carotene in the emulsion was increased

432

with the rise in Rha level due to the smaller droplet size and compact interfacial layer.

433

3.4.3. Thermal stability. 21

434

In order to expand the applications of Pickering emulsions in food industry, the

435

impact of thermal treatment on droplet size and β-carotene content of Pickering

436

emulsions with different interfacial compositions were evaluated.

437

As shown in Fig. 4A, the droplet size of ZPNPs-stabilized Pickering emulsion

438

was increased from 6.73 ± 0.36 µm to 9.66 ± 0.08 µm after thermal treatment. When

439

the low concentrations of LF were added, there was a slight increase in the droplet

440

size (Z-P/0.10LF and Z-P/0.25LF). However, thermal stability of Pickering emulsions

441

was greatly reduced with the rise in LF level. The highest increase in the droplet size

442

was ranging from 17.01 ± 0.08 µm to 77.07 ± 6.77 µm (Z-P/0.50LF), indicating that

443

thermal processing caused the denaturation of the adsorbed protein at the interface

444

with the exposure of hydrophobic residues. The hydrophobic attraction facilitated the

445

aggregation and coalescence between the droplets, resulting in the instability of the

446

emulsions.(Liu et al., 2018) Besides, excessive LF molecules in the continuous phase

447

led to the depletion flocculation between the droplets, which could be more serious

448

due to thermal denaturation of the protein.

449

The ZPNPs and Rha co-stabilized Pickering emulsions kept stable at a low level

450

of Rha (< 0.50%, w/v), indicating that Rha and ZPNPs had a synergetic effect on

451

stabilizing the Pickering emulsion. At a low level of Rha, its molecules could rapidly

452

adsorb onto the interfacial pores and reduced the interfacial tension rapidly, thereby

453

elevating the strength and stability of interfacial membrane. With further a rise in Rha

454

level, the emulsions became unstable and the droplet size fluctuated obviously,

455

interpreting that the high concentration of Rha exhibited a detrimental effect on the 22

456

particle-Rha mixed interface. On the one hand, Rha and particles had a competitive

457

absorption onto the droplet surface, which caused the displacement of particles at the

458

interface and the loss of steric hinderance against the aggregation of

459

droplets.(Dickinson, 2011) On the other hand, the existence of solid particles hindered

460

the adsorption and movement of Rha on the surface of droplets, which was

461

disadvantageous to lower the interfacial tension and stabilize the emulsions.(Mackie

462

& Wilde, 2005; Wilde et al., 2004)

463

The thermal stability of β-carotene was negatively correlated with the

464

concentration of LF or Rha, the lower the concentration of the emulsifier added led to

465

the higher the retention rate of β-carotene and vice versa. The smaller droplet size

466

with a larger specific surface area resulted in more exposure of β-carotene entrapped

467

in oil core to environment. However, the lower concentration (0.10-0.50%, w/v) of

468

emulsifiers (either LF or Rha) and ZPNPs formed a stronger and denser interfacial

469

membrane against thermal degradation than Z-P. As depicted in Fig. 4B, the addition

470

of LF at low concentrations improved thermal stability of β-carotene in the Pickering

471

emulsion. The highest retention rate of β-carotene was 83.96 ± 1.15% in Z-P/0.25LF

472

compared to 63.41 ± 1.00% in Z-P. As the LF concentration exceeded 0.25% (w/v),

473

the retention rate of β-carotene in the emulsions decreased with the rise of LF level.

474

The β-carotene remained in Z-P/1.25LF was decreased to 19.38 ± 0.19% after thermal

475

treatment at 85 ℃ for 1 h, which was consistent with the increased droplet size.

