Kafirin Nanoparticle-Stabilized Pickering Emulsions as Oral Delivery

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Kafirin nanoparticles-stabilized Pickering emulsions as oral delivery vehicles: physicochemical stability and in vitro digestion profile Jie Xiao, Chao Li, and Qingrong Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04385 • Publication Date (Web): 05 Nov 2015 Downloaded from http://pubs.acs.org on November 6, 2015

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Journal of Agricultural and Food Chemistry

Kafirin nanoparticles-stabilized Pickering emulsions as oral delivery vehicles: physicochemical stability and in vitro digestion profile Jie Xiao†, Chao Li‡ and Qingrong Huang†,§,* †

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick,

New Jersey 08901, USA ‡

College of Light Industry and Food Science, South China University of Technology,

Wushan Road 381, Guangzhou 510640, China §

College of Food Science and Engineering, Wuhan Polytechnic University, 68 Xuefu

South Road, Wuhan 430023, China

*To whom correspondence should be addressed. Tel: (848)-932-5514. Fax (732)-9326776. Email:[email protected]

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ACS Paragon Plus Environment


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Abstract

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Kafirin nanoparticles-stabilized Pickering emulsions (KPEs) were used to encapsulate curcumin.

3

The stability of KPEs under processing conditions, their protective effects against photo-

4

oxidation of curcumin and lipid oxidation of oil in emulsions, as well as the digestion profiles in

5

gastrointestinal tract were investigated. KPEs were found to be more stable under acidic than

6

basic environment, and elevated temperature induced their structural instability. The protective

7

effect of KPE on chemical stability of curcumin was manifested when subjected to UV radiation

8

as compared to other comparable formulations, such as bulk oil or Tween 80 stabilized

9

emulsions (TE). Meanwhile, the lipid oxidation rate was retarded in KPE as compared to those

10

of TE. Due to hydrolysis of pepsin, KPE could not survive through the gastric digestion process.

11

After the intestinal digestion process, the extent of lipolysis of KPE and the curcumin

12

bioaccessibility fell in between those of TE and bulk oil. Our results will fill the gap between the

13

physicochemical properties of protein particles based Pickering emulsions and their realistic

14

applications in the oral delivery of functional food ingredients.

15 16

Keywords: Pickering emulsion; kafirin; curcumin; encapsulation; lipid oxidation; in vitro

17

digestion

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18


INTRODUCTION

19

Emulsions have received enduring research interest in fields of food, dietary supplement,

20

pharmaceutical and cosmetic industries, since large proportion of products function through the

21

emulsion based formulations. Small molecular weight surfactants or amphiphilic polymers have

22

long been utilized in industry to stabilize emulsion droplets through either reducing interfacial

23

tension or forming a viscoelastic interfacial film. Although well understood and widely applied,

24

they are not the only possible sources for emulsion stabilization. Colloidal particles with proper

25

partial wettability in dispersed and continuous phases can function as Pickering-type stabilizer by

26

providing a physical barrier at droplet interface. This phenomenon was first known to the

27

academic world since the publication of Pickering at the beginning of 19th century 1. After

28

continuous research accumulation in the past one hundred years, distinctive characteristics of

29

Pickering emulsifiers compared with conventional emulsifiers were recognized, such as,

30

irreversible interfacial adsorption 2, outstanding stability against coalescence and Oswald

31

ripening 3, the ability to stabilize emulsions with large droplet size (up to several millimeters) or

32

high internal phase 4, peculiar rheological properties, etc. A rich array of accessible formulations

33

including droplet dispersions, concentrated emulsions

34

bicontinuous microstructures

35

above mentioned progresses were achieved via model Pickering systems stabilized by inorganic

36

(e.g. silica nanoparticles) or synthetic polymer based particles 9.

8

5

, gels

6

, colloidosomes

7

, and

evolved from Pickering emulsions-based systems. Most of the

37

In contrast to the significant progress of Pickering emulsions in the application fields of

38

material science, research efforts aiming at exploring their applications in food and

39

pharmaceutics are relatively small. Theoretically, the robust interfacial particle layers of

40

Pickering emulsions promise their advantages in serving as novel encapsulation or delivery

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vehicles for bioactive compounds. To be specific, as for storage compartment, the thick

42

interfacial particle layer would enhance the oxidation stability of lipophilic compounds by

43

dramatically decreasing interaction incidences between oxygen and transitional metals in

44

aqueous phase and lipophilic compounds in oil phase. As for delivery vehicle, oil-in-water (O/W)

45

type Pickering emulsions have the potential of providing improved stability as well as controlled

46

release in gastrointestinal tract for lipophilic compounds by encapsulation them within the inner

47

oil phase. Recent research efforts in fulfilling such potentials have included silica particles

48

stabilized Pickering emulsion for oral drug delivery

49

oxidation 12. Edible colloidal particles, microcrystalline cellulose and modified starch, stabilized

50

Pickering emulsion were also investigated to enhance the oxidative stability of oil-in-water

51

emulsions 13.

