Vitamin E Encapsulation within Oil-in-Water Emulsions: Impact of

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Food and Beverage Chemistry/Biochemistry

Vitamin E encapsulation within oil-in-water emulsions: Impact of emulsifier type on physicochemical stability and bioaccessibility Shanshan Lv, Yanhua Zhang, Haiyan Tan, Ruojie Zhang, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06347 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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Journal of Agricultural and Food Chemistry

Vitamin E encapsulation within oil-in-water emulsions: Impact of emulsifier type on physicochemical stability and bioaccessibility Shanshan Lv1,2, Yanhua Zhang*1, Haiyan Tan1, Ruojie Zhang2, David Julian McClements*2 1

Key Laboratory of Bio-based Material Science and Technology (Ministry of Education),

College of Material Science and Engineering, Northeast Forestry University, Harbin, 150040, P.R. China 2

Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

Corresponding Author: Yanhua Zhang; David Julian McClements Corresponding author emails: [email protected]; [email protected]

1 ACS Paragon Plus Environment


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Abstract

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The influence of plant-based (gum arabic and quillaja saponin) and animal-based (whey

3

protein isolate, WPI) emulsifiers on the production and stability of vitamin E-fortified emulsions

4

was investigated. Their impact on lipid digestibility and vitamin bioaccessibility was also

5

studied utilizing an in vitro gastrointestinal tract. WPI and saponin produced smaller emulsions

6

than gum arabic. All emulsions had good storage stability at room temperature (4 weeks, pH 7).

7

Saponin- and gum arabic-emulsions were resistant to droplet aggregation from pH 2 to 8 because

8

these emulsifiers generated strong electro-steric repulsion. WPI-coated droplets flocculated

9

around pH 5 due to a reduction in charge near their isoelectric point. Lipid digestion was slower

10

in saponin-emulsions, presumably because the high surface-activity of saponins inhibited their

11

removal by bile acids and lipase. Vitamin bioaccessibility was higher in WPI- than in saponin-

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or gum arabic-emulsions. This information may facilitate the design of more efficacious

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vitamin-fortified delivery systems.

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Keywords: natural emulsifier; vitamin E; lipid digestion; bioaccessibility; nanoemulsions

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Introduction With the recent improvement in global living standards, more people are paying attention to

18

the incorporation of essential nutrients and nutraceuticals into their diets. Consequently, many

19

companies are developing a new generation of functional food and beverage products that are

20

fortified with these bioactive components. Vitamin E is a group of isomeric micronutrients, with

21

α-tocopherol having the strongest biological activity.1-2 Daily intake of α-tocopherol may benefit

22

human health due to its antioxidant activity and ability to inhibit various diseases.3-5 However, it

23

is a strongly hydrophobic molecule, making it hard to disperse directly into foods and beverages

24

that have an aqueous continuous phase. Moreover, exposure to light, heat, and oxygen promotes

25

the chemical degradation of α-tocopherol during storage, leading to a reduction in its biological

26

activity and nutritional benefits. To overcome these challenges, α-tocopherol can be

27

encapsulated and protected using colloidal delivery systems.6-7

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Oil-in-water emulsions are particularly suitable for encapsulating and delivering lipophilic

29

vitamins because they can be designed to have good physicochemical stability and to promote

30

vitamin bioavailability.8 Emulsifiers play an essential role in the production and stabilization of

31

emulsions, as well as in determining their functional performance. Selection of an appropriate

32

emulsifier is therefore crucial to the success of any emulsion-based delivery system. Consumers

33

are increasingly demanding more ethical and sustainable food products, which has promoted the

34

food industry to search for plant-derived ingredients to replace synthetic or animal-derived

35

ones.9-10 There has, therefore, been great interest in the identification of natural plant-derived

36

emulsifiers that can be used in functional foods and beverages.11

37 38

In the current study, three types of natural emulsifier were tested to establish their impact on the production, stability, and performance of vitamin E-fortified emulsions. Quillaja saponin



39

(QS) consists of a group of surface-active substances extracted from the Quillaja saponaria

40

Molina tree.12-13 Gum arabic (GA) consists of a blend of amphiphilic glycoproteins and

41

polysaccharides extracted from the exudate of two species of acacia tree.14-16 Whey protein

42

isolate (WPI) consists of a blend of amphiphilic globular proteins isolated from bovine milk,

43

such as -lactoglobulin, -lactalbumin, and bovine serum albumin.17-18 These three emulsifiers

44

vary in their molecular weights, structures, polarities, and electrical characteristics, which affect

45

their performance in emulsions.

46

Previous studies have reported that QS and WPI are better at producing emulsions

47

containing fine oil droplets than GA because of their higher surface activity and faster adsorption

48

rate during homogenization.19 Under neutral pH conditions, the emulsions formed using QS and

49

WPI were more stable to creaming than those formed using GA because they contained smaller

50

oil droplets. Conversely, the GA-emulsions had better stability to droplet aggregation when

51

exposed to alterations in environmental conditions, such as pH variations, high salt levels, or

52

elevated temperatures. The WPI-emulsions tended to aggregate around their isoelectric points, at

53

high ionic strengths, and when heated due to changes in the colloidal interactions acting amongst

54

the oil droplets. The QS-emulsions were shown to have good aggregation stability over most of

55

the pH range found in foods but were unstable at pH 2, which was attributed to a reduction in

56

their negative surface potential in this strongly acidic environment.20-21

57

Most previous studies on these emulsifiers have focused on their influence on the

58

physicochemical stability of emulsions. There is a much poorer understanding of the influence

59

of these emulsifiers on the behavior of vitamin-fortified emulsions under gastrointestinal

60

conditions. The current study was therefore carried out to determine the influence of these

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natural emulsifiers on the gastrointestinal behavior of vitamin-fortified emulsions. We


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hypothesized that it is important to understand this process because encapsulated vitamins should

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be released and solubilized inside the lumen of the human gut prior to being transported and

64

absorbed by the intestinal epithelium cells.22 Previous researchers have shown that the

65

bioaccessibility and/or bioavailability of oil-soluble vitamins encapsulated within emulsion-

66

based delivery systems depends on numerous factors, including droplet size, oil level, carrier oil

67

type, and interfacial properties 23-28. Consequently, it is important to optimize the composition,

68

structure, and physicochemical properties of these systems to ensure good performance. Some

69

previous studies have measured the bioaccessibility of emulsified vitamin E stabilized by

70

different kinds of emulsifier 29-31, but they have not focused on a direct comparison of plant- and

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animal-based emulsifiers and the differences in the mechanisms involved. The insights gained

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form the current research should therefore be helpful for improving the nutritional quality of

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functional foods and beverages.