476

The degradation of β-carotene in the Pickering emulsion co-stabilized by

477

particles and Rha was similar to that of the ZPNPs and LF co-stabilized Pickering 23

478

emulsion. The thermal stability of β-carotene in Z-P/0.10Rha, Z-P/0.25Rha and

479

Z-P/0.50Rha became better than that of Z-P, indicating that the low concentration of

480

Rha indeed improved thermal stability of β-carotene. The highest retention rate of

481

β-carotene in the ZPNPs and Rha co-stabilized Pickering emulsion was 94.50 ± 1.20%

482

(Z-P/0.25Rha). As the Rha level was over 0.50% (w/v), chemical stability of

483

β-carotene was greatly reduced due to the competitive displacement between the

484

particles and Rha. Commonly, surfactants are less effective than biopolymers or

485

biopolymer-based solid particles in the protection of nutraceuticals entrapped.(Mao,

486

Yang, Xu, Yuan, & Gao, 2010)

487

3.4.4. pH stability

488

The physical stability of delivery systems under various pH values has an

489

important guiding significance for their applications in processed foods and complex

490

human digestive environments.(Dai, Sun, et al., 2018) As demonstrated in Fig. 5A,

491

the influence of different pH values (2.5, 6.0 and 8.5) on the droplet size and

492

zeta-potential of novel Pickering emulsions co-stabilized by particles and emulsifiers

493

was investigated. The Pickering emulsion solely stabilized by particles (Z-P)

494

remained stable as they experienced pH fluctuation. The largest increase of droplet

495

size occurred in the ZPNPs and LF co-stabilized Pickering emulsion at pH 2.5.

496

Besides, the zeta-potential of ZPNPs and LF co-stabilized Pickering emulsions was

497

increased from negative charge (-10 ~ 0 mV) to positive charge (0 ~ +10 mV),

498

indicating a charge reversal when pH was changed from 4.0 to 2.5. As aforementioned,

499

ZPNPs and LF could form a layer-by-layer interfacial structure through electrostatic 24

500

deposition with the opposite charges. However, the compact multilayered interface

501

was difficult to be generated without electrostatic attraction at pH 2.5, which

502

undermined the steric repulsion between the droplets. A zeta-potential value over |30|

503

mV was necessary to provide sufficient electrostatic repulsion among oil droplets in

504

traditional emulsions and progressive flocculation might occur between |5| and |15|

505

mV.(Fernanda S. PolettoRuy C. R. BeckSílvia S. GuterresAdriana R. Pohlmann, 2011)

506

Therefore, the reduced steric and electrostatic repulsion between the droplets made

507

the emulsions lose their physical stability. Although the stability of the ZPNPs and LF

508

co-stabilized Pickering emulsion at pH 8.5 was better than that at pH 2.5, an obvious

509

increase was observed in the droplet size. At pH 8.5, the negative zeta-potential was

510

mainly derived from ZPNPs, while the LF molecules were electrically neutral because

511

the pH was close to the pI of the protein. This phenomenon might affect LF to provide

512

the steric hindrance on the surface of droplets as an outer layer. Besides, the charge of

513

LF influenced its molecular structure, thereby affecting the attractive force between

514

the droplets.

515

Compared to the acid and alkaline environments, the particles and LF

516

co-stabilized Pickering emulsion was the most stable at pH 6.0. The droplet size of the

517

emulsions kept constant and zeta-potential of the droplets was more negative with the

518

rise in pH. Therefore, the sufficient electrostatic and steric repulsion guaranteed the

519

excellent stability of Pickering emulsions and prevented the droplets from aggregation

520

and coalescence. On the other hand, the ZPNPs and Rha co-stabilized Pickering

521

emulsion could keep stable at different pH values. 25

Rha molecules at the mixed

522

interface improved physical stability of Pickering emulsions in response to the

523

fluctuation of pH in the surrounding environment. This distinction might be attributed

524

to the fact that Rha molecules could enter into the gaps between the particles freely,

525

and adsorb at the interface rather than being limited by proteins, such as LF.(Mackie

526

& Wilde, 2005) In addition, the functional properties of Rha are not as susceptible as

527

proteins.

528

3.4.5. Ionic strength stability

529

Food products usually need to go through environments with different ionic

530

strengths, and therefore the stability of Pickering emulsions was investigated under

531

various NaCl concentrations (50 - 200 mM). The Z-P was very unstable at high levels

532

of ionic strength (Fig. 5B). As the concentration of NaCl was elevated from 50 mM to

533

100 mM, the droplet size of Z-P was increased from 48.45 ± 1.39 µm to 108.11 ± 2.04

534

µm. French, Taylor, Fowler and Paul (2015) reported that the addition of small

535

quantity (< 40 mM) of sodium chloride to the silica dispersion prevented the

536

aggregation of oil droplets.(French, Taylor, Fowler, & Clegg, 2015) However, the

537

higher level of salt would decrease the contact angle (ϴo/w) of particles and strengthen

538

their hydrophilicity, which further promoted the formation of particle bridges between

539

the droplets. Besides, the addition of salt screened the electrostatic repulsion between

540

the particles, which induced the aggregation of droplets.