10

, topical delivery

11

and reduce lipid

52

In our previous study, kafirin, an alcohol-soluble prolamin protein from sorghum grain 14,

53

was assembled into spherical nanoparticles to stabilize Pickering emulsions. The O/W type of

54

Pickering emulsion obtained under our operation conditions exhibited long-term stability against

55

coalescence. The effects of physical parameters (e.g., protein particle concentration, oil phase

56

ratio, ionic strength) on the microstructure, rheological properties and stability of emulsion were

57

systematically studied 15. In the present study, O/W type Pickering emulsion formulations were

58

selected as a model system to justify the feasibility of being functioned as a novel encapsulation

59

as well as oral delivery vehicle. Comprehensive studies related to encapsulation of lipophilic

60

compounds in the inner oil phase will be conducted to examine the stability of emulsion under

61

common storage or processing conditions, to test the protection effect towards lipophilic

62

encapsulate as well as the lipid phase, and to clarify its digestion profile in simulated

63

gastrointestinal tract.

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Curcumin, a natural coloring and flavoring agent from turmeric (Curcuma longa), exhibited

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multiple evidenced health promoting properties, such as anti-inflammatory, anti-carcinogenic,

66

and antioxidant activities 16. It shows no toxicity to human up to several grams consumption per

67

day. However, the chemical structure of curcumin, which consists of 2 hydroxy methoxyphenyl

68

rings connected by two β-diketone groups, easily undergoes degradation under alkaline condition

69

or exposure to light/UV radiation during storage and processing, resulting in loss in stability and

70

bioactivities

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solubility (11 ng/ml) 18. Among various delivery systems targeting to address this issue, lipid-

72

based formulations are able to generate mixed micelles in the lumen of the digestive tract along

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with lipid digestion, through which the originally dissolved curcumin in lipid will be solubilized

74

in the micelle core and thus become bioaccessible. Up to now, such formulations have involved

75

conventional O/W emulsions

76

organogels-derived systems

77

and the protection as well as delivery capacity of kafirin particles stabilized Pickering emulsions

78

were examined.

17

. In addition, its oral bioaccessibility is quite poor due to its low intrinsic water

22

19

, self-emulsifying system

20

, solid lipid particles

21

and

. For the above-mentioned reasons, curcumin was encapsulated,

79

The physical stability of kafirin particles stabilized Pickering emulsions (KPE) was

80

investigated by monitoring the “oiling off” effect and changes in microstructure under different

81

pH values and temperatures. Their capacity of preserving bioactive compound during storage

82

was evaluated by comparing the residual level of curcumin under UV exposure treatment with

83

other comparable formulations. The protection effect against lipid oxidation of the lipid phase

84

was also investigated. The digestion profile of the formulation was investigated in simulated

85

gastric fluid and simulated intestinal fluid by monitoring their structural integrity as well as the

86

extent of lipolysis.

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MATERIALS AND METHODS

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Materials. Curcumin (82% curcumin, 15% demethoxycurcumin (D-Cur) and 3%

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bisdemethoxy-curcumin (BD-Cur), Mw 361.05) was a gift from Sabinsa Corporation

90

(Piscataway, NJ) and used without further purification. Kafirin protein with a purity of 90% was

91

extracted from whole sorghum grain and characterized in our lab

92

(each 100 g contains 14 g of saturated fat, 21 g of mono-unsaturated fat, and 57 g of poly-

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unsaturated fat) (ConAgra Foods. Inc., USA) was purchased from a local market and used

94

without further purification. Glacial acetic acid, HPLC-grade acetonitrile and analytical grade

95

HCl and NaOH were purchased from Alfa Aesar (Ward Hill, MA). Sodium chloride, bile salts,

96

Tween 80, Pepsin from porcine gastric mucosa (P7125), pancreatin with 8 × USP specification,

97

Tris maleate iron (II) chloride, ammonium thiocyanate, cumene hydroperoxide, 2-thiobarbituric

98

acid, 1,1,3,3-tetraethoxypropane were purchased from Sigma-Aldorich (St. Louis, MO, USA).

99

Sodium taurodeoxycholate (Na TDC) was purchased from CalBiochem (La Jolla, CA). Water

100

14

. Pure Wesson vegetable oil

purified by Milli-Q system was used for sample preparation.

101

Fabrication of curcumin encapsulated emulsions stabilized by kafirin nanoparticles.

102

Curcumin was dissolved in vegetable oil under heat to the final concentration of 3 mg/mL.

103

Kafirin protein was first dissolved in acetic acid and then added drop-wise into bulk water to

104

form nanoparticles suspension, dialysis was then conducted in Spectra/Por® 7 RC dialysis tubes

105

(MWCO: 10 kDa) to remove excess acetic acid. Pickering emulsions (total volume 8 mL) with

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different oil fraction ø (0.2, 0.4, 0.6, 0.8) were prepared by mixing the vegetable oil with kafirin

107

particle suspension (1% w/v) in a glass vial (1.3 cm in internal diameter and 6 cm in length).

