Gastrointestinal Fate of Fluid and Gelled Nutraceutical Emulsions

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Food and Beverage Chemistry/Biochemistry

Gastrointestinal Fate of Fluid and Gelled Nutraceutical Emulsions: Impact on Proteolysis, Lipolysis, and Quercetin Bioaccessibility Xing Chen, David Julian McClements, Yuqing Zhu, Liqiang Zou, Ziling Li, Wei Liu, Ce Cheng, Hongxia Gao, and Chengmei Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03003 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Journal of Agricultural and Food Chemistry

Gastrointestinal Fate of Fluid and Gelled Nutraceutical Emulsions: Impact on Proteolysis, Lipolysis, and Quercetin Bioaccessibility Xing Chen a, David Julian McClements b, Yuqing Zhu a, Liqiang Zou a*, Ziling Li a,c, Wei Liu a*, Ce Cheng, Hongxia Gao a, Chengmei Liu a a

State Key Laboratory of Food Science and Technology, Nanchang University, No.

235 Nanjing East Road, Nanchang 330047, Jiangxi, China b

Biopolymers & Colloids Research Laboratory, Department of Food Science,

University of Massachusetts, Amherst, MA 01003, USA c

School of Life Science, Jiangxi Science and Technology Normal University,

Nanchang, 330013, Jiangxi, PR China

Corresponding Authors *Liqiang Zou (L.Z.), Tel: + 86 791 88305872 8106. Fax: + 86 791 88334509. E-mail: [email protected]. *Wei Liu (W.L.), Tel: + 86 791 88305872 8106. Fax: + 86 791 88334509. E-mail [email protected].

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ACS Paragon Plus Environment


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ABSTRACT: Fluid and gelled nutraceutical emulsions were formulated from

2

quercetin-loaded caseinate-stabilized emulsions by addition of gellan gum with or

3

without acidification with glucono-delta-lactone (GDL).

4

increased the viscosity or gel strength of the fluid and gelled emulsions, respectively.

5

The behavior of the nutraceutical emulsions in a simulated gastrointestinal tract

6

depended on their initial composition.

7

gum levels (0 to 0.2%) had similar protein and lipid hydrolysis rates, as well as

8

similar quercetin bioaccessibility (~ 51%). Conversely, proteolysis, lipolysis, and

9

quercetin bioaccessibility decreased with increasing gellan gum level in the gelled

10

emulsions. Compared with gelled emulsions, fluid emulsions were digested more

11

rapidly and led to higher quercetin bioaccessibility. There was a good correlation

12

between quercetin bioaccessibility and lipolysis rate. These findings are useful for

13

designing nutraceutical-loaded emulsions that can be used in a wide range of food

14

products with different rheological properties.

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KEYWORDS: Emulsions, Emulsion gels, Quercetin, Proteolysis, Lipolysis,

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Bioaccessibility

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Gellan gum addition

Fluid emulsions containing different gellan

2


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INTRODUCTION

18

The potential health benefits of many hydrophobic nutraceuticals are not

19

realized in practice because of their low water-solubility, chemical instability, and

20

low oral bioavailability

21

designing the composition and structure of food matrices.

22

nutraceutical bioavailability can be improved in the gastrointestinal tract (GIT) by

23

increasing bioaccessibility, reducing degradation, and enhancing absorption by

24

creating food matrices that enhance these different phenomena

25

emulsions are particularly good platforms for the development of delivery systems

26

for hydrophobic nutraceuticals because of the considerable flexibility in controlling

27

their compositions and structures 5.

28

carbohydrates, lipids and minerals in emulsions can easily be controlled, as well as

29

the droplet size distribution of emulsions.

30

shown to be high effective at enhancing the bioaccessibility of various types of

31

hydrophobic nutraceuticals, including curcumin

32

quercetin 10, 11.

1, 2

.

These challenges can often be overcome by carefully In particular,

3, 4

. Oil-in-water

For instance, the type and level of proteins,

Fluid emulsions have already been

6, 7

, lycopene 8, β-carotene 9, and

33

In the current study, we focus on using emulsions to improve the

34

bioaccessibility of quercetin (3,3′,4′,5,7-pentahydroxyflavone), which is a major

35

source of flavonoids in the human diet 12.

36

a number of biological properties that may maintain or improve human health

37

including antioxidant, anti-carcinogenic, antidiabetic, anti-inflammatory, anti-obesity,

Quercetin has been reported to exhibit 13-16

,

3



17-19

.

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38

antibacterial and hepato-protective effects.

Like other hydrophobic

39

nutraceuticals, the utilization of quercetin as a bioactive agent in foods is limited by

40

its poor water-solubility, chemical instability, and low oral bioavailability

41

previous studies, fluid emulsions stabilized by either synthetic surfactants or natural

42

biopolymers have been shown to improve the water-solubility, chemical stability,

43

and bioaccessibility of quercetin 10, 11.

