Modulation of Lipid Digestion Profiles Using Filled Egg White Protein

Subscriber access provided by UOW Library

Article

Modulation of lipid digestion profiles using filled egg white protein microgels Luping Gu, YUJIE SU, Zipei Zhang, Bingjing Zheng, Ruojie Zhang, David J McClements, and Yan-Jun Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02674 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 26, 2017

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

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

Page 1 of 43

Journal of Agricultural and Food Chemistry

Modulation of lipid digestion profiles using filled egg white protein microgels Luping Guab, Yujie Sua, Zipei Zhangb, Bingjing Zhengb, Ruojie Zhangb, David Julian McClementsb*, Yanjun Yanga*

a

Key Laboratory of Food Science and Technology, School of Food Science and Technology,

Jiangnan University, Wuxi 214122, People’s Republic of China b

Department of Food Science, University of Massachusetts, Amherst, MA 01003, United States

*Corresponding author: Email address: [email protected]; [email protected]

ACS Paragon Plus Environment

1


1

Page 2 of 43

Abstract

2

Colloidal delivery systems are required to encapsulate, protect, and release active food

3

ingredients, such as vitamins, nutraceuticals, and minerals. In this study, lipid droplets were

4

encapsulated within biopolymer microgels fabricated from egg white proteins using an injection-

5

gelation process. Confocal fluorescence microscopy indicated that lipid droplets were dispersed

6

within a network of crosslinked proteins within the microgels. The properties of the lipid-loaded

7

microgels were compared to those of simple oil-in-water emulsions stabilized by egg white

8

proteins. Light scattering and microscopy measurements indicated both delivery systems

9

exhibited good stability under acid conditions (pH 3 to 5), but aggregated at higher pH values

10

due to a reduction in electrostatic repulsion. Simulated gastrointestinal tract studies indicated that

11

lipid droplets encapsulated within protein microgels were digested more slowly than free lipid

12

droplets. Our results therefore suggest that egg white protein microgels may be useful for

13

encapsulation and controlled release of hydrophobic bioactive agents.

14

Keywords: egg white proteins; emulsions; microgels; hydrogel beads; digestion; controlled

15

release


2

Page 3 of 43

16


Introduction

17

Bioactive agents are substances that exhibit anti-inflammatory, antioxidant, antibacterial,

18

antiviral, anticancer and/or tissue regenerative activity. There has been increasing interest in the

19

encapsulation of bioactive agents in various fields, including the pharmaceutical, food,

20

agrochemical, and cosmetics industries 1. Encapsulation is being utilized to improve the

21

dispersibility, stability, bioactivity, release profile, and safety of bioactive substances

22

Numerous different kinds of colloidal delivery systems have been designed to encapsulate

23

bioactive agents, including microemulsions, nanoemulsions, emulsions, liposomes, solid lipid

24

nanoparticles, biopolymer nanoparticles, and microgels

25

particularly suitable for the encapsulation, protection, and release of lipophilic bioactive agents

26

10-12

27

have limited scope to control the retention and release of encapsulated bioactive components

28

because of the relatively small size and large specific surface area of the droplets they contain 13.

29

Consequently, new forms of delivery systems are required to more precisely control the retention

30

and release profiles of encapsulated components 14.

31

6-9

2-5

.

. Emulsion-based systems are

. However, one of the current limitations of using emulsions for this purpose is that they only

Recently, there has been interest in the design and fabrication of biopolymer microgels as a 15-17

32

means of extending the functional attributes of emulsion-based delivery systems

33

Biopolymer microgels consist of microscopic soft particles containing a network of cross-linked

34

biopolymer molecules inside 15. They typically contain a relatively large amount of water trapped

35

through capillary forces (> 80%), which distinguishes them from biopolymer nanoparticles.

36

Lipid droplets can be encapsulated within the interior of the biopolymer microgels, which

37

enables one to modulate their stability, retention and release behavior by varying the pore size,

38

dimensions, and network characteristics of the microgels. For example, the release of


.

3


Page 4 of 43

39

encapsulated components can be retarded by decreasing the pore size, increasing the microgel

40

dimensions, or ensuring that there are strong attractive interactions with the biopolymer network

41

18

42

advantage for the development of more “label-friendly” consumer products

43

microgels are most commonly formed from food-grade polysaccharides, such as alginate,

44

chitosan and carrageenan

45

proteins can also be used 24-27.

. Biopolymer microgels are typically assembled from proteins or polysaccharides, which is an

20-23

19

. Biopolymer

, and/or food-grade proteins, such as milk, meat, fish, and plant

46

There have been a number of previous studies on the application of protein microgels as

47

delivery systems for pharmaceuticals or nutraceuticals. For instance, riboflavin has been

48

encapsulated in microgels assembled from whey protein

49

has been encapsulated in protein microgels formed from a mixture of β-lactoglobulin and

50

lysozyme

51

gliadin or zein

52

33, 34

53

microgels formed from egg white proteins.

30

28

, soy protein, and zein

29

. Vitamin D

. Resveratrol has been encapsulated in protein microgels fabricated from either 31, 32

. Zein microgels have also been used to encapsulate curcumin and quercetin

. In the current study, we examine the possibility of encapsulating lipid droplets within

54

Egg white proteins are common food ingredients that are widely used in food products due

55

to their high nutritional value and excellent functional properties 35. Egg white contains a mixture

56

of globular proteins, such as ovalbumin, ovotransferrin and lysozyme 36. These globular proteins

57

unfold when they are heated above their thermal denaturation temperature (around 70-85 °C),

58

which exposes non-polar and sulfhydryl-containing amino acids. The exposure of these reactive

59

amino acids may lead to protein aggregation and cross-linking due to attractive hydrophobic

60

interactions and disulfide bond formation. At sufficiently high protein levels, the cross-linking of

61

the protein molecules leads to the formation of a strong viscoelastic hydrogel. Compared to whey


4

Page 5 of 43


62

and soy proteins, a lower temperature is typically required to denature and cross-link egg white

63

proteins. Moreover, egg white proteins are also effective emulsifiers that can be used to form and

64

stabilize oil-in-water emulsions

65

properties of egg white proteins enables the fabrication of lipid-droplet loaded biopolymer

66

microgels, which could be used as delivery system for lipophilic bioactive agents.