108

Mixtures

109

ULTRATURRAX T25 digital, IKA 190 Works, Inc., Wilmington, NC, USA) with an 8 mm

were

then

homogenized

by

high-speed

6

homogenizer


unit

(model

IKA-

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110

dispersion probe at 13,000 rpm for 3 min. The final pH after preparation was between 3.3 and

111

3.6. Experiments were performed in triplicate for each formulation.

112

Characterization of emulsions. Right after emulsions were formed, emulsion type of the

113

resultant formulations was determined by dilution of the emulsion droplets with water or oil: if

114

the emulsion dispersed readily in the water phase, it was of O/W type; if the emulsion dispersed

115

readily in the oil phase, it was assessed as W/O type. The height of serum phase (Hs) and total

116

height of formulation (Ht) were recorded along with the incubation time at room temperature and

117

the creaming/sedimentation index was reported as (Hs/Ht) × 100. Optical microscopy observation

118

of emulsions was visualized using a Nikon Eclipse TE 2000-U with a Q Imaging camera.

119

Emulsion samples were first diluted with the continuous phase and then dripped onto the glass

120

slice and imaged at 100 or 1000 magnification. Image J2x 2.1.4.7 was used to estimate the mean

121

droplet diameter by measuring a minimum of 20 drops from each slide. The formulation with oil

122

phase ratio of 0.6 (refer to as KPE) was selected for further investigation, and the encapsulation

123

efficiency of curcumin within was determined to be 90.3 ± 3.8 %.

124

Effect of pH and temperature on emulsion stability. Freshly prepared KPE emulsions

125

were first diluted with DI water for four times. Then the pH values were adjusted to 1.5, 3.3, 7.4

126

and 8.0 respectively by adding either hydrochloric acid (1M) or sodium hydroxide (1 M) drop-

127

wise with a gentle stirring speed of 230 rpm. After one week’s storage at room temperature, 50

128

µL of emulsion was pipetted at the center of the glass vial after stirring and the droplet

129

morphology was recorded by optical microscopy. Emulsion of each pH value was then

130

centrifuged at 4,000 rpm for 5 min, and the volume of the oiling layer was recorded. The

131

released oil percentage was reported as volume of the oiling layer divided by the initial

132

encapsulated oil. The stability of emulsion under different storage temperatures was conducted

7



133

by storing freshly prepared emulsion aliquots at 4, 20, 37, 60

134

morphology after storage and released oil percentage were recorded as described above.

for 4 h. The emulsion droplets

135

Effect of KPE on chemical stability of curcumin under UV radiation. For UV radiation

136

treatment, KPE was poured into a 60 × 15 mm (diameter × height) polystyrene petri dish (Fisher

137

Scientific, Pittsburgh, PA, USA). UV radiation (4W, 365 nm) was performed using the UV

138

radiation equipment (Spectroline Model ENF-260C, 115 V, 60 Hz, Spectronics Corporation,

139

Westbury, New York, USA) at room temperature. Fifty µL aliquot was sampled at designed time

140

point. To recover curcumin, 1 mL of methanol was mixed with the aliquot, sample then

141

underwent vortex and was centrifuged at 10,000 rpm for 5 min to fully separate the supernatant

142

layer. Absorbance of methanolic extract after proper dilution was measured at 425 nm using a

143

microplate reader (BioTek Instruments, Winooski, VT) in conjunction with KC4 data reduction

144

software. The measured absorbance was converted to curcumin concentration using a standard

145

curve prepared by measuring the absorbance of a series of known concentrations of curcumin in

146

methanol. The effect of formulation on the kinetics curcumin degradation was evaluated by

147

plotting the residual level of curcumin against treatment time. For comparison, degradation

148

profiles of curcumin under UV exposure in Tween 80 stabilized emulsion (TE) (3 mL vegetable

149

oil with 3 mg/mL curcumin was homongenized with 7 mL DI water with 2% Tween 80), and

150

curcumin dissolved in vegetable oil (3 mg/mL) were reported.

151

Effect of Pickering emulsion on the lipid oxidation of vegetable oil. This section was

152

designed to investigate the effect of Pickering emulsion formulation on the lipid oxidation of oil

153

phase. For comparison, lipid oxidation of bulk oil and tween 80 stabilized emulsions were also

154

investigated. To exclude the influence of curcumin, which is an antioxidant compound itself,

155

soybean oil without curcumin encapsulation was selected as the oil phase of the emulsions. Five

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156

mL sample was stored in open glass vials with a surface area of 19.6 cm2 exposed to air at room

157

temperature. Sampling was conducted at 1st, 3rd, 5th, 7th, 9th, 11th, 13th and 15th days after

158

preparation. Peroxide value (PV) was determined according to the method of Shantha and

159

Decker

160

centrifuged at 10, 000 rpm for 5 min to precipitate the protein particles, if any. The organic

161

solvent phase (50 µL) was added to 2 mL of a methanol: 1-butanol mixture (2:1, v/v), followed

162

by the addition of 15 µL of 3.94 M ammonium thiocyanate and 15 µL of ferrous iron solution

163

(freshly mixed 0.144 M BaCl2 with 0.144 M FeSO4). The absorbance of resultant solution was

164

measured 20 min after the addition of iron at 510 nm. Lipid hydroperoxide concentration

165

(µmol/g oil) was determined using a standard curve made from cumene hydroperoxide.