541

The incorporation of LF into the ZPNPs-stabilized interface significantly

542

improved the stability of the emulsions under high ionic strength. Compared to Z-P,

543

there was a much smaller increase in the droplet size of the ZPNPs and LF 26

544

co-stabilized Pickering emulsion. Although electrostatic attraction was reduced with

545

the rise in NaCl level, the addition of LF provided a protection for the stability of

546

Pickering emulsions with the sufficient steric repulsion. As NaCl concentration was

547

elevated from 50 mM to 200 mM, the droplet size of the emulsions was still stable

548

even smaller. In terms of the ZPNPs and Rha co-stabilized Pickering emulsion, when

549

the Rha concentration was lower than 0.50% (w/v), an obvious increase was observed

550

in the droplet size at different concentrations of NaCl. With the rise in NaCl level, the

551

droplet size of the emulsions was increased gradually, revealing that the low

552

concentration of Rha could not effectively stabilize the emulsions. As the

553

concentration of Rha was increased (> 0.50 %, w/v), the stability of the emulsions

554

was visibly improved. Although partial competitive displacement might occur, the

555

diffusion of Rha molecules into the interfacial pores could form a compact interface

556

and keep the emulsions stable.

557

3.5.Morphological observation

558

The morphology of the ZPNPs-stabilized Pickering emulsion was observed

559

through optical microscopy (Fig. 6A). The individual and spherical droplets were

560

scarcely aggregated in Z-P dispersion. The influence of surfactant or protein on the

561

visual appearance of β-carotene Pickering emulsions was presented in Fig. S3.

562

Although the Pickering emulsion stabilized by particles alone showed a homogeneous

563

state without creaming, the dimension of droplets was gradually increased with the

564

rise in LF level, which was consistent with the result of laser scattering. When the low

565

concentrations of Rha was applied (0.10% and 0.25%, w/v), the droplet size still kept 27

566

constant. As the concentration of Rha was elevated to 0.50% and 0.75% (w/v), there

567

was an apparent aggregation among the droplets in the Pickering emulsions. When the

568

higher concentration of Rha was applied, there existed a reduction in the droplet size

569

of the emulsions with ununiform droplets.

570

3.6.CLSM

571

CLSM is a strong and versatile tool to visualize the morphology of the emulsion

572

droplets. The ZPNPs-stabilized Pickering emulsion showed the spherical and small

573

droplets (Fig. 7). When the low concentration of LF (0.10-0.50%, w/v) was added,

574

partial particles still adsorbed onto the surface of oil droplets, but the remaining

575

particles were desorbed from the droplet surface and entered into the continuous

576

phase, which was consistent with the cloudy aqueous layer (Fig. S3). As the

577

concentration of LF continued to increase, the interfacial structure of the Pickering

578

emulsion was greatly influenced. It was observed that the particles became aggregated

579

and flocculated into larger particles at the interface induced by LF.(Dickinson, 2011;

580

Mackie & Wilde, 2005) In terms of ZPNPs and Rha co-stabilized Pickering emulsions,

581

a low level of Rha elevated the droplet size of the emulsions. Nevertheless, the

582

Pickering emulsions remained stable and the droplet size was decreased when the

583

concentration of Rha was over 0.50% (w/v). It was speculated that the excessive

584

surfactant replaced the particles at the interface and adsorbed on the surface of oil

585

droplets, which was beneficial to generate the smaller droplets but not conducive to

586

the long-term stability of the emulsion.