37

. As a consequence, the good gelling and emulsifying

67

Various approaches are available to encapsulate lipophilic bioactive agents into protein

68

microgels including injection methods, templating methods, phase separation methods,

69

antisolvent precipitation, and particle reduction methods

70

biopolymer microgels were formed using an injection-gelation method that involved a number of

71

steps. First, an oil-in-water emulsion was formed by homogenizing oil, water, and egg white

72

protein together using a high-pressure homogenizer. Second, this emulsion was injected into a

73

hot oil phase using an encapsulation unit to crosslink the egg white proteins and form lipid-

74

loaded microgels. The stability and potential gastrointestinal fate of the lipid-loaded microgels

75

were compared to those of simple oil-in-water emulsions stabilized by egg white protein.

76

Materials and Methods

77

Materials

18

. In the present study, lipid-loaded

78

Hen eggs and sunflower oil were purchased from a local supermarket. Polyglycerol

79

polyricinoleate ester (PGPR) was obtained from Palsgaard USA (Morristown, NJ, USA). Nile

80

red, fluorescein isothiocyanate (FITC), n-hexane, mucin from porcine stomach, pepsin from

81

porcine gastric mucosa, lipase from porcine pancreas, and porcine bile extract were purchased

82

from Sigma Chemical Company (St. Louis, MO, USA). All other chemicals and reagents used in

83

this study were of analytical grade. Double distilled water was used to prepare all aqueous

84

solutions.


5


85

Sample preparation

86

Preparation of Egg White Proteins

87

Page 6 of 43

The egg white proteins were extracted from fresh eggs by an isoelectric precipitation 37

88

method, which has been described in detail in our previous study

. The protein content was

89

determined using the Kjeldahl method (10%, w/v) and the determination of proteins composition

90

showed that ovomucoid was removed from the solutions by isoelectric precipitation, leaving

91

lysozyme, ovalbumin, and ovotransferrin. A major reason for removing the insoluble matter from

92

the protein solutions was to prevent the homogenizer and encapsulation devices from becoming

93

blocked.

94

Preparation of Emulsions

95

The oil-in-water emulsions were fabricated by homogenizing 80% of aqueous phase (10%

96

egg white proteins, pH 3.0) with 20% of oil phase (sunflower oil). This pH was selected for

97

aqueous phase because it was found to be the optimum one for forming stable emulsions.

98

Initially, the aqueous and oil phases were mixed together using a high-speed blender for 2 min

99

(M133/1281-0, Biospec Products, Inc., ESGC, Switzerland). This led to the formation of some

100

foam on top of the coarse emulsions, which was discarded because it would no pass through the

101

homogenizer. The coarse emulsions were then further homogenized using a two-stage high-

102

pressure valve homogenizer (15MR-8TA, APV Gaulin Inc., Wilmington, MA, USA) for three

103

cycles. The operating pressures of first and second stages of the homogenizer were 4500 psi and

104

500 psi, respectively. The resulting emulsions were then naturally cooled down to room

105

temperature.

106

Preparation of Microgel Beads

107

Egg white protein microgels were fabricated using a commercial extrusion device


6

Page 7 of 43


108

(Encapsulator B-390, BUCHI, Switzerland). Initially, sunflower oil containing an oil-soluble

109

surfactant-PGPR (4%, w/w) was heated to 90 °C as an external oil phase. Subsequently, oil-in-

110

water emulsions were extruded through a 120 µm nozzle into the external oil phase with

111

continuous stirring. The emulsion droplets suspended in oil phase were then kept at 90 °C for 30

112

min to promote protein gelation and microgel formation 38. After that, the microgel beads formed

113

were left in the oil phase at room temperature for 24 h. The hardened microgels were then

114

collected by vacuum filtration and washed with n-hexane and distilled water to remove any

115

excess external oil phase or PGPR from their surfaces. Finally, the dried microgels were placed

116

at 4 °C before further analysis. It was anticipated that most of the lipophilic PGPR and external

117

oil phase would be removed from the microgels during the solvent washing process, but this

118

should be confirmed in future studies.

119

Determination of Particle Size and ζ-potential

120

The mean particle size of samples and particle size distributions were measured using a laser

121

light

scattering

instrument

(Mastersizer

2000,

Malvern

Instruments

Ltd.,

Malvern,

122

Worcestershire, UK). Prior to measurements, samples were diluted with pH-adjusted phosphate

123

buffer (5 mM, pH 3~8) to minimize multiple scattering effects and stirred continuously during

124

analysis to ensure the systems were homogeneous. The refractive index values used in the

125

calculations were 1.45 and 1.33 for oil phase and aqueous phase, respectively. ζ-potential was

126

tested by a particle electrophoresis device (Zetasizer Nano, Malvern Instruments, Worcestershire,

127

UK).

128

In Vitro Digestion

129

Before digestion, samples were diluted with phosphate buffer (5 mM, pH 3.0) to obtain the

130

same initial lipid content (0.2 g) and total weight (7.5 g), and then were subjected to a simulated


7


Page 8 of 43

131

gastrointestinal tract (GIT) model, which consists of mouth, stomach and small intestine phases.

132

Since the GIT mode used has been described previously in detail by our research group 39, it was

133

briefly summarized in the present study. The compositions of the simulated GIT fluids used are

134

listed in Table 1 40, 41.