23, 24

with some modifications: aliquot was mixed with 1 mL of isobutanol and then

166

TBARS content of the emulsions upon storage was determined using the procedure described

167

in a previous paper 25 with a few modifications. In brief, 0.2 mL sample was mixed with 0.3 mL

168

distilled water (isooctane/isobutanol), and the diluted samples were mixed with 1.5 mL of TBA

169

reagent (15% (w/v) trichloroacetic acid and 0.2% (w/v) thiobarbituric acid in 0.25 M HCl) in test

170

tubes. The resultant mixtures were heated in a boiling water bath for 15 min, and then cooled in

171

the air to room temperature for approximately 10 min. After cooling, supernatant was recovered

172

by centrifugation at 10, 000 rpm for 5 min and the absorbance was measured at wavelength of

173

532 nm. TBARS concentration (µmol/g oil) was determined according to a standard curve made

174

from 1,1,3,3-tetraethoxypropane (TEP).

175

Digestion profile in simulated gastric fluid. Simulated gastric fluid (SGF) was prepared

176

according to the procedure described by Tikekar, et al 10: 1 L of SGF was prepared by adding 5 g

177

sodium chloride and then adjusting pH to 1.5 by using 5 M HCl. Sample (bulk oil, KPE and TE)

178

containing 250 mg oil with 3 mg/mL curcumin was mixed with 9 mL SGF and incubated at 37

9



179

°C under magnetic stirring (230 rpm) for 10 min. 16 mg pepsin was dissolved in 1 mL SGF and

180

added to the mixture to start the digestion process. The emulsion was sampled (200 µL) at 0, 15,

181

30 and 60 min after enzyme addition to monitor the integrity of emulsion droplet structure during

182

digestion. Digestions were stopped by raising the pH to 7.5 to inactive the pepsin.

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Digestion profile in simulated small intestine. The digestion profiles of samples in

184

stimulated small intestine fluid (SIF) were collected according to our previous work 22. Briefly, 1

185

g pancreatin was mixed with 5 mL fed-state buffer (50 mM Tris maleate, 150 mM NaCl, 5mM

186

CaCl2, 20 mM NaTDC, 5 mM Phosphatidylcholine), centrifuged and kept on ice. Sample (bulk

187

oil, KPE and TE containing 250 mg vegetable oil) was mixed with 9.0 mL fed state buffer under

188

a water bath at 37.0

189

pancreatin preparation was added to start the lipolysis. During lipolysis, the pH was maintained

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at 7.50 ± 0.02 by adding 0.25 N NaOH manually. The volume of consumed NaOH over time was

191

recorded throughout the lipolysis experiments, and was used to calculate the concentration of

192

free fatty acids generated by lipolysis. Blanks were carried out in the absence of oil and

193

subtracted from the reported values. After digestion, the end products of digestion were

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ultracentrifuged at 50,000 rpm (Type 60 Ti rotor, 180,000 g, Beckman Coulter) for 40 min. The

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aqueous phase was then filtered through 0.22 µm filters and the amount of curcumin in the

196

aqueous phase was determined using HPLC. Microstructure changes of KPE and TE along with

197

the digestion process was observed under optical microscopy observation.

for 10min and then adjusted to pH = 7.5 using 1M NaOH. One mL

198

The extent of lipolysis was defined as the percentage of triglycerides digested in the in vitro

199

lipolysis experiments. It was assumed that digestion of one mole of triglycerides released exactly

200

two moles of free fatty acids (FFA) and consumed two moles of NaOH. Consequently, FFA can

201

be calculated as 26:

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% = 100 ×

× × × 2

202

Here VNaOH is the volume of sodium hydroxide required to neutralize the FFA produced (L),

203

mNaOH is the molarity of the sodium hydroxide solution used (in M), wlipid is the total mass of

204

triacylglycerol oil initially present in the digestion cell (in g), and Mlipid is the molecular mass of

205

the triacylglycerol oil (in g/mol).

206

The bioaccessibility of curcumin after digestion was calculated as: %Bioaccessibility =

!"#

$#% × "#&$#%

207

Where the numerator and denominator was mass of solubilized curcumin and mass of

208

curcumin in lipid. The mass of solubilized curcumin was the product of the concentration of

209

solubilized curcumin in the aqueous phase after lipolysis and the volume of the aqueous phase.

210

The mass of curcumin in lipid was calculated from the concentration of the curcumin in oil, the

211

density of oil (0.92 g/mL) was used. An UltiMate 3000 HPLC system equipped with a 25D UV-

212

VIS absorption detector (Dionex) and a Nova-Pak C18, 3.9× 150 mm column (Waters) was used

213

to analyze the concentration of curcumin in aqueous phase. Mobile phase solvents were: (A)

214

water with 0.02% acetic acid, and (b) acetonitrile. Ten microliters of samples were injected.