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Materials and Methods

75

Materials

76

Vitamin E (α-tocopherol, purity 95%) was provided by Fisher Scientific (Waltham, MA).

77

Corn oil (Mazola) was obtained from a local commercial supplier. Quillaja saponin (Q-Naturale

78

200) was kindly supplied by Ingredion Inc. (Westchester, IL). Whey protein isolate (WPI) was

79

bought from Davisco Foods International (Le Sueur, MN). Gum Arabic (GA) was purchased

80

from TIC Gums (Belcamp, MD). All these emulsifiers were used without further purification.

81

The mucin, pepsin, lipase, and bile extract (from porcine) were purchased from the Sigma-

82

Aldrich Chemical Company (St. Louis, MO).

83

purchased either from Fisher Scientific or Sigma-Aldrich. All concentrations are reported as

84

weight percentages (w/w), unless otherwise stated.

All other reagents and chemicals were



85 86

Emulsion Preparation Aqueous phases were prepared by dissolving the natural emulsifiers (1.5% w/w) in 5 mM

87

phosphate buffer (pH 7.0). Oil phases were prepared by mixing vitamin E (20% w/w) and corn

88

oil (80% w/w). Emulsions were prepared by blending the oil phase (10% w/w) and aqueous

89

phase (90% w/w) together and then passing the resulting coarse emulsion through a

90

microfluidizer (M110Y, Microfluidics, Newton, MA) three times at 12,000 psi.

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Emulsion Stability

92

The storage stability of the emulsions was investigated by incubating them in the dark at

93

room temperature for 4 weeks. The pH stability of the emulsions was determined by preparing a

94

series of systems with different pH values (2.0-8.0) using HCl or NaOH solutions.

95

In vitro Digestion

96

A three-stage simulated GIT, consisting of mouth, stomach, and small intestine, was

97

employed to explore the potential gastrointestinal behavior of the emulsions. Briefly, emulsions

98

(2 wt.% oil level) were mixed with simulated saliva fluid (containing 3 mg/mL mucin) at a ratio

99

of 1:1, the pH was adjusted to 6.8 and then the samples were incubated for 2 min to mimic the

100

mouth phase. After 2 min, the mouth phase was mixed with simulated gastric fluid (with 3.2

101

mg/mL pepsin) at a ratio of 1:1, the pH was adjusted to 2.5 and the system was incubated for 2 h

102

to mimic the stomach phase. Finally, 30 mL of the stomach fluid contents were collected and

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subjected to the simulated small intestinal digestion condition. At this stage, the pH was

104

adjusted to 7.0, then 1.5 mL simulated small intestinal fluid (containing 3.75 M NaCl and 0.25 M

105

CaCl2) and 3.5 mL bile extract solution was added. Afterward, the pH was adjusted back to 7.0

106

and 2.5 mL lipase solution (24 mg/mL) was added to mimic the small intestine digestion. The

107

temperature of the entire digestion process was controlled at 37 °C. A pH-stat method was used


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to monitor lipolysis in the small intestine phase. The free fatty acids (FFA) released were

109

calculated as follows 32:

110

𝐹𝐹𝐴(%) =

𝑉𝑁𝑎𝑂𝐻 × 𝐶𝑁𝑎𝑂𝐻 × 𝑀𝑙𝑖𝑝𝑖𝑑 2𝑊𝑙𝑖𝑝𝑖𝑑

× 100

111

Here, VNaOH is the NaOH consumption during the small intestinal digestion process, CNaOH is the

112

NaOH concentration (0.25 M), Mlipid is the molar mass of digestible lipid (824 g·mol-1), and

113

Wlipid is the digestible lipid weight in the initial digestion system. The GIT model used in our

114

study is closely related to the standardized INFOGEST international consensus procedure

115

developed for in vitro digestion studies33, but was optimized for application to emulsions by our

116

group some years ago 32. This method was employed so that the results of this study could be

117

directly compared to our previous studies on related systems.

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Particle Characterization

119

The characteristics of the particles in the various systems were measured using static light

120

scattering and electrophoresis. A Mastersizer 3000 and Zetasizer NanoZS (Malvern,

121

Worcestershire, UK) were used to measure the particle size and charge, respectively. Before

122

analysis, the samples from the stomach phase were diluted with pH 2.5 phosphate buffer, while

123

the other samples were diluted with pH 7.0 phosphate buffer to avoid multiple scattering effects.

124

Confocal fluorescence microscopy (Nikon D-Eclipse C1 80i, USA) was performed to observe

125

the samples’ microstructures. Prior to observation, Nile Red (1 mg mL-1 ethanol) was added to

126

stain the lipid phase.

127

Vitamin Bioaccessibility

128

The mixed micelle phase was obtained by centrifuging (4°C, 41 657 g, 50 min) the digest

129

that remained after digestion of the samples within the small intestine. Vitamin bioaccessibility

130

was determined by measuring the level of vitamin E in the mixed micelle and digest phases 7 ACS Paragon Plus Environment


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utilizing high-performance liquid chromatography (HPLC, Agilent 1100, Agilent Technologies,

132

USA) with a C18 column (250 × 4.6 mm, 5 μm). Before HPLC analysis, the oil-soluble vitamin

133

was extracted from the samples using a hexane/ethanol mixture (1/1, v/v). Briefly, 3 mL

134

samples were mixed with the mixed organic solvent, and then centrifuged at 2500 g for 2 min to

135

obtain a supernatant layer. This extraction process was repeated three times. Afterward, the

136

supernatant layers were combined together and dried under nitrogen. Finally, the dried samples

137

were dissolved in methanol and filtered through a 0.45 μm filter before carrying out HPLC

138

analysis. The details of the HPLC analysis conditions have been reported in our previous

139

work.34 Briefly, a mixture of 95% methanol and 5% double distilled water was used as the

140

mobile phase. An isocratic elution running at 1.0 mL/min was carried out to separate the vitamin

141

E at a wavelength of 295 nm. Finally, the vitamin E bioaccessibility was calculated as follows:

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Bioaccessibility(%) =

𝐶𝑚𝑖𝑐𝑒𝑙𝑙𝑒 𝐶𝐷𝑖𝑔𝑒𝑠𝑡𝑎

× 100

143

Here, Cmicelle and CDigesta represent the vitamin concentrations in the mixed micelle fraction and

144

total digesta collected after the small intestine phase.