135

Briefly, samples and simulated saliva fluid containing mucin were preheated to 37 °C before

136

use. The digestion started by mixing samples (7.5 g) and saliva (7.5 g) and incubating for 2 min

137

at 37 °C. Then pepsin solution (15 g) was added to the mixture and the value of pH was adjusted

138

to 2.5. Subsequently, the mixture was incubated for 2 h at 37 °C. Finally, simulated intestine

139

fluid (1.5 mL), bile salts (3.5 mL) and lipase (2.5 mL) were added to the mixture and incubated

140

for 2 h at 37 °C. During incubation, the pH was maintained at 7.0.

141

Microstructural Analysis

142

Microstructural analysis of the different samples was performed using optical microscopy

143

(Nikon Eclipse E400, Nikon Corp., Japan). Samples were diluted with phosphate buffer in a

144

glass test tube. For emulsions, a drop of diluted sample was placed on a microscope slide and

145

then covered with a thin glass slip. For microgels, a drop of diluted sample was placed on the

146

microscope slide and then analyzed without a cover slip, since microgels were relatively soft and

147

easily deformed. The microstructural images were then captured using digital image processing

148

software (Micro Video Instruments Inc., Avon, MA, USA). Samples were observed at a

149

magnification of ×600 (×60 objective lens and ×10 eyepiece lens).

150

The confocal fluorescence microscopy images of samples were acquired using a Nikon

151

confocal microscope (Nikon D-Eclipse C1 80i, Nikon, Melville, NY, USA). To observe the

152

internal structure of microgel beads, a freezing microtome (Cryostar NX70, Thermo Electron

153

Corporation, MA, USA) was used to cut them into thin slices. The temperature and section


8

Page 9 of 43


154

thickness were set to -21 °C and 5 µm, respectively. Prior to confocal microscopy observation,

155

samples were dyed with Nile red and FITC solution to obtain red (lipids) and green (protein)

156

fluorescence images, respectively. The image analysis software (NIS-Elements, Nikon, Melville,

157

NY) was used to analyze the resulting confocal microscopy of samples.

158

Statistical Analysis

159

Each measurement was conducted in triplicate. One-way variance analysis was performed

160

using SPSS 19.0 package and Duncan’s Multiple Range Test (p < 0.05) was used to detect

161

significant difference between mean values.

162

Results and Discussion

163

Formation of Emulsions and Microgel Beads

164

In the present study, two delivery vehicles were designed: (i) emulsions and (ii) microgels. A

165

schematic overview of the procedure used to prepare the lipid-droplet loaded microgels is shown

166

in Fig. 1.

167

Initially, oil-in-water emulsions were produced through homogenization of 20% oil phase

168

(sunflower oil) and 80% aqueous phase (10% egg white proteins, pH 3.0). The mean particle size

169

of fresh emulsions was 339 nm (Table 2) and particle size distribution was monomodal, i.e., it

170

contained a single peak (Figs. 2a and 4). Preliminary experiments indicated that the pH of

171

aqueous phase during homogenization was a key factor in determining stability of emulsions

172

formed. When emulsions were prepared in the pH range 5.0 to 8.0, rapid phase separation was

173

observed immediately after homogenization. The most likely reason for the poor emulsion

174

stability observed in this pH range is the relatively weak electrostatic repulsion between the

175

protein-coated lipid droplets. The main components in egg white proteins are ovalbumin,

176

ovotransferrin and lysozyme, which have isoelectric points of approximately 4.7, 6.1, and 10.7,


9


Page 10 of 43

177

respectively 37, 38. Consequently, the surface potential on the oil droplets would be expected to be

178

fairly low in this pH range. Conversely, relatively stable emulsions could be formed when the pH

179

during homogenization was in the range 3.0 to 4.0, which was far from the isoelectric point of

180

egg white proteins.

181

A relatively high level of proteins (10%) was included in the emulsions so as to ensure that

182

they would form a gel after heating. Preliminary experiments showed that when the protein level

183

was below this value the microgels formed were very fragile and easily broken by simple stirring

184

(data not shown). Conversely, when the protein level was too high, the emulsions were too

185

viscous to pass through the extrusion device used for the preparation of the microgels. Around

186

10% protein was therefore used as a compromise to ensure that the emulsions would easily flow

187

through the encapsulator, but that relatively strong microgels were formed after injection.

188

Protein microgels were produced using the emulsification-gelation method by injecting an

189

aliquot of the emulsion into hot oil so as to promote protein cross-linking and gel formation. The

190

temperature (90 °C) used was sufficient to promote rapid gelation of the egg white proteins. The

191

aggregation of the microgels formed within the oil was inhibited by including a lipophilic

192

surfactant within the oil phase (4% PGPR). This surfactant would be expected to adsorb to

193

hydrophilic microgels surfaces and form a protective coating that inhibited their close

194

association. Under the optimized conditions, the mean diameter of microgels formed was ~260

195

µm (Table 2) and the particle size distribution was relatively narrow (Fig. 2b). In addition, the

196

microgels formed were spherical and had smooth surfaces (Fig. 5). It should be noted that we

197

also tried to form microgels by simply injecting emulsions into a hot oil phase using a syringe or

198

pipette, but we could not form microgels with uniform sizes and shapes using this method.

199

Influence of pH on Particle Stability


10

Page 11 of 43


200

The colloidal stability of emulsions and microgels would be expected to play a key role in

201

their commercial applications within food products. And thus, pH sensitivity of the two delivery

202

systems was analyzed from pH 3.0 to 8.0 since this covers most food applications. The

203

emulsions and beads were dispersed in phosphate buffer solutions with different pH values for

204

24 h before analysis.