215

Elution condition was: 0 to 2 min, 65% A and 35% B; 2 to 17 min, linear gradient from 35% B

216

to 55% B; 17 to 22 min, held at 55% B; 22 to 23 min, B went back to 35% linearly. Flow rate

217

was 1 mL/min. Detection wavelength was set at 425 nm.

218

Statistical analysis. At least triplicate experiments were performed for each experiment.

219

Origin 9.0 software was used to perform all the statistical analysis. One-way analysis of variance

220

(ANOVA) procedure followed by Tukey’s mean comparison test was used for establishing the

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221

significance of differences among mean values at p < 0.05. The results were reported as the mean

222

standard ± deviation (SD).

223

RESULTS AND DISCUSSION

224

Characteristics of kafirin particles stabilized emulsions (KPE) with curcumin

225

encapsulated. Right after emulsification, emulsified phases were formed in all the formulations

226

and they were all determined as oil-in-water (O/W) type of emulsion. Upon storage, formulations

227

with oil phase of 0.2, 0.4 and 0.6 underwent different degree of fast creaming process during the

228

first 3 hours, and then gradually reached the plateau creaming index value afterward as shown in

229

Fig. 1 (A). Visual examination of emulsion with oil phase ratio of 0.2 and 0.4 showed phase

230

boundary after creaming process, while nearly fully emulsified phase was observed when the oil

231

phase ratio was lifted up to 0.6. The yellowish serum phase after creaming could be explained by

232

the combined results of the existence of smaller emulsion droplets in the water suspension phase,

233

and the absorption of free curcumin onto excess kafirin particles remaining in the water

234

suspension phase after the emulsification process. It was reported that the kinetic energy barriers

235

associated with the restricted movement through highly viscous networks of oil droplets at high

236

oil phase ratio diminished the rate and extent of phase separation in creaming process

237

Emulsion with 80% oil phase fraction changed from O/W type to W/O type after 1 h storage,

238

probably due to insufficient amount of kafirin particles to stabilize the emulsion, leading to the

239

creaming of the oil phase.

27

.

240

Microstructure of the formulations with different oil phase fraction after 96 h storage is

241

shown in Fig. 1(B). The emulsion droplet size increased as the oil phase ratio increased from 0.2

242

to 0.6, while large aggregations without distinct dispersed droplets were observed in formulation

243

with oil phase ratio of 0.8. When observed under higher magnification, typical individual droplet

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244

of emulsion with oil fraction of 0.6 was surrounded by a distinct ring shadow, indicating the

245

formation of polymeric coating layer. Besides, bridging phenomena among adjacent droplets

246

with shared interface were typical in this formulation, and these flocculated droplets could

247

remain their individual integrity without coalescence for several hours, a typical characteristic of

248

Pickering emulsion.

249

Our previous study has suggested that KPE would form the gel-like network after up to 312

250

hour incubation time period

251

particles stabilized emulsion was taken out with a spatula, and the morphology and flow

252

behaviors of our formulation were like stirred type yogurt (see supplemental data in Fig. S1).

253

Similar phenomenon was also reported by Tang et al. in the preheated soy protein isolate

254

stabilized emulsions

255

and flocs of oil droplets contributed to the formation of gel-like network structure 29, 30.

28

. In this study, after 5-day storage, the creaming layer of kafirin

. We believed that the kafirin particles provided rigidity to the interface

256

Physical stability of KPE. To guide future utilization of kafirin particles stabilized

257

emulsions, it is quite relevant to understand its physical stability under common processing and

258

storage conditions. For this purpose, the structure integrity of formulation with oil phase ratio of

259

0.6 was evaluated under different pH as well as temperature conditions. In both cases, the

260

released oil percentage and microstructure were monitored since they served as more reliable and

261

straightforward indicators for emulsion coalescence.

262

There have been several investigations on effects of initial pH of aqueous suspension on the

263

formation and stability of Pickering emulsions 31, 32, in which the pH of particle dispersion phase

264

was adjusted before the emulsification process. However, there have been few research efforts

265

focusing on the effect of pH changes on the stability of Pickering emulsion after emulsion

266

formation, which is a more relevant circumstance when products undergo blending, mixing and

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digestion in the human gastrointestinal tract. As shown in Fig. 2 (A), the coalescence induced

268

oiling off was much more significant under basic conditions than under acidic conditions. The

269

average released oil fractions after 7-day storage under stomach pH (1.5) and the pH right after

270

formulation (3.3) were 17.2% and 17.0%, respectively, and no distinguishable difference could

271

be found between their microstructures for emulsion droplets in the bulk emulsified phase. For

272

emulsions stored under intestinal pH (7.4) and alkaline (8.5) conditions, the average released oil

273

fractions after 7-day storage were 33.5% and 44.1%, respectively. Microstructures in both cases

274

showed large-scale flocculation and droplets with large droplet size or irregular shape emerged,

275

indicating the progress of coalescence. This result suggested that kafirin particles stabilized

276

emulsions were more stable in acidic pH and changes in partial wettability of kafirin particles

277

induced by basic environment might lead to structural instability.