587

3.7.Cryo-SEM 28

588

Cryo-SEM can be utilized to observe the original interfacial structure of the

589

emulsions. As depicted in Fig. 6C, the dense solid particles were adsorbed onto the

590

surface of the droplets. The particle network was generated in the emulsion and the

591

particle bridges were observed between the droplets. The tendency of the Pickering

592

emulsion to form particle bridges was influenced by the wettability of particles. The

593

hydrophilicity of ZPNPs facilitated the particles to enter into the continuous phase and

594

had a tendency to form the bridges.(French et al., 2015)

595

The influence of the LF level on the interfacial structure and particle distribution

596

of Pickering emulsions was clearly presented in Fig. 7. At a low level of LF, the

597

ZPNPs were still closely packed onto the droplet surface without any flocculation.

598

The particle bridges and the network could be observed between the droplets, which

599

was similar to Z-P. However, when the LF concentration was elevated above 0.50%

600

(w/v), the zeta-potential of droplets was increased from a negative value, passed

601

through a zero value at 0.75% (w/v), and became an increasingly positive charge (Fig.

602

1), attributing to the adsorption of LF onto the surface of particles. As previously

603

reported, ZPNPs were initially hydrophilic and became hydrophobic with increasing

604

LF concentration, near the condition of zero charge, where they flocculated the most

605

and then hydrophilic again once recharged.(Binks et al., 2007; Binks & Rodrigues,

606

2007) Therefore, it was observed that the particles that uniformly distributed at the

607

interface underwent a severe aggregation and flocculation due to the hydrophobic

608

interaction between the particles. These findings in the ZPNPs and LF co-stabilized

609

Pickering emulsion were in accordance with the result of CLSM. 29

610

In terms of the microstructure of the ZPNPs and Rha co-stabilized Pickering

611

emulsion, the densely packed particles were adsorbed onto the droplet surface at a low

612

level of Rha. However, there was no particle bridge occurring between the droplets,

613

which was different from the ZPNPs and LF co-stabilized Pickering emulsion. As

614

previously reported, the adsorption of Rha onto the surface of particles altered the

615

wettability of particles (probably became more hydrophobic), which broke the bridges

616

between the particles and inhibited the formation of particle network.(Binks &

617

Rodrigues, 2007; French et al., 2015) When the concentration of Rha was elevated

618

above 0.25% (w/v), a lot of small holes appeared on the surface of droplets. The size

619

of these holes was consistent with that of particles adsorbed previously, revealing that

620

the presence of Rha indeed resulted in a competitive displacement between ZPNPs

621

and Rha at the mixed interface. Furthermore, the particles were embedded deeply into

622

the interface, indicating that the hydrophobicity of particles became elevated.

623

3.8. Storage stability

624

The storage stability of the β-carotene Pickering emulsions was assessed to

625

mimic the shelf-life of food products (Fig. 8). Z-P kept stable in the first two weeks,

626

but an obvious increase was observed in the droplet size when the storage time

627

exceeded two weeks. As the low concentration of LF was applied, physical stability of

628

the Pickering emulsions was significantly (p<0.05) improved compared to Z-P (Fig.

629

8A). According to the observation through cryo-SEM, the particle bridges between

630

the droplets still remained in the Pickering emulsions at a low level of LF. The

631

formation of particle network could enhance the storage stability of the emulsions by 30

632

reducing the frequency of collisions between oil droplets. When the higher level of LF

633

was incorporated, the droplet size increased continuously with the extension of

634

storage period, attributing to the reduced electrostatic repulsion and depletion

635

flocculation caused by excessive LF in the continuous phase.

636

Similarly, storage stability of the emulsions was reduced gradually with the rise

637

in Rha level (Fig. 8B). ZPNPs and Rha could co-stabilize the Pickering emulsion at a

638

low level of Rha.(Binks et al., 2007; Binks & Rodrigues, 2007) Besides, the

639

adsorption of Rha onto the particle-stabilized surface could increase the θo/w of

640

particles and facilitate the ZPNPs to adsorb at the interface. However, it was

641

disadvantageous to stabilize the Pickering emulsions with the addition of surfactant at

642

a higher level (≥

643

surfactant severely reduced the steric repulsion between the droplets and limited the

644

long-term stability of the emulsions.

0.75%, w/v).(Xu et al., 2018) The competitive displacement of

645

The chemical stability of β-carotene entrapped in the emulsions was dependent

646

on the level of LF, which was consistent with the physical stability (Fig. 8C).