205

As shown in Table 2, the mean particle diameters of oil droplets in emulsions were relatively

206

small and stable between pH 3.0 and 6.0 (< 0.4 µm), but increased significantly from pH 7.0 to

207

8.0 (> 17 µm). The particle size distributions of the emulsions broadened appreciably when the

208

pH was increased to around 6.0 (Fig. 2a), suggesting that they had become unstable to droplet

209

aggregation. An increase in particles size was also observed in microscopy images of these

210

systems, which was attributed to extensive droplet flocculation (Fig. 4). Conversely, emulsions

211

exhibited good aggregation stability under more acidic conditions, which may be because the pH

212

value was well below the isoelectric point of adsorbed proteins. Consequently, the droplets had a

213

high positive surface potential, which would generate a strong electrostatic repulsion between

214

them

215

values support this hypothesis (Fig. 3): the ζ-potential changes from highly positive to fairly

216

negative from pH 3.0 to 8.0 and the zero-charge point appears around pH 6.6.

42, 43

. Results from electrophoresis measurements made on the emulsions at different pH

217

For microgels, with the value of pH increasing, the mean particle size decreased slightly

218

(Table 2), and slight change was observed in particle size distribution (Fig. 2b). The optical

219

microscopy images suggested the shape of the microgels was spherical with smooth surfaces at

220

all pH values (Fig. 5a). Confocal microscopy were utilized for the determination of locations of

221

oil droplets (stained red) and protein molecules (stained green) within the microgels. Initially, the

222

fluorescence dyes were added to a suspension of the microgels, and then the images were taken.


11


Page 12 of 43

223

The confocal microscopy images exhibited that dyed oil droplets were fairly evenly distributed

224

throughout microgels at lower pH values, but that they tended to be located primarily at the

225

edges of the microgels at higher pH values (Fig. 5b). Interestingly, the confocal microscopy also

226

images suggested that the dyed protein molecules tended to be located at the exterior of the

227

microgels at lower pH values, but that they tended to leach out of the microgels at higher pH

228

values (Fig. 5b). The phenomenon may have been due to differences in the location of the

229

proteins and lipids within the microgels at different pH values, or they may simply have been due

230

to a restriction in the ability of the fluorescence dyes to diffuse into the protein microgels. For

231

this reason, the potential impact of dye diffusion into the microgels on their confocal microscopy

232

images was investigated. A microtome was used to cut the microgels into thin slices that were

233

then dyed to observe their internal structure. Since the internal microstructure images of the

234

microgels obtained at different pH values were similar, only the images at pH 3.0 are shown as

235

an example (Fig. 6). These images clearly show that both the oil droplets and proteins were

236

relatively uniformly distributed throughout the interior of microgels, which indicates that the

237

heterogeneous distribution of the fluorescence dyes observed within the intact microgels (Fig. 5)

238

was due to restriction of dye diffusion rather than differences in protein or lipid distribution.

239

Nevertheless, the fact that an appreciable amount of proteins was observed in aqueous phase

240

surrounding microgels under neutral and alkaline conditions (Fig. 5) would account for the

241

decrease in mean particle diameter that was also observed at high pH values (Table 2).

242

The ζ-potential-pH profiles of emulsions and microgels was fairly similar (Fig. 3), which

243

could be explained by the fact that both types of colloidal particles would be covered by egg

244

white protein molecules. However, the microgels did not appear to be prone to extensive

245

flocculation at higher pH conditions, whereas emulsions were. This may have been because the


12

Page 13 of 43


246

microgels were so large that they did not move around due to Brownian motion, and so they did

247

not encounter each other frequently.

248

Influence of GIT Conditions on Particle Characteristics

249

The two different delivery systems were designed to encapsulate and deliver bioactive

250

agents, and thus it is important to analyze their behavior when they are subjected to the various

251

stages of the GIT mode: mouth; stomach; and, small intestine. Consequently, changes in particle

252

diameter, ζ- potential and microstructure of emulsion droplets and microgels were measured as

253

they passed through the simulated GIT conditions.

254

The initial emulsions had relatively small mean particle size and the particle size distribution

255

was monomodal (Fig. 7a). After they were exposed to the three stages of GIT mode, their

256

particle size distributions remained monomodal, but the mean particle diameters increased

257

considerably, which indicated some droplet aggregation had occurred. The extensive droplet

258

aggregation was also observed in confocal microscopy images of emulsions, after exposure to

259

the simulated mouth and stomach phases (Fig. 9). Since interfacial properties of emulsions were

260

related to the changes in particle size when they were exposed to various stages of the GIT

261

conditions, changes in surface potential (ζ-potential) of particles were also evaluated (Fig. 8).

262

The initial emulsions had relatively high positive surface potentials (+35 mV), which can be

263

explained by the fact that the pH is far from their isoelectric point. As a result, the initial

264

emulsions had good aggregation stability due to the relatively strong electrostatic repulsion

265

between lipid droplets. As microgels passed through mouth phase, the surface potential became

266

negative (-13.2 mV) because of the change in pH condition to neutral one (see Fig. 3) and the

267

presence of anionic mucin molecules that adsorbed to surfaces of lipid droplet. As they passed

268

through stomach phase, ζ-potential became close to zero (-1.8 mV), which was unexpected since


13


Page 14 of 43

269

the protein molecules would have been expected to have a high positive charge in the highly

270

acidic gastric fluids. This phenomenon may have occurred due to the fact that some anionic

271

species (such as mucin) adsorbed to the surfaces of protein-coated lipid droplet and some

272

positively charged residues in proteins exfoliated from surfaces of oil droplet inducing by pepsin

273

44, 45

274

intestine phase (-40.8 mV), which may due to adsorption of surface-active anionic species to

275

their surface, such as peptides, bile salts, phospholipids, and free fatty acids 46.

. The ζ-potential of droplets became strongly negative as they passed through to the small

276

The microgels exhibited quite different behavior in the various regions of GIT compared to

277

emulsions. The mean particle size of microgels decreased slightly from the initial systems (269

278

µm) to the ones that had been exposed to simulated oral conditions (263 µm) (Table 3). This

279

decrease may have been caused by some protein exfoliation from protein microgels surfaces

280

under neutral conditions of simulated saliva. When microgel beads moved from mouth to

281

stomach stage, a further reduction had occurred in the mean particle diameter and a population of

282

relatively small particles formed (Fig. 7b), which was mainly because of the digestion of proteins

283

by pepsin in simulated gastric phase. In addition, the degradation of the microgels may also have

284

been induced by other factors, such as alternations in ionic strength, pH, or agitation as exposure

285

to simulated stomach conditions. After incubation in the small intestine fluids, there was a

286

further decrease in particle diameters to only a few microns and the particle size distributions

287

became broad, indicating that a large-scale of different-sized colloidal particles were present in

288

the digestive fluids.