278

As for the effect of storage temperature on the stability of KPE, our results suggested that

279

emulsions were more stable under refrigeration (4 °C) and room temperatures (20 °C) (Fig. 2

280

(B)), with both released oil percentages lower than 5% and no noticeable changes in

281

microstructure of bulk emulsion. When stored under body temperature (37 °C) for 4 hours,

282

40.5% oil was released and the population of small droplets was large, which was probably due

283

to the loss of large oil droplets. The most severe oiling off happened at 60 °C, where over 50%

284

oil was released and considerable flocculation happened within the bulk emulsion system.

285

Although protein particles were believed to be irreversibly adsorbed onto the interface, our result

286

suggested that the increase in temperature had outweighed their resistance capacity against

287

coalescence.

288

Protection effect of KPE on curcumin under UV treatment. To investigate whether

289

Pickering emulsion encapsulation can introduce protection effects to curcumin under UV

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290

radiation, the residual level of curcumin during treatment with KPE was compared with Tween

291

80-stabilized emulsion (TE) and curcumin in oil solution. The residual level of curcumin as a

292

function of UV treatment time was shown in Fig. 3A. The protective effect of formulation upon

293

curcumin decreased in the order of KPE, TW and bulk oil with residual levels of curcumin being

294

56.3 ± 4.9%, 42.5 ± 2.9 and 6.5 ± 3.1%, respectively. The degradation of curcumin has been

295

reported as: energy gained from UV light breaks adjacent carbonyl groups to generate ferulic

296

aldehydes, which were then oxidized into ferulic acid, vanillin or vanillic acid

297

bulk oil, free curcumin molecules were exposed directly to the UV radiation, it gained the

298

highest energy efficiency for degradation. In emulsion systems, the curcumin-enriched disperse

299

phase was separated in the water continuous phase leaving only the interface area as the direct

300

reaction area for UV degradation. Compared to TE, additional protection effects were gained

301

through the physical barrier of kafirin protein particles, which sheltered the encapsulated

302

curcumin from direct UV radiation. This was supported by evidence gained from cryo-SEM

303

images of kafirin protein particles covered emulsion droplets as compared to the smooth surface

304

of Tween 80 stabilized emulsion droplet as reported in one of our previous paper

305

conjugated double bonds in tyrosine, phenylalanine and tryptophan residues of kafirin protein 14

306

would absorb UV light hence protecting curcumin from degradation. In addition, larger emulsion

307

droplet size (49.0 ± 16.9 µm), and thus smaller interfacial area as compared to TE (7.0 ± 3.5 µm)

308

might also contribute to its better protection effect.

33, 34

. In case of

15

. Besides,

309

Effect of KPE on lipid oxidation. Lipid oxidation limits the shelf-life of emulsion products

310

and it leads to oxidation degradation of bioactive compounds in oil phase such as vitamin,

311

antioxidants and anti-inflammatory compounds

312

the effect of Pickering encapsulation on the lipid oxidation of oil phase by comparing with those

35

15

. Therefore, this section aimed to investigate



Page 16 of 33

313

of bulk oil and TE. In all formulations, positive correlations between TBARS and PV level were

314

found. Peroxide value and TBARS value increased significantly faster in TE, while KPE had

315

fairly the same pace of lipid oxidation as in bulk oil (Fig. 3B). The lipid oxidation rate in

316

conventional emulsion system was expected to be faster than that in bulk oil partly due to the

317

higher water activity (beyond 0.4), and also the accessibility of oil droplets to pro-oxidant agents

318

in water phase. And several factors contributed to the less severe lipid oxidation in KPE as

319

compared to that of TE. Firstly, the droplets size range of TE was appreciably smaller than that

320

of KPE, resulting in a significantly larger interfacial area in bulk water, where pro-oxidants

321

reside. Secondly, KPE formed thicker interfacial barrier as compared to TE (usually a few

322

nanometers in thickness). The interfacial layer then acts as an efficient physical barrier to limit

323

the diffusion of lipid oxidation initiators into oil droplets. Thirdly, since kafirin contained amino

324

acids in side chain with antioxidant activity, such as methionine, ½ cysteine, tryptophan, proline

325

and histidine, the protein particles at the interface as well as those remain in the bulk water phase

326

would scavenge free radicals to inhibit the propagation of lipid oxidation 24, 36. Finally, since the

327

pH of KPE was lower than the pI of kafirin protein, cationic droplet surface would repel

328

transitional metals away from the droplets

329

lipid oxidation rate of kafirin protein particles stabilized emulsion was fairly offset, keeping the

330

oxidation rate similar to that of bulk oil. The result validated that structural differences between

331

Pickering emulsion and small molecular weight surfactant stabilized emulsion can significantly

332

influence oxidative degradation of encapsulated lipid phase.