647

Z-P/0.25LF exhibited the best chemical stability during whole storage period,

648

followed by Z-P/0.10LF. The result indicated that the addition of LF effectively

649

improved physicochemical stability of the Pickering emulsions due to the formation

650

of particle bridges and then networks of particles. With the rise of LF, the chemical

651

stability of β-carotene in the emulsions was reversely decreased. The poorer the

652

physical stability of the emulsion was, the faster the β-carotene entrapped degraded.

653

Similarly, the low concentrations of Rha and particles had a synergetic effect on the 31

654

protection of β-carotene in the emulsion during different storage periods (Fig. 8D).

655

Nevertheless, the chemical stability of β-carotene was decreased with the Rha

656

concentration over 0.50% (w/v), attributing to the aggregation of oil droplets by

657

competitive absorption between particles and Rha molecules.

658

3.9. Rheological property

659

Most previous studies focused on the rheological properties of traditional

660

emulsions stabilized by small molecular emulsifiers or particles solely. However, the

661

rheological properties of Pickering emulsions using the particle-emulsifier mixed

662

interface have been scarcely reported.

663

3.9.1. Apparent viscosity

664

Among all the samples, the ZPNPs stabilized Pickering emulsion exhibited the

665

lowest apparent viscosity due to its small droplet size (Fig. S4A). When LF at a level

666

of 0.10% (w/v) was added, there was a significant (p<0.05) increase in the viscosity,

667

attributing to the bridging flocculation between the droplets. When the concentrations

668

of LF were elevated to 0.25% and 0.50% (w/v), the viscosity of the emulsions was

669

slightly decreased due to the deflocculation of droplets. At the higher level of LF, the

670

emulsion became more viscous. As aforementioned, the addition of LF at a high level

671

resulted in the flocculation of particles (diffusing into the interfacial pores) and

672

depletion flocculation of droplets (entering into the continuous phase), which further

673

led to the coalescence of droplets and increased the viscosity of the emulsion.

674

As depicted in Fig. S4B, Z-P/0.10Rha exhibited the highest viscosity among the

675

particles and Rha co-stabilized Pickering emulsions, which was consistent with the 32

676

synergetic effect of particles and Rha in the stabilization of the emulsions with the

677

low level of Rha. As the concentration of Rha was elevated from 0.25% to 1.00%

678

(w/v), the viscosity of the emulsion was decreased continuously due to the

679

competitive displacement of particles with Rha, which reduced the steric hinderance

680

and particle bridges. However, when the concentration of Rha reached 1.25% (w/v),

681

an obvious increase in the emulsion viscosity was observed, which might be ascribed

682

to the depletion flocculation in the presence of excessive Rha molecules (Wei, Tong,

683

et al., 2019).

684

3.9.2. Viscoelastic properties

685

Both G' and G'' of the emulsions were increased with the rise in frequency, and

686

they were almost frequency dependent (Fig. S4C and D). Among all the emulsions, G'

687

was obviously higher than G'' in the frequency ranging from 0.1 to 100 rad/s,

688

indicating that an elastic particulate gel-like structure was formed.(Dai, Sun, et al.,

689

2018; Dai, Zhan, et al., 2018; Wei, Sun, Dai, Mao, et al., 2018) The G' of Z-P

690

exhibited the lowest value, revealing a liquid-like behavior of the emulsion solely

691

stabilized by ZPNPs. When LF at the levels of 0.10% and 0.25% (w/v) was added, the

692

G' of the emulsion was increased. The addition of LF induced the bridging

693

flocculation between the droplets, leading to the aggregation of droplets and the

694

transformation from liquid-like to solid-like properties. The G' of the emulsion was

695

slightly reduced at the higher level of LF, which promoted the particle flocculation at

696

the interface but reduced the particle bridges between the droplets.

697

In terms of particles and Rha co-stabilized Pickering emulsions, the highest G' 33

698

was observed in Z-P/0.10Rha, which was consistent with its apparent viscosity.