145

Statistical Analysis

146

All experiments were repeated at least two or three times and the mean and standard

147

deviation values were obtained. ANOVA analysis was employed to analyze the significant

148

difference at a significance level of 0.05.

149

Result and Discussion

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Impact of emulsifier type on emulsion formation

151 152

In these experiments, the influence of emulsifier type on the characteristics of the oil droplets produced using standardized homogenization conditions was measured (Figure 1). The 8 ACS Paragon Plus Environment

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emulsions formed using QS and WPI had much smaller droplets than those formed using GA.

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This may have been because of differences in the surface activity and adsorption kinetics of the

155

different emulsifiers. The ability of emulsifiers to reduce the interfacial tension is a critical

156

factor in the formation of fine droplets during microfluidization, because the breakup of oil

157

droplets is facilitated when the interfacial tension is reduced.35 As reported in our previous

158

study, QS and WPI reduced the interfacial tension more effectively than GA.19 Consequently,

159

they should generate finer oil droplets inside the homogenizer by promoting a higher degree of

160

droplet disruption.35 Moreover, the relatively small QS and WPI emulsifiers are likely to adsorb

161

to the lipid droplet surfaces more quickly than the relatively large GA emulsifiers, thereby

162

inhibiting droplet coalescence inside the homogenizer.

163

Under neutral pH conditions, the ζ-potential values for the droplets in all three emulsions

164

were strongly negative (Figure 1b), thereby creating a strong electrostatic repulsion between

165

them.34 As a result, all three emulsions were relatively stable to aggregation after they were

166

prepared.

167

Effect of pH on emulsion stability

168

The pH-stability of emulsions plays an important role in determining their application in

169

many foods and beverages. Consequently, the influence of pH on the electrical properties and

170

aggregation stability of the different emulsifier-coated oil droplets was studied. The mean

171

particle diameter of QS- and GA-emulsions did not change appreciably from pH 2 to 8 (Figure

172

2a), indicating they had relatively good pH-stability. Even so, there was a small increase in

173

particle size for the QS-emulsions at the most acidic condition used (pH 2), which suggests that

174

some particle aggregation occurred due to the reduction in droplet charge (Figure 2b).

175

Consequently, the electrostatic repulsive forces acting amongst the QS-coated droplets was



176

reduced, thereby leading to flocculation 29. Interestingly, the GA-emulsions did not exhibit any

177

change in particle size across the full pH range used, even though the droplet charge also

178

decreased notably under acidic conditions. This is because the droplets in GA-emulsions are

179

mainly prevented from aggregating by steric forces, rather than electrostatic ones.36 Unlike the

180

other two emulsions, the size of the particles in the WPI-emulsions was highly sensitive to pH,

181

being relatively large at pH 5 but small at lower (pH 2~4) and higher (pH 6~8) values. This

182

phenomenon is a result of changes in the electrostatic forces acting between the droplets as the

183

pH was varied 9. At relatively low and high pH values, there is a high net surface potential

184

associated with the droplets, leading to intense electrostatic repulsive forces. Conversely, around

185

the isoelectric point of the adsorbed proteins, the net surface potential on the droplets is fairly

186

low, thereby generating only a weak repulsion.

187

After storage, the appearance of the emulsions was consistent with the results of the light

188

scattering analysis. Briefly, no changes in appearance were observed in the QS- or GA-

189

emulsions over the pH range used, whereas droplet creaming was observed at pH 5 in the WPI-

190

emulsions (Figure 3). In particular, a droplet-enriched layer was seen at the top of the test tubes

191

and a droplet-depleted layer was observed at the bottom.

192

The QS- and GA-emulsions had fairly similar ζ-potential versus pH patterns, with the

193

surface potential decreasing from highly negative at pH 8 to slightly negative at pH 2 (Figure

194

2b). A fairly similar pH-dependence of the surface potential has previously been reported for

195

these two emulsifiers.20, 37 The ζ-potential of these systems became less negative below 4, which

196

is due to carboxylic acid protonation (-COOH) of the emulsifiers around their pKa values.20 QS

197

and GA have been reported to have pKa values around pH 3.2 and 2.2, respectively.38-39 As

198

mentioned earlier, the ζ-potential of the GA-coated lipid droplets was near zero at pH 2 but the


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droplets were stable to aggregation and gravitational separation (Figures 2a and 3). This is

200

because the large GA molecules create a thick interfacial coating around the droplets.15-16 Under

201

neutral conditions, the GA is highly charged and so the droplets are stabilized by a blend of steric

202

and electrostatic repulsive forces. Under highly acidic conditions, the GA loses most of its

203

charge but is still able to stabilize the droplets through a strong steric repulsion. This is not the

204

case for the QS-coated droplets, because the interfacial layer is too thin to generate a strong long-

205

range steric repulsion.

206

The ζ-potential of the WPI-emulsions went from strongly negative at neutral pH to strongly

207

positive at acidic pH, with a zero charge around pH 5 (Figure 2b). The instability of these

208

emulsions around pH 5 is because the protein layer is too thin to generate a strong steric

209

repulsion and the electrostatic repulsion is not strong enough to outweigh the van der Waals and

210

hydrophobic attractive forces. 40

211

Impact of emulsifier type on the storage stability

212

The impact of the three emulsifiers on the stability of the emulsions during storage at room

213

temperature was also studied (pH 7.0). The particle size of the QS- and WPI-emulsions did not

214

exhibit any significant changes throughout 28-days storage (Figure 4a). Moreover, no creaming

215

or flocculation was observed by visual inspection (Figure S1) or microscopy analysis (data not

216

shown), respectively. The good storage stability of emulsions formed using these emulsifiers has

217

also been reported previously.19, 34 This phenomenon is mainly due to the relatively small

218

dimensions of the oil droplets present in these emulsions, which made them more stable to

219

aggregation and creaming.41 Moreover, the ζ-potential of both these emulsions remained fairly

220

constant and highly negative throughout storage (Figure S2), suggesting there was little change

221

in interfacial composition during this period.