37

. Based on the above-mentioned factors, the fast

333

In vitro digestion profile of KPE. Lipids in food may be consumed in a variety of forms,

334

such as structural oil, bulk oil or emulsified oil, and the subsequent digestion and adsorption of

335

lipid and lipid soluble nutraceuticals are quite essential in guiding products design. In stomach,

16


Page 17 of 33


336

main digestion occurs due to hydrolysis of protein by pepsin, we thus first looked into the

337

physicochemical events happened in pre-digestion process under simulated gastric fluid.

338

As can be seen in Fig. 4, after mixing with gastric fluid, KPE droplets underwent

339

coalescence, and large-scale droplet collapse occurred 30 min after mixing. At the end of gastric

340

treatment, the majority of oil droplets lost their integrity and resulted in macrophase separation.

341

While TE droplets maintained their integrity until the end of treatment, although slight increase

342

in the population of larger droplets could be observed, probably due to coalescence and/or

343

Ostwald ripening. The digestion of kafirin particles by pepsin was the primary reason for the fast

344

collapse of oil droplets, since much less severe droplet coalescence was observed under

345

experimental conditions without addition of pepsin (data provided in supporting information Fig.

346

S2). It was reported that the main hydrolysis degradation of whey protein and BSA in emulsion

347

interface happened within the first 10 min after gastric fluid treatment

38

348

protein has been long considered as one of the least digestible protein

39

349

through the gastric digestion process. The fast release property of protein particles stabilized

350

Pickering emulsion can be either a desirable or undesirable property depending on the purpose

351

and format of products. To be specific, if protein particles functioned as the outermost emulsifier

352

for a w/o/w double emulsion, fast digestion in gastric fluid would enable programed release of

353

the oil phase and facilitate sufficient release and adsorption in subsequent intestinal fluid. For

354

protein particles stabilized single o/w emulsion, additional capsular or co-stabilizers might be

355

needed to ensure a prolonged release profile.

. Although kafirin

, it cannot survive

356

To investigate the possible effect of protein particles layer on the lipid digestion as well as

357

bioaccessibility of lipid soluble compounds, bulk oil, TE and KPE were subjected to SIF

358

digestion escaping the gastric digestion. This would resemble circumstances where outside

17



Page 18 of 33

359

capsular were involved when orally consumed. Results suggested that the digestion rates varied

360

among different formulations (Fig. 5): TE exhibited a sharp release of free fatty acid (FFA)

361

during the fist 20 min, and the FFA reached to a plateau after 60 min digestion; KPE showed a

362

slightly slow release kinetics during the first 50 min digestion before reaching to its plateau

363

value; For bulk oil, the release of FFA performed in a constant yet slow manner till the end of the

364

digestion test. The maximum FFA release decreased in the order of TE (46.5%) > KPE (37.9%)

365

> bulk oil (18.2%). We then fitted the release of free fatty acids with the hyperbola of ' = '() ∗

366

+/(+ + /( ), where r and t are the measured rate and time of digestion. The results suggested that

367

rmax decreased in the order of TE (46.47%) > KPE (45.25%) > bulk oil (21.90%), and Km

368

increased in the order of TE (4.36 min) > KPE (13.23 min) > bulk oil (23.45 min). Fitted

369

parameter of rmax represents the maximum rate of degradation, km is an indicative value for

370

reaction velocity 40. Thus, the maximum FFA-release of TE and KPE are appreciably higher than

371

that of bulk oil with much faster reaction velocities.

372

When subjected to SIF, bulk oil was broke down into oil in water dispersions due to the

373

mechanical impact as well as presence of surface-active components (lecithin, bile salts, etc.).

374

Meanwhile, emulsion droplets underwent destabilization process due to the dilution effect of

375

SIF. In all cases, lipid substrates dispersed in separated colony, surrounding by either bare

376

interface (bulk oil), or small molecular weight surfactant or protein nanoparticles. The lipase

377

then migrated and adsorbed onto the oil-water interface so as to react with the lipid substrate.

378

The area as well as the physicochemical properties of interfacial layer surrounding the oil

379

droplets thus played an important role in determining the rate and extent of oil digestion. For

380

bulk oil, since the bare interface was readily exposed to bile salt and lipase, the rate limiting

381

process for lipid digestion was the remarkably low reactive area available for lipase due to large

18


Page 19 of 33


382

oil droplet size (in the range of micrometers to millimeters). While for KPE, lipase had to

383

penetrate or diffuse through the particle interfacial layer before reacting with the lipid subjects,

384

the reaction efficiency of lipase was thus retarded. Initially, Tween 80 stabilized smaller

385

emulsion droplets than those stabilized by kafirin particles, and the thickness of the interfacial

386

layer was appreciably lower than that KPE, TE thus gained a faster lipolysis rate during the early

387

stage of lipolysis. Overall, the FFA release fraction (less than 50% in all cases) suggested that the

388

lipolysis efficiency for soybean oil under our experimental condition was not very high, which

389

were in agreement with other comparable long chain oils (e.g. corn oil). Intact emulsion oil

390

droplets can be seen in microscopy images of droplets at the end of lipolysis (Fig. 5).