699

Although a low level of Rha promoted the bridging flocculation of droplets, ZPNPs

700

and Rha could co-stabilize the emulsion at the interface without competitive

701

displacement. However, the G' of the emulsion was decreased continuously with the

702

rise in Rha level and reached the minimum in Z-P/1.00Rha. When Rha at the

703

concentration of 1.25% (w/v) was added, an obvious increase was found in the

704

viscoelasticity due to the depletion flocculation between the droplets in the presence

705

of excessive Rha molecules.

706

3.10.

707

In vitro digestion fate of Pickering emulsions and bioaccessibility of

β-carotene

708

The droplet size, free fatty acid (FFA) release, and bioaccessibility of β-carotene

709

in the emulsions were monitored during in vitro gastrointestinal digestion. As shown

710

in Fig. 9A, the ZPNPs stabilized Pickering emulsion kept stable after 60 min in the

711

gastric digestion, while the aggregation and flocculation of oil droplets occurred in the

712

ZPNPs and LF co-stabilized Pickering emulsion. The phenomenon was explained by

713

that LF was digested by pepsin and caused the deconstruction of protein conformation,

714

resulting in an increase in the droplet size of the emulsions. After the gastric digesta

715

passed into the small intestine, there was a significant (p<0.05) increase in the droplet

716

size of Z-P as well as ZPNPs and LF co-stabilized Pickering emulsions at low levels

717

of LF (0.10% and 0.25%, w/v). Nevertheless, the droplet size of the emulsions at a

718

higher concentration of LF still kept stable during the intestinal digestion. Comparably,

719

the Pickering emulsion in the presence of Rha became more stable in the stomach, 34

720

especially at a high level of Rha (Fig. 9B), which was consistent with better physical

721

stability of particles and Rha co-stabilized Pickering emulsions at pH 2.5. While the

722

digesta passed into the intestinal phase, the droplet size was increased significantly

723

(p<0.05). The result indicated that the digestion of the emulsion mainly occurred in

724

the small intestine rather than the stomach in the presence of pancreatin and lipase.

725

As shown in Fig. 9C, the ZPNPs stabilized Pickering emulsion exhibited the

726

highest FFA release (25.84%), but much lower than conventional emulsions stabilized

727

by surfactants (40%-90%).(Liu et al., 2017; Sarkar, Ye, & Singh, 2016) The high

728

desorption energy of particles at the interface of Pickering emulsions made it difficult

729

to be displaced with bile salts in the small intestine, limiting the lipolysis and

730

generation of FFA.(Sarkar et al., 2016, 2019) As the concentration of LF was

731

increased from 0.10% to 1.00% (w/v), the FFA release was reduced progressively

732

from 24.80% to 13.02%, which suggested that the addition of LF into the

733

particle-stabilized interface effectively reduced the lipolysis. The LF molecules would

734

occupy the interfacial gaps between the particles and further adsorb onto the outer

735

layer of particles to form a thicker interface, thereby preventing the adsorption of bile

736

salts at the interface, which further inhibited the adsorption of lipase/colipase.(Sarkar

737

et al., 2019) Among ZPNPs and Rha co-stabilized Pickering emulsions, the highest

738

FFA release was 18.05% (Z-P/0.10Rha), which was much lower than that of ZPNPs

739

and LF co-stabilized Pickering emulsions (Fig. 9D). There were two possible

740

explanations to illustrate this phenomenon: on the one hand, the droplet size of ZPNPs

741

and LF co-stabilized Pickering emulsions was smaller than that of ZPNPs and Rha 35

742

co-stabilized Pickering emulsions, which provided a larger specific surface area with

743

more binding sites for lipase. On the other hand, the zeta-potential of droplets in

744

ZPNPs and LF co-stabilized Pickering emulsions was close to zero even positive

745

value. The electrostatic attraction could be generated between the droplets and

746

negatively-charged bile salts, which facilitated the adsorption of lipase.(Sarkar et al.,

747

2019) While both the lipid droplet and bile salts possessed the same negative charge,

748

the electrostatic repulsion would be disadvantageous to the lipid hydrolysis. With a

749

continuous rise in Rha level, the FFA release of the emulsion was reduced greatly to

750

12.40% in Z-P/0.75Rha. Rha molecules could diffuse into the inter-particle gaps at

751

the interface and occupy the sites where the bile salts and lipase could adsorb. As