222

Interestingly, the GA-emulsions exhibited a small but noticeable rise in particle size during

223

storage (Figure 4a), even though the surface charge on the droplets did not change appreciably

224

(Figure S2). Moreover, some creaming was observed in these emulsions after 7 days storage

225

(Figure S1). Finally, confocal microscopy indicated that the extent of droplet aggregation in

226

these emulsions increased progressively during storage (Figure 4c). The level of GA used in our

227

study (1.5 wt.%) was relatively low and may therefore not have been sufficient to saturate the

228

surfaces of the lipid droplets. As a result, some coalescence may have occurred when two

229

partially covered oil droplets collided. Alternatively, a single GA molecule may have desorbed

230

from the surface of one droplet and then reattached itself to the surface of a different droplet,

231

leading to bridging flocculation.42 A schematic representation of this process is shown in Figure

232

4b.

233

In summary, the emulsions stabilized by the saponin and protein appeared to have good

234

storage stability, whereas those stabilized by the polysaccharide were prone to flocculation. In

235

practice, this problem would be overcome by increasing the GA level employed to produce the

236

original emulsions. However, in this study we wanted to compare emulsifiers at similar usage

237

levels.

238

Influence of emulsifier type on simulated gastrointestinal behavior

239

A static three-stage GIT model was utilized to investigate the influence of emulsifier type on

240

the gastrointestinal behavior of the emulsions. Each emulsion was subjected to a simulated

241

human gut by exposing it sequentially to artificial oral, gastric, and small intestinal conditions.

242

Alterations in the structural and physicochemical properties of the emulsions were determined

243

after being incubated in each GIT phase.


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Fresh emulsions

245

Emulsions stabilized by the three emulsifiers were produced by microfluidization as

246

described earlier. The QS- and WPI-emulsions contained smaller droplets than the GA-

247

emulsions (Figure 5a). All three systems had particle size distributions (PSDs) that were

248

monomodal (Figure S3) and contained oil droplets were uniformly distributed throughout them

249

(Figure 6). The fresh emulsions all had a relatively strong negative charge (Figure 5b),

250

accounting for their good stability. The origin of these differences was discussed in an earlier

251

section and so will not be repeated here.

252

Mouth stage

253

After being incubated in simulated saliva, the mean particle size of any of the emulsions did

254

not change significantly as determined by static light scattering (Figure 5a). The PSDs of all the

255

emulsions remained monomodal and similar to those of the initial emulsions (Figure S3).

256

Confocal microscopy analysis showed that widespread droplet aggregation did not occur under

257

artificial mouth conditions (Figure 6). Taken together, our data suggests that all emulsions

258

remained relatively resistant to flocculation and coalescence in the mouth, which is in agreement

259

with previous studies on emulsions stabilized by these emulsifiers.34, 43 Other studies, however,

260

have reported that appreciable levels of bridging and/or depletion flocculation can be induced in

261

artificial saliva for other types of emulsifiers.44-45 The relatively good stability of the emulsions

262

prepared in our study may have been due to various reasons. First, the electrostatic and steric

263

repulsive forces acting amongst the lipid droplets are sufficiently strong to inhibit their

264

aggregation. Second, the incubation time in the oral phase (2 min) used in our work was

265

relatively short compared to that used in many previous studies (10 min), so the mucin did not

266

have sufficient time to act on the lipid droplets.



267

The electrical properties of the emulsifier-coated oil droplets were altered appreciably after

268

encountering the simulated oral fluids (Figure 5b). The absolute value of the -potential

269

decreased appreciably on the QS-coated droplets, but only slightly on the WPI- and GA-coated

270

droplets. This reduction in negative charge could have occurred because of ion binding or

271

electrostatic screening effects linked to the existence of mucin or inorganic salts in the artificial

272

saliva.45-46

273

Gastric stage

274

There were obvious alterations in the dimensions and charge of the particles within the

275

emulsions after they had been incubated in the artificial gastric fluids for 2h (Figure 5). The

276

particle size of the GA-emulsions stayed fairly constant, that of the QS-emulsions increased

277

somewhat, and that of the WPI-emulsions increased appreciably (Figure 5a). Similarly, the PSD

278

of the GA-emulsions remained relatively unchanged, while those of the QS- and WPI-emulsions

279

shifted upward (Figure S3). These results indicate that the GA-coated droplets were highly

280

resistant to aggregation under simulated gastric conditions, the GS-coated droplets were

281

moderately resistant, and the WPI-coated droplets were strongly prone to aggregation. The light

282

scattering data were supported by the microscopy images, which indicated the QS- and GA-

283

emulsions had relatively good stability to droplet aggregation but the WPI-emulsions exhibited

284

severe droplet flocculation (Figure 6). Our results agree with prior work that has also shown

285

that QS-emulsions are relatively stable to aggregation in simulated stomach conditions, whereas

286

protein-stabilized emulsions are unstable.34, 43

287 288

The tendency for protein-coated droplets to aggregate under stomach conditions has been attributed to several mechanisms: (i) reduced electrostatic repulsion caused by pH changes; (ii)


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increased electrostatic screening caused by salts; (iii) proteolysis of absorbed proteins by pepsin;

290

and, (iv) bridging flocculation caused by mucin.46-49

291

After incubation within the gastric fluids, the ζ-potential values became slightly positive for

292

all the emulsions (Figure 5b). The strong acidity and high ionic strength of gastric fluids largely

293

account for this effect.34, 44 The low pH means the adsorbed emulsifiers should have a positive

294

or slightly negative charge (Figure 1b). The high salt concentration means the surface potential

295

will be reduced as a result of electrostatic screening. Moreover, the existence of anionic mucin

296

within the gastric fluids may have altered the surface potential by adsorbing to any positively

297

charged regions on the droplet surfaces, particularly for the protein-coated droplets.49 Moreover,

298

the partial digestion of the proteins adsorbed to the lipid droplet interfaces by pepsin may also

299

have altered the surface potential.6, 46, 50

300

Small intestine stage

301

After being incubated in the artificial small intestinal fluids, all of the digested emulsions

302

contained particles with fairly similar mean particle diameters and surface potentials (Figure 5)

303

as well as broad particle size distributions (Figure S2). Moreover, the confocal microscopy

304

images showed that they all contained relatively large lipid-rich particles (Figure 6). After

305

digestion, there are various kinds of molecular species in the gastrointestinal fluids that can

306

assemble into numerous types of colloidal particles. For instance, triacylglycerols, free fatty

307

acids, monoglycerides, bile salts, phospholipids, peptides, and undigested emulsifiers may be

308

present as lipid droplets, micelles, vesicles, liquid crystals, or calcium soaps.34, 44 This diversity

309

of particles accounts for the wide range of dimensions seen in the PSDs (Figure S2).