289

The surface potentials of microgel particles were fairly similar to those of emulsions during

290

incubation in different GIT regions (Fig. 8). This phenomenon can be explained by the fact that

291

both kinds of colloidal particles were initially coated by egg white proteins, which dominated


14

Page 15 of 43


292

their electrical characteristics. Confocal laser scanning microscopy showed that the microgels

293

maintained their spherical shape after exposure to the oral and gastric phases, but then were

294

largely disrupted under small intestinal condition (Fig. 10). Lipid-rich particles observed in the

295

small intestine phase were probably undigested lipid droplets, vesicles, micelles and insoluble

296

calcium salts 47.

297

Influence of Delivery System Types on Lipid Digestion

298

Finally, an automatic titration (pH stat) method was used to evaluate the influence of

299

delivery system types on the rate and extent of lipid digestion in the simulated small intestine

300

condition. The amount of free fatty acids (FFA) released from oil phase was calculated based on

301

the dosage of NaOH titrated into the reaction chamber to maintain a constant pH value (7.0). The

302

FFA release profiles indicated the initial rate of lipid digestion was considerably slower in

303

microgel beads than in emulsions (Fig. 11). For the emulsions, FFAs released rapidly throughout

304

the first 15 min, subsequently, more slowly at longer times. This rapid rate of lipid digestion can

305

be attributed to small droplet sizes and large surface area of lipid droplets, which would enable

306

lipase molecules to easily absorb to the lipid droplets surfaces and initiate lipid digestion. In

307

converse, the rate of lipid digestion was noticeably slower in microgels throughout the small

308

intestine phase. There was an initial burst of lipid digestion (~20% FFA released) during the first

309

1 min of digestion, which may because that some of microgels were disrupted in stomach phase

310

by pepsin hydrolysis, thereby releasing a fraction of lipid droplets into the surrounding aqueous

311

phase. In consequence, when exposed to lipase molecules, these free oil droplets were rapidly

312

digested. There was a more gradual release of FFAs at longer incubation time, which suggested

313

that digestion of lipid droplets was retarded when they were encapsulated within microgels.

314

Presumably, protein matrix surrounding the lipid droplets had to be digested before the lipid


15


Page 16 of 43

315

droplets could be digested in this system. Interestingly, after exposure to small intestine phase,

316

full digestion occurred for both types of colloidal particles. Consequently, the microgels could be

317

used to provide a delayed or sustained release of the lipids, without preventing their full

318

digestion.

319

In summary, we have shown that lipid-loaded egg white protein microgels can be fabricated

320

using an emulsification-injection-gelation method. Initially, an oil-in-water emulsion is prepared

321

that contains egg white proteins as both an emulsifier and gelling agent. This emulsion is then

322

injected into a hot oil phase, which promotes thermal gelation of proteins and the formation of

323

lipid-loaded microgels. The microgels are then collected and washed to remove the residual

324

external oil phase. The emulsions and microgels had good stability at values ranging from pH 3.0

325

to 5.0 due to strong electric repulsion between cationic protein-coated surfaces. However, they

326

were unstable at higher pH values due to a change in their charge characteristics around the

327

protein isoelectric point. Simulated GIT studies suggested that the digestion rate of lipid droplets

328

encapsulated in microgels beads was significantly slower than those encapsulated in emulsions.

329

Consequently, microgel beads fabricated by egg white proteins may be appropriate for

330

encapsulation, retention and controlled release of lipophilic bioactive components.

331

References

332

1.

333

protein beads by an emulsification/cold gelation process: application for the protection of

334

retinol. Biomacromolecules 2002, 3, 239-248.

335

2.

336

nanoemulsions and emulsions: comparison of physicochemical stability, lipid oxidation, and

337

lipase digestibility. J. Agric. Food Chem. 2011, 59, 415-427.

Beaulieu, L.; Savoie, L.; Paquin, P.; Subirade, M., Elaboration and characterization of whey

Lee, S. J.; Choi, S. J.; Li, Y.; Decker, E. A.; McClements, D. J., Protein-stabilized


16

Page 17 of 43


338

3.

Bansal, D.; Gulbake, A.; Tiwari, J.; Jain, S. K., Development of liposomes entrapped in

339

alginate beads for the treatment of colorectal cancer. Int J Biol Macromol 2016, 82, 687-695.

340

4.

341

colon specific delivery of self-emulsifying curcumin. J. Drug Deliv. Sci. Technol. 2015, 29, 159-

342

166.

343

5.

344

bioactive oils: From alimentary to pharmaceutical perspectives. Food Res. Int. 2016, 83, 41-59.

345

6.

346

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

347

7.

348

based delivery systems. Trends Food Sci. Technol. 2012, 23, 13-27.

349

8.

350

delivery of bioactive components in nutraceuticals and functional foods. Crit. Rev. Food Sci.

351

Nutr. 2009, 49, 577-606.

352

9.

353

Interface Sci. 2010, 15, 61-72.

354

10. Liu, F.; Ma, C.; McClements, D. J.; Gao, Y., Development of polyphenol-protein-

355

polysaccharide ternary complexes as emulsifiers for nutraceutical emulsions: Impact on

356

formation, stability, and bioaccessibility of β-carotene emulsions. Food Hydrocolloids 2016, 61,

357

578-588.

358

11. Qian, C.; Decker, E. A.; Xiao, H.; McClements, D. J., Inhibition of β-carotene degradation

359

in oil-in-water nanoemulsions: Influence of oil-soluble and water-soluble antioxidants. Food

360

Chem. 2012, 135, 1036-1043.