752

previously reported, Rha exhibited the best emulsifying activity with a high negative

753

charge at netural pH.(Lovaglio et al., 2011) Therefore, the presence of Rha created a

754

stronger electrostatic barrier to the access of negatively-charged bile salts to the

755

vicinity of a negatively-charged mixed particle-surfactant interface. At the higher

756

concentration of Rha, the FFA release of the emulsion was reversely elevated. As

757

discussed above, excessive Rha molecules would adsorb competitively with particles

758

at the interface, which impeded the steric barrier to the adsorption of bile salts and

759

delay the lipid digestion. After the displacement of particles, Rha could hardly

760

provide sufficient steric hinderance to limit the lipid digestion solely, which was

761

similar to traditional emulsions.

762

As shown in Fig. 9E, the bioaccessibility of β-carotene in ZPNPs stabilized

763

Pickering emulsion was 11.13 ± 0.52%, which was much lower than that in traditional 36

764

emulsions stabilized by surfactants. Lin et al. (2017) reported that the bioaccessbility

765

of β-carotene in modified starch stabilized nanoemulsion was about 30%, attributing

766

to the nanoscale size and digestible emulsifiers.(Lin, Liang, Ye, Singh, & Zhong,

767

2017) The microscale size and irreplaceable particle stabilizers of Pickering

768

emulsions greatly limited the degree of lipolysis and release of FFA, thereby reducing

769

the generation of micelles where solubilized the bioaccessible hydrophobic

770

nutraceuticals.(Sarkar et al., 2019) The bioaccessibility of β-carotene was slightly

771

increased with the addition of 0.10% (w/v) LF, due to the enhanced electrostatic

772

attraction to negatively charged bile salts. However, the bioaccessibiliy of β-carotene

773

in the Pickering emulsions was then reduced with the rise in LF level, which was

774

mainly attributed to the limited FFA release during the digestion, thereby resulting in

775

the lower bioaccessibility of β-carotene. The level of FFA release directly determined

776

the capacity of the micelle phase to solubilize β-carotene.(Lin et al., 2017; Sarkar et

777

al., 2019) It was noted that although the FFA release of the emulsion was increased

778

obviously in Z-P/1.25LF, there was no significant increase in the bioaccessibility of

779

β-carotene. Excessive LF molecules in the continuous phase might combine with the

780

negative bile salts to form precipitates, which reduced the amount of bile salts in the

781

micelle phase and bioaccessibility of β-carotene.(R. Van der Meer, D. S. Termont,

782

1991)

783

In terms of ZPNPs and Rha co-stabilized Pickering emulsions (Fig. 9F), the

784

bioaccessibility of β-carotene in Z-P/0.10Rha was 8.05 ± 0.62% and reduced

785

continuously with the rise of Rha level until it reached the minimum in Z-P/0.75Rha 37

786

(5.38 ± 0.75%). This result demonstrated that the bioaccessibility of β-carotene was

787

positively correlated with the FFA release. The FFA release and the amount of bile

788

salts binding in the mixed micelle phase were two of dominators influencing the

789

bioaccessibility of nutrients in the emulsions. As the Rha level was above 0.75%

790

(w/v), the bioaccessibility of β-carotene was elevated to 7.95 ± 0.32% and 9.76 ±

791

0.36% in Z-P/1.00Rha and Z-P/1.25Rha, respectively. The higher level of Rha

792

resulted in the competitive displacement of particles adsorbed at the interface, which

793

promoted the magnitude of lipolysis, following the rise of β-carotene bioaccessibility.

794

The microstructure of different Pickering emulsions in gastric and intestinal

795

phases was also observed through CLSM. As shown in Fig. S5, the oil droplets

796

remained stable after the digestion in SGF. After being digested in SIF, the oil

797

droplets were severely aggregated, and a clear interfacial structure was scarcely to be

798

distinguished, indicating that the digestion process of the emulsions mainly occurred

799

in the small intestine.

800

4. Conclusions

801

In the present study, novel β-carotene Pickering emulsions were prepared using

802

the composite particle-emulsifier interface. The Pickering emulsions co-stabilized by

803

particles and LF or Rha kept stable against different environmental stresses (light,

804

heat, pH and ionic strength) as well as for a storage period of 28 days at 55 ℃.