20

. In

44

Commercial food emulsions come in various textures, including low viscosity

45

liquids (dairy, soy, or almond milk), viscous fluids (creams, dips, dressings), or gels

46

(yoghurts, cheeses, desserts).

47

low viscosity nutraceutical emulsions, which may not be suitable for application in

48

all food products.

49

different forms of nutraceutical emulsions that are more suitable for other food

50

applications.

51

creating emulsions with different textural properties.

52

with different viscosities were fabricated by adding a thickening agent (gellan gum)

53

to the systems.

54

Sphingomonas elodea, which is used in foods as a thickener, gelling agent, or

55

stabilizer

56

aggregation of the casein-coated droplets and gellan gum mixture by acidification

57

using glucono-delta-lactone (GDL) 23.

58

Previous studies have focused on the development of

For this reason, there is a need to examine the development of

In this study, we used casein-coated oil droplets as a platform for Fluid nutraceutical emulsions

Gellan gum is a linear anionic polysaccharide produced by

21, 22

. Gelled nutraceutical emulsions were formed by promoting

We hypothesized that the initial composition, structure, and rheology of the 4


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nutraceutical emulsions would influence their gastrointestinal fates by interfering

60

with digestion or release processes.

61

shown that these properties alter the gastrointestinal fate of nutrients and

62

nutraceuticals.

63

starch suspensions 24.

64

viscosity and interact with digestive enzymes 25. Xanthan or carrageenan decreased

65

digestion of soybean proteins due to gelation of gastric fluids

66

reduced the bioaccessibility of β-carotene encapsulated within lipid droplets

27

67

Pectin caused significantly reduced bioaccessibility of lycopene in tomato paste

28

68

These studies suggest that the incorporation of texturing biopolymers into emulsions

69

may alter their gastrointestinal fates.

Indeed, a number of studies have already

β-glucan increases the viscosity and decreases the hydrolysis of oat Pectin decreases starch digestion due to its ability to increase

26

. Carrageenan . .

70

In the present study, nutraceutical emulsions with different textural properties

71

were fabricated by addition of gellan gum and GDL. Rheological and morphological

72

analyses were conducted to provide understanding of the impact of biopolymer

73

addition and acidification on the properties of the emulsions. Additionally, an in

74

vitro gastrointestinal model was used to characterize the gastrointestinal fate of the

75

emulsions.

76

MATERIALS AND METHODS

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Materials

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Corn oil was obtained from Yi Haikerry Grain and Oil Food Company

79

(Nanchang, China). Quercetin (97%) and D-(+)-Gluconic acid δ-lactone were 5



80

purchased from Aladdin Industrial Corporation (Shanghai, China). Gellan gum (high

81

acyl KELCOGEL), high ester pectin, carrageenan, and xanthan gum were provided

82

by CP Kelco (Shanghai, China). Casein (sodium salt) from bovine milk (C8654),

83

gum arabic from acacia tree (G9752), pepsin from porcine gastric mucosa (P7125,

84

enzymatic activity of ≥ 400 units/mg protein), lipase from porcine pancreas type II

85

(L3126; enzymatic activity of 100−500 units/mg protein using olive oil), pancreatin

86

from porcine pancreas (P1750; 4 × US Pharmacopeia (USP) specifications), mucin

87

from porcine stomach type II (M2378), Nile Blue A (N0766) and Nile red (72485)

88

were obtained from the Sigma Chemical Company (St. Louis, MO). All other

89

reagents used were of analytical grade.

90

Emulsion preparation

91

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The fluid emulsions were prepared based on the method described in a previous 29

92

study (Figure 1)

93

sodium caseinate in ultrapure water, and then sodium azide (0.02 wt%) was added to

94

protect the protein from microbial spoilage during storage. To ensure complete

95

protein hydration, the casein solutions were continuously stirred (150 rpm) for 2 h at

96

40 °C, and then left overnight at ambient temperature. A coarse oil-in-water

97

emulsion was then prepared by blending corn oil (10 wt%) with emulsifier solution

98

(90 wt%) at 15,000 rpm for 2 min using a high shear mixer (T18 digital,

99

ULTRATURRAX®, IKA, Staufen, Germany). The droplet size was then reduced

100

further by passing the coarse emulsions through a microfluidizer 3 times at 12,000

.

An emulsifier solution was prepared by dispersing 4 wt%

6


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psi (M-110EH30, Microfluidic Corp., Newton, MA, USA). The emulsions were then

102

mixed with gellan gum (0.1, 0.2, 0.3 wt%) at 80 °C for 30 min, followed by cooling

103

to ambient temperature (25 °C) to obtain emulsions with different viscosities. These

104

samples were then all adjusted to pH 6.5 before further use. Quercetin-loaded

105

emulsions were then prepared by dissolving 0.4 mg/g quercetin into the emulsions

106

under continuous stirring at 100 °C for 10 min.

107

Gelled emulsions were prepared by adding a fixed amount (0.8 wt%) of GDL to

108

the fluid nutraceutical emulsions.