Sookkasem, A.; Chatpun, S.; Yuenyongsawad, S.; Wiwattanapatapee, R., Alginate beads for

Rodríguez, J.; Martín, M. J.; Ruiz, M. A.; Clares, B., Current encapsulation strategies for

Chen, L.; Remondetto, G. E.; Subirade, M., Food protein-based materials as nutraceutical

Fathi, M.; Mozafari, M. R.; Mohebbi, M., Nanoencapsulation of food ingredients using lipid

McClements, D. J.; Decker, E. A.; Park, Y.; Weiss, J., Structural design principles for

Sagalowicz, L.; Leser, M. E., Delivery systems for liquid food products. Curr. Opin. Colloid


17


Page 18 of 43

361

12. Yi, J.; Lam, T. I.; Yokoyama, W.; Cheng, L. W.; Zhong, F., Controlled release of β-Carotene

362

in β-lactoglobulin–dextran-conjugated nanoparticles’ in vitro digestion and transport with Caco-2

363

monolayers. J. Agric. Food Chem. 2014, 62, 8900-8907.

364

13. Zhang, Z.; Zhang, R.; Zou, L.; Chen, L.; Ahmed, Y.; Al Bishri, W.; Balamash, K.;

365

McClements, D. J., Encapsulation of curcumin in polysaccharide-based hydrogel beads: Impact

366

of bead type on lipid digestion and curcumin bioaccessibility. Food Hydrocolloids 2016, 58, 160-

367

170.

368

14. McClements, D. J., Nanoscale nutrient delivery systems for food applications: improving

369

bioactive dispersibility, stability, and bioavailability. J. Food Sci. 2015, 80, N1602-N1611.

370

15. McClements, D. J., Recent progress in hydrogel delivery systems for improving

371

nutraceutical bioavailability. Food Hydrocolloids 2017, 68, 238-245.

372

16. Shimanovich, U.; Bernardes, G. J. L.; Knowles, T. P. J.; Cavaco-Paulo, A., Protein micro-

373

and nano-capsules for biomedical applications. Chem. Soc. Rev. 2014, 43, 1361-1371.

374

17. Zhang, Z.; Zhang, R.; Chen, L.; Tong, Q.; McClements, D. J., Designing hydrogel particles

375

for controlled or targeted release of lipophilic bioactive agents in the gastrointestinal tract. Eur.

376

Polym. J. 2015, 72, 698-716.

377

18. McClements, D. J., Designing biopolymer microgels to encapsulate, protect and deliver

378

bioactive components: Physicochemical aspects. Adv. Colloid Interface Sci. 2017, 240, 31-59.

379

19. Devi, N.; Sarmah, M.; Khatun, B.; Maji, T. K., Encapsulation of active ingredients in

380

polysaccharide–protein complex coacervates. Adv. Colloid Interface Sci. 2017, 239, 136-145.

381

20. Yuan, D.; Jacquier, J. C.; O'Riordan, E. D., Entrapment of protein in chitosan-

382

tripolyphosphate beads and its release in an in vitro digestive model. Food Chem. 2017, 229,

383

495-501.


18

Page 19 of 43


384

21. Lohani, A.; Singh, G.; Bhattacharya, S. S.; Rama Hegde, R.; Verma, A., Tailored-

385

interpenetrating polymer network beads of κ-carrageenan and sodium carboxymethyl cellulose

386

for controlled drug delivery. J. Drug Deliv. Sci. Technol. 2016, 31, 53-64.

387

22. Balanč, B.; Trifković, K.; Đorđević, V.; Marković, S.; Pjanović, R.; Nedović, V.; Bugarski,

388

B., Novel resveratrol delivery systems based on alginate-sucrose and alginate-chitosan

389

microbeads containing liposomes. Food Hydrocolloids 2016, 61, 832-842.

390

23. Zhang, Z.; Zhang, R.; Chen, L.; McClements, D. J., Encapsulation of lactase (β-

391

galactosidase) into κ-carrageenan-based hydrogel beads: Impact of environmental conditions on

392

enzyme activity. Food Chem. 2016, 200, 69-75.

393

24. Guo, J.; Zhou, Q.; Liu, Y.-C.; Yang, X.-Q.; Wang, J.-M.; Yin, S.-W.; Qi, J.-R., Preparation of

394

soy protein-based microgel particles using a hydrogel homogenizing strategy and their interfacial

395

properties. Food Hydrocolloids 2016, 58, 324-334.

396

25. Sağlam, D.; Venema, P.; de Vries, R.; Sagis, L. M. C.; van der Linden, E., Preparation of

397

high protein micro-particles using two-step emulsification. Food Hydrocolloids 2011, 25, 1139-

398

1148.

399

26. Liao, L.; Luo, Y.; Zhao, M.; Wang, Q., Preparation and characterization of succinic acid

400

deamidated wheat gluten microspheres for encapsulation of fish oil. Colloids Surf. B.

401

Biointerfaces 2012, 92, 305-314.

402

27. Can Karaca, A.; Low, N. H.; Nickerson, M. T., Potential use of plant proteins in the

403

microencapsulation of lipophilic materials in foods. Trends Food Sci. Technol. 2015, 42, 5-12.

404

28. O'Neill, G. J.; Jacquier, J. C.; Mukhopadhya, A.; Egan, T.; O'Sullivan, M.; Sweeney, T.;

405

O'Riordan, E. D., In vitro and in vivo evaluation of whey protein hydrogels for oral delivery of

406

riboflavin. J. Funct. Foods 2015, 19, Part A, 512-521.


19


Page 20 of 43

407

29. Chen, L.; Hébrard, G.; Beyssac, E.; Denis, S.; Subirade, M., In vitro study of the release

408

properties of soy−zein protein microspheres with a dynamic artificial digestive system. J. Agric.