310 311

Influence of emulsifier on lipid digestion We hypothesize that the lipid digestion rate would impact the speed of mixed micelle

312

generation, as well as the kinetics of vitamin release from the lipid droplets, which would be

313

expected to affect the final vitamin bioaccessibility. The influence of emulsifier type on lipid

314

digestion was thus determined using the pH-stat method to establish FFA-time profiles for the

315

various emulsions (Figure 7). Generally, FFA generation occurred rapidly throughout the first

316

10 minutes of lipolysis but then more gradually at later times. The rapid generation of FFAs in

317

the initial period suggests that lipase quickly attached itself to the surfaces of the oil droplets and

318

then hydrolyzed the triacylglycerols.51 The slower release of FFAs at longer times was probably

319

because most of the triacylglycerols had already been hydrolyzed thereby making it harder for

320

the lipase to access the few remaining undigested triacylglycerols inside the droplet interiors.

321

The WPI- and GA-emulsions released very similar levels of FFAs throughout the entire

322

digestion process. But there were some distinct differences in the FFA-time relationships for the

323

QS-emulsions. First, a short-lag phase was observed before rapid lipid digestion occurred.

324

Second, the rate of lipid digestion in the initial stages was slower than for the WPI- and GA-

325

emulsions. Third, the level of FFAs generated by the end of the small intestinal stage was lower

326

than for the other two emulsions. This data suggests that the saponins slightly suppressed lipid

327

digestion. The hydrolysis of lipids involves a series of physicochemical processes that mainly

328

occur at the oil droplet surfaces and so interfacial phenomena are critical.22 The lag-phase,

329

slower digestion rate, and lower digestion extent for the QS-emulsions may therefore be

330

associated with the behavior of the saponins at the oil/water interface. As mentioned earlier, QS

331

reduces the interfacial tension more than WPI or GA, indicating that it is more surface-active. It

332

is therefore possible that the saponins formed a strong interfacial film at the surfaces of the oil


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droplets that inhibited the adsorption of the bile salts and/or lipase, thereby suppressing

334

digestion. Another possible reason is that the saponins formed electrostatic complexes with the

335

calcium ions in the gastrointestinal fluids. The highly anionic saponins could have bound

336

strongly to the cationic calcium ions thereby reducing the level of calcium ions available to

337

precipitate and remove long-chain FFAs from the lipid droplet surfaces.44

338

The faster digestion for the WPI- and GA-coated droplets is probably because the interfacial

339

layers formed by these emulsifiers were less effective at preventing the attachment of bile salts

340

and lipase to the surfaces of the oil droplets. The whey proteins are likely to have been partially

341

digested by pepsin in the stomach, thereby forming a relatively weak interfacial layer. The GA

342

molecules have a relatively low surface activity and would therefore be displaced more easily

343

from the surfaces of the oil droplets. Previous studies have also demonstrated that lipid digestion

344

depends on the characteristics of the emulsifiers used to coat the oil droplets.52-53

345

Influence of emulsifier on vitamin bioaccessibility

346

Finally, the influence of emulsifier type on the bioaccessibility of the encapsulated vitamin

347

was evaluated after the emulsions were incubated in simulated small intestine conditions. The

348

WPI-emulsions led to the highest bioaccessibility, whereas the QS- and GA-emulsions had fairly

349

similar bioaccessibilities (Figure 8). Nevertheless, in all cases, the measured bioaccessibility

350

was relatively high (65-85%).

351

In general, the bioaccessibility of lipophilic bioactives depends on the fraction of digested

352

triacylglycerols and the absolute amount of FFAs generated during lipid digestion. Lipophilic

353

bioactives are typically located in the interior of the lipid droplets and so the surrounding

354

triacylglycerols have to be digested before they can be released. Moreover, once they are

355

released from the droplets they have to be incorporated into the hydrophobic domains within the



356

mixed micelles, otherwise they will simply precipitate or form a separate layer. In our study, we

357

did not find a strong correlation between the final level of lipid digestion and vitamin

358

bioaccessibility. The highest level of FFAs generated was for both the WPI- and the GA-

359

emulsions (Figure 7), but the highest bioaccessibility was only for the WPI-emulsions (Figure

360

8).

361

This phenomenon cannot be simply explained, as lipid digestion and bioactive solubilization

362

are complex processes influenced by multiple factors. It is possible that the lipids were digested

363

in the GA-emulsions, but for some reason, the vitamin was not fully solubilized in the mixed

364

micelles or the mixed micelles that were formed precipitate. It is known that polysaccharides

365

can also interact with various digestive components, including lipase, bile acids, and calcium

366

ions.54 Therefore, the GA may interact with bile salts and/or FFAs, thereby reducing the

367

incorporation of vitamin E into the mixed micelles or causing the vitamin-enriched mixed

368

micelles to precipitate and so not be measured. These results suggest that the bioaccessibility of

369

lipophilic bioactive compounds is not only influenced by the final amount of lipid digestion

370

products generated but also by other factors. Clearly, further studies are required to better

371

understand this complex phenomenon.

372

It is interesting to compare the in vitro bioaccessibility of vitamin E determined in this study

373

(65-85%) to its reported in vivo bioavailability. A recent feeding study using rats reported that

374

the relative bioavailability of vitamin E (tocopherol) delivered in palm oil-in-water emulsions

375

was around 82.5% 55, which is in reasonable agreement with our bioaccessibility data.