391

Along with the lipid digestion process, hydrolysis products such as diacylglycerols,

392

monoglycerols and free fatty acid will release from the interface, forming micelles together with

393

bile salts and phospholipids. Through which the originally dissolved curcumin in lipid will be

394

solubilized in the micelle core and thus become bioaccessible. The contents of solubilized

395

curcumin after lipolysis were determined by HPLC, and the bioaccessiblity of curcumin

396

decreased in the same order of TE > KPE > bulk oil (Table 1). This indicated that increase in the

397

extent of lipolysis would enhance the extent of bioaccessibility. The percentage of each

398

component of the curcuminoids in the aqueous solution obtained after lipolysis was also

399

presented. Compared to raw curcuminoids without the treatment in SIF, all the digested

400

formulations showed lower percentage of curcumin and higher percentage of D-Cur and BD-

401

Cur, probably due to the sensitiveness of curcumin to the weak alkaline conditions

402

significant differences among percentages of each component in difference formulations (One-

403

way ANOVA) suggested that after curcuminoids were solubilized into micelles its fate of

404

degradation was independent of the initial formulation formats.

19


41

. No


Page 20 of 33

405

In summary, edible particles stabilized emulsion exhibited distinctive processing

406

properties as compared to conventional emulsions. During processing, KPEs were more stable

407

under acidic than basic environment, and elevated temperature would induce their structural

408

instability. KPEs showed protective effect upon chemical stability of encapsulate when subjected

409

to UV radiation as compared to bulk oil or Tween 80 stabilized emulsions (TE). Meanwhile,

410

KPEs retard the lipid oxidation rate as compared to those of TE. During oral consumption, KPEs

411

collapsed in the gastric fluid due to hydrolysis of kafirin particles by pepsin. The extent of

412

lipolysis of KPE during intestinal digestion process fell in between those of TE and bulk oil.

413

Results obtained in this work will broaden our current knowledge of edible Pickering emulsions

414

and provide realistic applications in food, dietary supplement and pharmaceutical industries.

415

ACKNOWLEDGMENT

416

We thank Yan Wang from Department of Food Science, Rutgers University for insightful

417

suggestions in photo-degradation experiments. This project was in part supported by the

418

“Hundred Talents” Program of Hubei Province, China. The financial support from the China

419

Scholarship Council to the first author is gratefully acknowledged.

420 421

Supporting Information. Optical images of gel-like karifin nanoparticles-stabilized Pickering

422

emulsions (KPE) and evolution of microstructure of KPE treated with simulated gastric fluid

423

without pepsin at 0 min, 15 min, 30 min and 60 min. This material is available free of charge via

424

the Internet at http://pubs.acs.org

425

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Page 21 of 33


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Page 26 of 33

Table 1. Percentage of each curcuminoid component in the raw material and solubilized fraction after lipolysis. Formulations Raw curcumin

Bioaccessibility (%) -

Curcumin (%) 82.1 ± 1.0

D-Cur (%) 14.8 ± 0.5

BD-Cur (%) 3.1 ± 0.5

Bulk oil

5.3 ± 0.1

55.0 ± 0.8

29.4 ± 0.3

15.6 ± 0.4

TE KPE

11.7 ± 0.7 8.8 ± 0.5

61.6 ± 2.8 58.1 ± 1.5

27.8 ± 1.5 28.0 ± 1.3

10.5 ± 1.3 13.8 ± 0.2

26


Page 27 of 33


Figure captions Fig. 1. (A) Creaming/sedimentation index of KPE at varying oil fractions along with storage time. Inserted were typical visual images of formulations right after preparation and after storage of 96 h. (B) Optical microscopy of KPE droplets with oil phase fraction of 0.2, 0.4, 0.6, 0.8 from left to right under 100 × magnification, scale bar= 100 µm. Enlarged figure showed the shared interface. Fig. 2. The released oil percentage after (A) 7 days’ storage under different pH conditions; (B) Four hours’ storage under different temperature. Inserted are corresponding microstructures. Fig. 3. (A) Residual curcumin level in KPE, TE and oil; (B) Evolution of lipid hydroperoxides (left) and TBARS (right) in KPE, TE and bulk oil under room temperature storage up to 15 days. Lines with different letters are significantly different (p < 0.05). Fig. 4. Microstructure integrity observation of KPE (upper row) and TE (lower row) along with SGF digestion at 0, 15, 30 and 60 min Fig. 5. Release profile of FFA in bulk oil, TE and KPE. Solid curves are the best fits to experimental data using the mathematical model. Inserted images are microstructures at the end of lipolysis.

27



Figure 1.

28


Page 28 of 33

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Figure 2.

29



Figure 3.

30


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Figure 4.

31



Figure 5.

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TOC.

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Kafirin Nanoparticle-Stabilized Pickering Emulsions as Oral Delivery

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