409

Food Chem. 2010, 58, 9861-9867.

410

30. Diarrassouba, F.; Garrait, G.; Remondetto, G.; Alvarez, P.; Beyssac, E.; Subirade, M., Food

411

protein-based microspheres for increased uptake of vitamin D3. Food Chem. 2015, 173, 1066-

412

1072.

413

31. Joye, I. J.; Davidov-Pardo, G.; McClements, D. J., Encapsulation of resveratrol in

414

biopolymer particles produced using liquid antisolvent precipitation. Part 2: Stability and

415

functionality. Food Hydrocolloids 2015, 49, 127-134.

416

32. Huang, X.; Dai, Y.; Cai, J.; Zhong, N.; Xiao, H.; McClements, D. J.; Hu, K., Resveratrol

417

encapsulation in core-shell biopolymer nanoparticles: Impact on antioxidant and anticancer

418

activities. Food Hydrocolloids 2017, 64, 157-165.

419

33. Hu, S.; Wang, T.; Fernandez, M. L.; Luo, Y., Development of tannic acid cross-linked

420

hollow zein nanoparticles as potential oral delivery vehicles for curcumin. Food Hydrocolloids

421

2016, 61, 821-831.

422

34. Penalva, R.; González-Navarro, C. J.; Gamazo, C.; Esparza, I.; Irache, J. M., Zein

423

nanoparticles for oral delivery of quercetin: Pharmacokinetic studies and preventive anti-

424

inflammatory effects in a mouse model of endotoxemia. Nanomed. Nanotechnol. Biol. Med.

425

2017, 13, 103-110.

426

35. Kovacs-Nolan, J.; Phillips, M.; Mine, Y., Advances in the value of eggs and egg components

427

for human health. J. Agric. Food Chem. 2005, 53, 8421-8431.

428

36. Drakos, A.; Kiosseoglou, V., Stability of acidic egg white protein emulsions containing

429

xanthan gum. J. Agric. Food Chem. 2006, 54, 10164-10169.


20

Page 21 of 43


430

37. Gu, L.; Su, Y.; Zhang, M.; Chang, C.; Li, J.; McClements, D. J.; Yang, Y., Protection of β-

431

carotene from chemical degradation in emulsion-based delivery systems using antioxidant

432

interfacial complexes: Catechin-egg white protein conjugates. Food Res. Int. 2017, 96, 84-93.

433

38. Mine, Y., Recent advances in the understanding of egg white protein functionality. Trends

434

Food Sci. Technol. 1995, 6, 225-232.

435

39. Zhang, Z.; Chen, F.; Zhang, R.; Deng, Z.; McClements, D. J., Encapsulation of pancreatic

436

lipase in hydrogel beads with self-regulating internal pH microenvironments: retention of lipase

437

activity after exposure to gastric conditions. J. Agric. Food Chem. 2016, 64, 9616-9623.

438

40. Sarkar, A.; Goh, K. K. T.; Singh, H., Colloidal stability and interactions of milk-protein-

439

stabilized emulsions in an artificial saliva. Food Hydrocolloids 2009, 23, 1270-1278.

440

41. Wooster, T. J.; Day, L.; Xu, M.; Golding, M.; Oiseth, S.; Keogh, J.; Clifton, P., Impact of

441

different biopolymer networks on the digestion of gastric structured emulsions. Food

442

Hydrocolloids 2014, 36, 102-114.

443

42. Zhang, R.; Zhang, Z.; Zhang, H.; Decker, E. A.; McClements, D. J., Influence of emulsifier

444

type on gastrointestinal fate of oil-in-water emulsions containing anionic dietary fiber (pectin).

445

Food Hydrocolloids 2015, 45, 175-185.

446

43. Liu, F.; Wang, D.; Xu, H.; Sun, C.; Gao, Y., Physicochemical properties of β-carotene

447

emulsions stabilized by chlorogenic acid–lactoferrin–glucose/polydextrose conjugates. Food

448

Chem. 2016, 196, 338-346.

449

44. Vingerhoeds, M. H.; Blijdenstein, T. B. J.; Zoet, F. D.; van Aken, G. A., Emulsion

450

flocculation induced by saliva and mucin. Food Hydrocolloids 2005, 19, 915-922.

451

45. Sarkar, A.; Goh, K. K. T.; Singh, R. P.; Singh, H., Behaviour of an oil-in-water emulsion

452

stabilized by β-lactoglobulin in an in vitro gastric model. Food Hydrocolloids 2009, 23, 1563-


21


Page 22 of 43

453

1569.

454

46. Zhang, R.; Zhang, Z.; Zhang, H.; Decker, E. A.; McClements, D. J., Influence of lipid type

455

on gastrointestinal fate of oil-in-water emulsions: In vitro digestion study. Food Res. Int. 2015,

456

75, 71-78.

457

47. Salvia-Trujillo, L.; Qian, C.; Martín-Belloso, O.; McClements, D. J., Influence of particle

458

size on lipid digestion and β-carotene bioaccessibility in emulsions and nanoemulsions. Food

459

Chem. 2013, 141, 1472-1480.

460

Funding Information

461

The work was supported by the National Natural Science Foundation of China [grant number

462

31501428 and 31671809] and Jiangsu province “Collaborative Innovation Center for Food safety

463

and quality control” industry development program. This material was also partly based upon

464

work supported by the National Institute of Food and Agriculture, USDA, Massachusetts

465

Agricultural Experiment Station (MAS00491) and USDA, AFRI Grants (2014-67021).


22

Page 23 of 43


466

Figure captions

467

Fig. 1: Scheme of preparation of O/W emulsions and microgel beads based on egg white

468

proteins.

469

Fig. 2: Influence of pH on the particle size distribution of different delivery systems: (a)

470

emulsions; (b) microgel beads.

471

Fig. 3: Influence of pH on the electrical characteristics (ζ-potential) of different delivery systems

472

under different pH conditions: (a) emulsions; (b) microgel beads.