376

In summary, emulsion-based delivery systems were fabricated from two plant-derived

377

emulsifiers (quillaja saponin and gum arabic) and one animal-derived emulsifier (whey protein

378

isolate). QS and WPI were more effective at producing emulsions containing small oil droplets,


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presumably due to the relatively high surface activity and rapid adsorption kinetics of these

380

emulsifiers. The good storage stability of these emulsions was linked to their relatively small

381

droplet size and the ability of the emulsifiers to generate strong repulsive interactions between

382

the droplets: mainly electrostatic for QS and WPI, and steric for GA. All the emulsions were

383

relatively stable to flocculation from pH 2 to 8, with the exception of the WPI-emulsions at pH 5,

384

which was linked to the reduction in electrostatic repulsion around the isoelectric point of the

385

protein. The WPI- and GA-emulsions had fairly similar digestion profiles, whereas the QS-

386

emulsions were digested more slowly. This phenomenon was linked to the ability of the

387

saponins to adsorb strongly to the lipid droplet surfaces, thereby inhibiting the attachment of the

388

bile salts and/or lipase. Interestingly, we did not find a strong correlation between the final level

389

of lipid digestion that had occurred and the vitamin bioaccessibility. The WPI-emulsions gave a

390

significantly higher bioaccessibility of the vitamin E than the other two emulsions. Our results

391

show that emulsifier type impacts the gastrointestinal fate of emulsions, which may have

392

important consequences for designing more effective vitamin-enriched delivery systems.

393

However, further studies are still needed to verify that the results obtained using in vitro

394

screening methods are translatable to industrial practice. In particular, the vitamin-fortified

395

delivery systems will have to be robust enough to survive during food production, storage, and

396

utilization and animal/human feeding studies are required to confirm the results of the simulated

397

GIT studies.

398

Acknowledgments

399

Shanshan Lv would like to thank the Chinese Scholarship Council for support.

400

Supporting Information: Additional Figures

401

Notes: The authors declare no competing financial interest. 19 ACS Paragon Plus Environment


402

References

403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446

1. Alqahtani, S.; Simon, L.; Astete, C. E.; Alayoubi, A.; Sylvester, P. W.; Nazzal, S.; Shen, Y.; Xu, Z.; Kaddoumi, A.; Sabliov, C. M., Cellular uptake, antioxidant and antiproliferative activity of entrapped αtocopherol and γ-tocotrienol in poly (lactic-co-glycolic) acid (PLGA) and chitosan covered PLGA nanoparticles (PLGA-Chi). Journal of Colloid and Interface Science 2015, 445, 243-251. 2. Sharif, H. R.; Sharif, M. K.; Zhong, F., Preparation, characterization and rheological properties of vitamin E enriched nanoemulsion. Pakistan Journal of Food Sciences 2017, 27 (1), 7-14. 3. Song, Y.-B.; Lee, J.-S.; Lee, H. G., α-Tocopherol-loaded Ca-pectinate microcapsules: Optimization, in vitro release, and bioavailability. Colloids and Surfaces B: Biointerfaces 2009, 73 (2), 394-398. 4. Schneider, C., Chemistry and biology of vitamin E. Molecular Nutrition & Food Research 2005, 49 (1), 7-30. 5. Laouini, A.; Fessi, H.; Charcosset, C., Membrane emulsification: A promising alternative for vitamin E encapsulation within nano-emulsion. Journal of Membrane Science 2012, 423-424, 85-96. 6. Parthasarathi, S.; Muthukumar, S. P.; Anandharamakrishnan, C., The influence of droplet size on the stability, in vivo digestion, and oral bioavailability of vitamin E emulsions. Food & Function 2016, 7 (5), 2294-2302. 7. Hategekimana, J.; Zhong, F., Degradation of vitamin E in nanoemulsions during storage as affected by temperature, light and darkness. International journal of food engineering 2015, 11 (2), 199206. 8. Huang, Q.; Yu, H.; Ru, Q., Bioavailability and delivery of nutraceuticals using nanotechnology. Journal of food science 2010, 75 (1), R50-R57. 9. McClements, D. J.; Gumus, C. E., Natural emulsifiers — Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Advances in Colloid and Interface Science 2016, 234, 3-26. 10. Can Karaca, A.; Low, N. H.; Nickerson, M. T., Potential use of plant proteins in the microencapsulation of lipophilic materials in foods. Trends in Food Science & Technology 2015, 42 (1), 512. 11. Cheung, L.; Wanasundara, J.; Nickerson, M. T., Effect of pH and NaCl on the Emulsifying Properties of a Napin Protein Isolate. Food Biophysics 2015, 10 (1), 30-38. 12. Kezwon, A.; Wojciechowski, K., Interaction of Quillaja bark saponins with food-relevant proteins. Advances in colloid and interface science 2014, 209, 185-195. 13. Pedebos, C.; Pol-Fachin, L.; Pons, R.; Teixeira, C. V.; Verli, H., Atomic model and micelle dynamics of QS-21 saponin. Molecules 2014, 19 (3), 3744-3760. 14. Salih, N. K. M., Applications of Gum Arabic in Medical and Health Benefits. In Gum Arabic, Mariod, A. A., Ed. Academic Press: 2018; pp 269-281. 15. Gulão, E. d. S.; de Souza, C. J. F.; Andrade, C. T.; Garcia-Rojas, E. E., Complex coacervates obtained from peptide leucine and gum arabic: Formation and characterization. Food Chemistry 2016, 194, 680-686. 16. Tan, C.-T., Beverage emulsions. Food Scinece and Technology-New York-Marcel Dekker- 2004, 485-524. 17. Sah, B.; McAinch, A.; Vasiljevic, T., Modulation of bovine whey protein digestion in gastrointestinal tract: A comprehensive review. International dairy journal 2016, 62, 10-18. 18. de Castro, R. J. S.; Domingues, M. A. F.; Ohara, A.; Okuro, P. K.; dos Santos, J. G.; Brexó, R. P.; Sato, H. H., Whey protein as a key component in food systems: Physicochemical properties, production technologies and applications. Food Structure 2017, 14, 17-29.