805

Compared with small molecule emulsifiers, the complex interface formed with LF at

806

a lower concentration (≤ 0.50%, w/v) became more surperior in stabilizing O/W 38

807

emulsions. But at a higher concentration, the Pickering emulsions co-stabilized by

808

small molecule emulsifiers and composite nanoparticles exhibited better physical

809

stability. The compact interfacial layer composed of particles and emulsifiers

810

effectively decreased the FFA release of Pickering emulsions along with the reduction

811

in the bioaccessibility of β-carotene, which was mainly attributed to the filling and

812

adsorption of emulsifiers between the particles, thereby reducing the exposure of oil

813

droplets to bile salts and lipase. These findings are meaningful for the design of

814

fat-reduced foods loaded with bioaccessible active ingredients to increase the satiation

815

perception and nutritional value.

816

ASSOCIATED CONTENT

817

AUTHOR INFORMATION

818

Corresponding Author

819

*E-mail: [email protected]

820

Notes

821

The authors declare no competing financial interest.

822

Acknowledgement

823

The research was funded by the National Natural Science Foundation of China

824

(No. 31871842). The authors are grateful to Tsinghua University Branch of China

825

National Center Protein Sciences (Beijing, China) for providing the facility support of

826

Cryo-SEM with the aid of Xiaomin Li.

827

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Figure captions Fig. 1 Droplet size and zeta-potential of Pickering emulsions co-stabilized by particles and Rha or LF. Fig. 2 Interfacial tension between the oil (MCT) and Rha or LF solution (A); interfacial tension between the ZPNPs stabilized Pickering emulsions (Z-P) and Rha or LF solution (B). Fig. 3 Photo stability of β-carotene entrapped in Pickering emulsions co-stabilized by particles and LF (A) or Rha (B). Fig. 4 Influence of thermal treatment on droplet size and zeta-potential (A) of Pickering emulsions and chemical stability of β-carotene (B) entrapped in Pickering emulsions co-stabilized by particles and LF or Rha. Fig. 5 Effects of different pH values (A) and ionic strengths (B) on droplet size and zeta-potential of Pickering emulsions co-stabilized by particles and LF or Rha. Fig. 6 Optical microscopy (A), CLSM image (B) and cryo-SEM microstructure (C) of Pickering emulsion solely stabilized by zein-PGA composite nanoparticles. Fig. 7 CLSM images and Cryo-SEM microstructures of Pickering emulsions co-stabilized by particles and LF or Rha. Fig. 8 Effect of storage period on droplet size of Pickering emulsions co-stabilized by particles and LF (A) or Rha (B), as well as retention rate of β-carotene entrapped in Pickering emulsions co-stabilized by particles and LF (C) or Rha (D). Fig. 9 Digestion time dependence of droplet size of Pickering emulsions co-stabilized by particles and LF (A) or Rha (B); digestion time dependence of FFA release (%) from Pickering emulsions co-stabilized by particles and LF (C) or Rha (D); bioaccessibility of β-carotene entrapped in Pickering emulsions co-stabilized by particles and LF (E) or Rha (F).

10

35

5

30

0

25

-5

20

-10

15 -15 10 -20 5 -25

Sample

a a a a a a Z-P .10LF .25LF .50LF .75LF .00LF .25LF 0Rh 5Rh 0Rh 5Rh 0Rh 5Rh 0 P/0 P/0 P/0 P/1 P/1 /0.1 /0.2 /0.5 /0.7 /1.0 /1.2 / P Z- Z- Z- Z- Z- Z- Z-P Z-P Z-P Z-P Z-P Z-P

Fig. 1

Zeta-potential (mV)

Droplet size (µm)

40

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Highlights Physicochemical stability of Pickering emulsions was enhanced using the particle-emulsifier mixed interface. Various interfacial structures were observed in Pickering emulsions. Lipid digestion in Pickering emulsions was modulated through interfacial engineering. The bioaccessibility of β-carotene entrapped in Pickering emulsions was influenced by the interfacial compositions.

Conflict of Interest The authors declare no competing financial interest in this study.