473

Fig. 4: Microstructures of emulsions under different pH conditions: (a) regular optical

474

microscopy; (b) laser confocal microscopy (scale bar is 1 µm).

475

Fig. 5: Microstructures of microgel beads under different pH conditions: (a) regular optical

476

microscopy; (b) laser confocal microscopy (scale bar is 200 µm).

477

Fig. 6: Laser confocal microscopy of internal microgel beads (scale bar is 200 µm). (a)

478

sunflower oil stained with Nile red; (B) egg white proteins stained with FITC; (c) merged image

479

of a, b.

480

Fig. 7: Particle size distribution of different delivery systems after exposure to successive GIT

481

stage: (a) emulsions; (b) microgel beads.

482

Fig. 8: The electrical characteristics (ζ-potential) of different delivery systems after exposure to

483

successive GIT stage: (a) emulsions; (b) microgel beads.

484

Fig. 9: Confocal microscopy of emulsions after exposing to different regions of the simulated

485

GIT (scale bar is 1 µm).

486

Fig. 10: Confocal microscopy of microgel beads after exposing to different regions of the

487

simulated GIT (scale bar is 200 µm).

488

Fig. 11: The amount of free fatty acids released from emulsions and microgel beads measured


23


489

using

a

pH-stat

in

vitro


Page 24 of 43

digestion

model.

24

Page 25 of 43


Table 1: Chemical Composition of Simulated GIT Fluids During Each in Vitro Digestion Region. Digestion Phase

Mouth Phase

Stomach Phase

Small Intestine Phase

Compound

Amount

Sodium chloride

13.64 mM

Ammonium nitrate

2.05 mM

Monopotassium phosphate

2.34 mM

Potassium chloride

1.35 mM

Potassium citrate

0.50 mM

Uric acid sodium salt

0.06 mM

Urea

1.65 mM

Lactic acid sodium salt

0.65 mM

Porcine gastric mucin (type II)

15 mg/mL

Sodium chloride

17.11 mM

Hydrochloric acid

41.91 mM

Pepsin

1.6 mg/mL

Calcium Chloride Dihydrate

10 mM

Sodium Chloride

150 mM

Bile Salts

5 mg/mL

Lipase

1.6 mg/mL


25


Page 26 of 43

Table 2: Influence of pH on the Mean Particle Diameter of Different Delivery Systems: Emulsions and Droplet-Loaded Microgel Beads. Mean Particle Diameter (µm) Samples pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

Emulsions

0.39±0.001a

0.335±0.001a

0.323±0.001a

0.351±0.004a

17.43±1.2b

17.65±0.61b

Microgel Beads

269.50±2.69a

264.47±2.91b

256.17±2.68cd

254.29±2.87cd

253.23±1.56c

258.89±1.97d

*

Data with different letters (a-d) in a row are significantly different (p< 0.05).


26

Page 27 of 43


Table 3: Mean Particle Diameter of Different Delivery Systems After Exposure to Different GIT Regions. Mean Particle Diameter (µm) Samples Initial

Mouth

Stomach

Intestine

Emulsions

0.339±0.001a

14.995±0.634b

23.407±1.923c

5.323±0.686d

Microgel Beads

269.504±2.687a

263.498±4.009b

44.488±5.009c

4.217±0.063d

*

Date with different letter (a-d) in a row mean significantly difference (p< 0.05).


27


Page 28 of 43

Fig. 1


28

Page 29 of 43


120 Emulsions

pH 3.0

Volume Fraction (%)

100 pH 4.0

80 pH 5.0

60 pH 6.0

40 pH 7.0

20 pH 8.0

0 0.01

0.1

1

10

100

1000

10000

Particle Diameter (µm) (a)


29


Page 30 of 43

140 Microgel beads

Volume Fraction (%)

120

pH 3.0

100 pH 4.0

80 pH 5.0

60 pH 6.0

40 pH 7.0

20 pH 8.0

0 10

100

1000

10000

Particle Diameter (µm) (b) Fig. 2


30

Page 31 of 43


40

ζ-potential (mV)

30 20 10 0 3

4

5

6

7

8

-10 Emulsions

-20

Microgel beads

-30

pH Fig. 3


31


Page 32 of 43

(a)


32

Page 33 of 43


pH 3.0

pH 4.0

pH 5.0

pH 6.0

pH 7.0

pH 8.0

(b) Fig. 4


33


Page 34 of 43

(a)


34

Page 35 of 43


pH 3.0

pH 4.0

pH 5.0

pH 6.0

pH 7.0

pH 8.0

(b) Fig. 5


35


(a)

(b)

Page 36 of 43

(c) Fig. 6


36

Page 37 of 43


80

Volume Fraction (%)

70

Emulsions Initial

60 50

Mouth

40 30

Stomach

20 10 0 0.01

Intestine

1

100

10000

Particle Diameter (µm) (a)


37


Page 38 of 43

90 80

Microgel beads

Volume Fraction (%)

Initial

70 60 Mouth

50 40

Stomach

30 20

Intestine

10 0 0.01

1

100

10000

Particle Diameter (µm) (b) Fig. 7


38

Page 39 of 43


40

ζ-potential (mV)

30 20 10 0 -10 -20 -30

Emulsions Microgel beads

-40 -50 Initial

Mouth

Stomach

Intestine

Fig. 8


39


Mouth

Stomach

Page 40 of 43

Intestine

Fig. 9


40

Page 41 of 43


Mouth

Stomach

Intestine

Fig. 10


41


Page 42 of 43

105

FFA Released (%)

90 75 60

Emulsions

45

Microgel beads

30 15 0 0

20

40

60

80

100

120

Digestion Time (min) Fig. 11


42

Page 43 of 43


Graphic for table of contents


43

Modulation of Lipid Digestion Profiles Using Filled Egg White Protein

Recommend Documents