Page 20 of 34

Page 21 of 34

447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492


19. Bai, L.; Huan, S.; Gu, J.; McClements, D. J., Fabrication of oil-in-water nanoemulsions by dualchannel microfluidization using natural emulsifiers: Saponins, phospholipids, proteins, and polysaccharides. Food Hydrocolloids 2016, 61, 703-711. 20. Ozturk, B.; Argin, S.; Ozilgen, M.; McClements, D. J., Formation and stabilization of nanoemulsion-based vitamin E delivery systems using natural surfactants: Quillaja saponin and lecithin. Journal of Food Engineering 2014, 142, 57-63. 21. Li, Z.; Dai, L.; Wang, D.; Mao, L.; Gao, Y., Stabilization and Rheology of Concentrated Emulsions Using the Natural Emulsifiers Quillaja Saponins and Rhamnolipids. Journal of Agricultural and Food Chemistry 2018, 66 (15), 3922-3929. 22. Yao, X.; Nie, K.; Chen, Y.; Jiang, F.; Kuang, Y.; Yan, H.; Fang, Y.; Yang, H.; Nishinari, K.; Phillips, G. O., The influence of non-ionic surfactant on lipid digestion of gum Arabic stabilized oil-in-water emulsion. Food Hydrocolloids 2018, 74, 78-86. 23. Kopec, R. E.; Failla, M. L., Recent advances in the bioaccessibility and bioavailability of carotenoids and effects of other dietary lipophiles. Journal of Food Composition and Analysis 2018, 68, 16-30. 24. Ozturk, B., Nanoemulsions for food fortification with lipophilic vitamins: Production challenges, stability, and bioavailability. European Journal of Lipid Science and Technology 2017, 119 (7). 25. Salvia-Trujillo, L.; Fumiaki, B.; Park, Y.; McClements, D. J., The influence of lipid droplet size on the oral bioavailability of vitamin D-2 encapsulated in emulsions: an in vitro and in vivo study. Food & Function 2017, 8 (2), 767-777. 26. Yang, Y.; Xiao, H.; McClements, D. J., Impact of Lipid Phase on the Bioavailability of Vitamin E in Emulsion-Based Delivery Systems: Relative Importance of Bioaccessibility, Absorption, and Transformation. Journal of Agricultural and Food Chemistry 2017, 65 (19), 3946-3955. 27. Salvia-Trujillo, L.; Verkempinck, S. H. E.; Sun, L.; Van Loey, A. M.; Grauwet, T.; Hendrickx, M. E., Lipid digestion, micelle formation and carotenoid bioaccessibility kinetics: Influence of emulsion droplet size. Food Chemistry 2017, 229, 653-662. 28. Salvia-Trujillo, L.; Verkempinck, S. H. E.; Zhang, X.; Van Loey, A. M.; Grauwet, T.; Hendrickx, M. E., Comparative study on lipid digestion and carotenoid bioaccessibility of emulsions, nanoemulsions and vegetable-based in situ emulsions. Food Hydrocolloids 2019, 87, 119-128. 29. Yang, Y.; McClements, D. J., Encapsulation of vitamin E in edible emulsions fabricated using a natural surfactant. Food Hydrocolloids 2013, 30 (2), 712-720. 30. Lv, S. S.; Gu, J. Y.; Zhang, R. J.; Zhang, Y. H.; Tan, H. Y.; McClements, D. J., Vitamin E Encapsulation in Plant-Based Nanoemulsions Fabricated Using Dual-Channel Microfluidization: Formation, Stability, and Bioaccessibility. Journal of Agricultural and Food Chemistry 2018, 66 (40), 10532-10542. 31. Ozturk, B.; Argin, S.; Ozilgen, M.; McClements, D. J., Formation and stabilization of nanoemulsion-based vitamin E delivery systems using natural biopolymers: Whey protein isolate and gum arabic. Food Chemistry 2015, 188, 256-263. 32. Li, Y.; Hu, M.; McClements, D. J., Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pH-stat method. Food Chemistry 2011, 126 (2), 498-505. 33. Minekus, M.; Alminger, M.; Alvito, P.; Ballance, S.; Bohn, T.; Bourlieu, C.; Carriere, F.; Boutrou, R.; Corredig, M.; Dupont, D.; Dufour, C.; Egger, L.; Golding, M.; Karakaya, S.; Kirkhus, B.; Le Feunteun, S.; Lesmes, U.; Macierzanka, A.; Mackie, A.; Marze, S.; McClements, D. J.; Menard, O.; Recio, I.; Santos, C. N.; Singh, R. P.; Vegarud, G. E.; Wickham, M. S. J.; Weitschies, W.; Brodkorb, A., A standardised static in vitro digestion method suitable for food - an international consensus. Food & Function 2014, 5 (6), 11131124.



493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539

34. Lv, S.; Gu, J.; Zhang, R.; Zhang, Y.; Tan, H.; McClements, D. J., Vitamin E Encapsulation in PlantBased Nanoemulsions Fabricated Using Dual-Channel Microfluidization: Formation, Stability, and Bioaccessibility. Journal of Agricultural and Food Chemistry 2018, 66 (40), 10532-10542. 35. Amine, C.; Dreher, J.; Helgason, T.; Tadros, T., Investigation of emulsifying properties and emulsion stability of plant and milk proteins using interfacial tension and interfacial elasticity. Food Hydrocolloids 2014, 39, 180-186. 36. Charoen, R.; Jangchud, A.; Jangchud, K.; Harnsilawat, T.; Naivikul, O.; McClements, D. J., Influence of Biopolymer Emulsifier Type on Formation and Stability of Rice Bran Oil-in-Water Emulsions: Whey Protein, Gum Arabic, and Modified Starch. Journal of Food Science 2011, 76 (1), E165-E172. 37. Nakauma, M.; Funami, T.; Noda, S.; Ishihara, S.; Al-Assaf, S.; Nishinari, K.; Phillips, G. O., Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids 2008, 22 (7), 1254-1267. 38. Busu, N. M.; Amonsou, E. O., Fractionation pH of bambara groundnut (Vigna subterranea) protein impacts the degree of complexation with gum arabic. Food Hydrocolloids 2019, 87, 653-660. 39. Mitra, S.; Dungan, S. R., Micellar properties of Quillaja saponin. 1. Effects of temperature, salt, and pH on solution properties. Journal of Agricultural and Food Chemistry 1997, 45 (5), 1587-1595. 40. Teo, A.; Goh, K. K. T.; Wen, J.; Oey, I.; Ko, S.; Kwak, H.-S.; Lee, S. J., Physicochemical properties of whey protein, lactoferrin and Tween 20 stabilised nanoemulsions: Effect of temperature, pH and salt. Food Chemistry 2016, 197, 297-306. 41. Xu, J.; Mukherjee, D.; Chang, S. K. C., Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization. Food Chemistry 2018, 240, 1005-1013. 42. Niu, F.; Zhou, J.; Niu, D.; Wang, C.; Liu, Y.; Su, Y.; Yang, Y., Synergistic effects of ovalbumin/gum arabic complexes on the stability of emulsions exposed to environmental stress. Food Hydrocolloids 2015, 47, 14-20. 43. Yang, Y.; McClements, D. J., Vitamin E bioaccessibility: Influence of carrier oil type on digestion and release of emulsified α-tocopherol acetate. Food Chemistry 2013, 141 (1), 473-481. 44. Zhang, R.; Zhang, Z.; Zhang, H.; Decker, E. A.; McClements, D. J., Influence of emulsifier type on gastrointestinal fate of oil-in-water emulsions containing anionic dietary fiber (pectin). Food Hydrocolloids 2015, 45, 175-185. 45. Sarkar, A.; Goh, K. K. T.; Singh, H., Colloidal stability and interactions of milk-protein-stabilized emulsions in an artificial saliva. Food Hydrocolloids 2009, 23 (5), 1270-1278. 46. Singh, H.; Sarkar, A., Behaviour of protein-stabilised emulsions under various physiological conditions. Advances in Colloid and Interface Science 2011, 165 (1), 47-57. 47. Sarkar, A.; Goh, K. K. T.; Singh, H., Properties of oil-in-water emulsions stabilized by βlactoglobulin in simulated gastric fluid as influenced by ionic strength and presence of mucin. Food Hydrocolloids 2010, 24 (5), 534-541. 48. Li, J.; Ye, A.; Lee, S. J.; Singh, H., Influence of gastric digestive reaction on subsequent in vitro intestinal digestion of sodium caseinate-stabilized emulsions. Food & Function 2012, 3 (3), 320-326. 49. Scheuble, N.; Schaffner, J.; Schumacher, M.; Windhab, E. J.; Liu, D.; Parker, H.; Steingoetter, A.; Fischer, P., Tailoring Emulsions for Controlled Lipid Release: Establishing in vitro–in Vivo Correlation for Digestion of Lipids. ACS Applied Materials & Interfaces 2018, 10 (21), 17571-17581. 50. Sarkar, A.; Goh, K. K. T.; Singh, R. P.; Singh, H., Behaviour of an oil-in-water emulsion stabilized by β-lactoglobulin in an in vitro gastric model. Food Hydrocolloids 2009, 23 (6), 1563-1569. 51. McClements, D. J.; Li, Y., Review of in vitro digestion models for rapid screening of emulsionbased systems. Food & Function 2010, 1 (1), 32-59. 22 ACS Paragon Plus Environment

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540 541 542 543 544 545 546 547 548 549 550 551


52. Verkempinck, S. H. E.; Salvia-Trujillo, L.; Moens, L. G.; Charleer, L.; Van Loey, A. M.; Hendrickx, M. E.; Grauwet, T., Emulsion stability during gastrointestinal conditions effects lipid digestion kinetics. Food Chemistry 2018, 246, 179-191. 53. Speranza, A.; Corradini, M. G.; Hartman, T. G.; Ribnicky, D.; Oren, A.; Rogers, M. A., Influence of Emulsifier Structure on Lipid Bioaccessibility in Oil–Water Nanoemulsions. Journal of Agricultural and Food Chemistry 2013, 61 (26), 6505-6515. 54. Verkempinck, S. H. E.; Salvia-Trujillo, L.; Denis, S.; Van Loey, A. M.; Hendrickx, M. E.; Grauwet, T., Pectin influences the kinetics of in vitro lipid digestion in oil-in-water emulsions. Food Chemistry 2018, 262, 150-161. 55. Harlen, W. C.; Muchtadi, T.; Palupi, N. S., Bioavailability of alpha-Tocopherol in Palm Oil Emulsion Drink on Rats (Rattus norvegicus) Blood Plasma and Liver. Agritech-Jurnal Teknologi Pertanian 2017, 37 (3), 352-361.

552 553

Funding

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This material was partly based upon work supported by the National Institute of Food and

555

Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00491) and USDA,

556

AFRI Grants (2016-25147, and 2016-08782).

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Figure Captions Figure 1. Impact of emulsifier type on the (a) mean particle diameter (d3,2) and (b) surface potential of oil-in-water emulsions (pH 7.0). Samples denoted with low case letter (a, b, c) were significantly different (p < 0.05). All emulsions were produced by microfluidization at 12,000 psi for 3 passes. Figure 2. Impact of pH on the (a) mean particle diameter (d3,2), and (b) particle charge of emulsions stabilized by different emulsifiers. Figure 3. Impact of pH on the visual appearances of emulsions stabilized by different emulsifiers: (a) QS, (b) WPI, and (d) GA. A schematic representation of the aggregation state of the droplets in the WPI-emulsions at different pH values is shown in (c). Figure 4. Storage stability of emulsions (pH 7): (a) particle size versus time; (b) schematic representation of changes in flocculation during storage; (c) confocal fluorescence microscopy images of GA-emulsions during storage. Figure 5. Impact of emulsifier type on (a) mean particle diameter (d3,2), (b) particle charge of emulsions after exposure to different simulated GIT stages. Samples denoted with different capital letters (A, B, C, D) were significantly different (p < 0.05) when compared with different digestion phase; samples denoted with different low case letters (a, b, c) were significantly different (p < 0.05) when compared at the same digestion phase. Figure 6. Microstructure of emulsions stabilized by different emulsifiers after digested in different simulated GIT stages (a) QS; (b) WPI; (c) GA. Figure 7. Free fatty acids (FFAs) released from emulsions stabilized by different emulsifiers during in vitro small intestinal digestion.


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Figure 8. Impact of emulsifier type on the bioaccessibility of vitamin E determined by measuring the fraction of the vitamin solubilized in the micelle phase after digestion. Samples denoted with low case letters (a, b) were significantly different (p < 0.05).



Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Figure 6



Figure 7


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Figure 8



Graphic for table of contents


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Vitamin E Encapsulation within Oil-in-Water Emulsions: Impact of

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