The samples were then stirred for 10 min using a

109

magnetic stirrer, and then maintained at 25 °C for gel formation to occur. The

110

change in pH with time was monitored using a digital pH meter (827 pH Lab,

111

Metrohm, Switzerland).

112

Zeta-potential measurements

113

The surface potential (zeta-potential) of the particles in the emulsions was

114

determined using a commercial particle electrophoresis instrument (Zetasizer Nano

115

ZSP, Malvern Instruments, Worcestershire, UK). Before each test, the samples were

116

diluted with ultrapure water and adjusted to different pH values using HCl or NaOH

117

solutions.

118

Rheology measurement

119

Time-sweep oscillatory measurements were performed using a dynamic shear

120

rheometer (MCR302, Anton Paar, Germany) with a plate-plate geometry (pp-50, 50

7



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mm diameter) as described elsewhere with some slight modifications 30. GDL (0.8

122

wt%) was mixed with fluid emulsions containing different gellan gum contents.

123

The mixtures were then stirred for 10 min using a magnetic stirrer and about 2 mL

124

of sample was collected and placed onto the measurement cell of the rheology

125

instrument maintained at 25 °C. The samples were then covered with a thin layer of

126

low viscosity silicone oil to prevent water evaporation and left to stand for 5 min to

127

allow thermal equilibrium before the measurement. The change in the elastic (G')

128

and loss (G'') modulus over time was measured at a constant frequency of 1 Hz and

129

strain amplitude of 0.5%.

130

which was established using a preliminary experiment.

131

Microstructure observation

These values were in the linear viscoelastic region,

132

The morphology of the fluid and gelled nutraceutical emulsions (after

133

acidification for 12 h) was determined using confocal laser scanning microscopy

134

(CLSM, Carl Zeiss LSM710, Jena, Germany) base on a earlier description

135

Emulsion droplets were stained using Nile red (oil phase) and Nile Blue A (aqueous

136

phase). Samples were placed into a glass bottom cell culture dish for observation

137

under a 63 × objective.

138

with wavelengths of 488 nm (Nile red) and 633 nm (Nile blue A), fluorescence

139

images were recorded at wavelengths of 570 nm for Nile red and 660 nm for Nile

140

blue A.

31

.

The fluorescence signal was excitated using laser sources

8


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141 142

In vitro digestion model The gastrointestinal fate of the nutraceutical emulsions was monitored using a

143

three-step simulated gastrointestinal model as described earlier 32.

144

0.3% gellan gum possessed relatively higher viscosity, which slowered the

145

dissolution of nutraceutical (quercetin) in emulsions.

146

gastrointestinal behavior of nutraceutical emulsions containing 0, 0.1 or 0.2% gellan

147

gum.

148

simulate oral breakdown

149

taken for lipolysis, proteolysis, and bioaccessibility measurements.

150

analysis, these samples were diluted with distilled water so that they had initial corn

151

oil contents of 2.5 wt% (for lipolysis analysis) and 5 wt% (for proteolysis and

152

bioaccessibility

153

gastrointestinal fluids were preheated to 37 °C prior to carrying out the simulated

154

GIT study.

155

stomach (120 min) and intestine (120 min) stages.

156

Emulsions with

We thus monitored the

Gelled emulsions after acidification for 12 h were cut into ∼5.0 mm cubes to 33, 34

analysis),

, and then fluid and gelled emulsion samples were

respectively.

All

the

samples

and

Prior to

simulated

The samples were then passed through the simulated mouth (10 min),

For proteolysis and lipolysis experiments, quercetin-free fluid and gelled

157

emulsions containing 0, 0.1 or 0.2% gellan gum were tested.

Changes in the

158

properties of the casein during gastric digestion were monitored by stopping the

159

proteolysis reaction at various digestion times by raising the pH to 7.0. These

160

samples were then kept at pH 7.0 with continuous stirring at 1000 rpm to obtain

161

uniform solutions for sodium dodecyl sulfate polyacrylamide gel electrophoresis 9



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(SDS-PAGE) analysis. During the simulated small intestine period, the free fatty

163

acids (FFA) released from the matrix versus time was measured to investigate the

164

rate and extent of triacylglycerol (corn oil) digestion. The small intestinal fluids

165

were maintained at pH 7.0 throughout lipolysis by addition of 0.1 mol/L NaOH

166

solution using a pH-stat automatic titration unit (Metrohm 907 Titrando, Metrohm,

167

Switzerland), and the volume of NaOH was recorded to calculate the percentage of

168

free fatty acids (FFA %) released from the emulsions 35.

169

The bioaccessibility of quercetin was measured in fluid and gelled nutraceutical

170

emulsions with different gellan gum levels (0, 0.1 or 0.2%).

171

small intestine stage, the digesta was immediately cooled in ice water, and the

172

undigested oil floating on the top of the samples was removed. The remainder of the

173

samples were then centrifuged (18,000 rpm) at 4 °C for 30 min. The clear middle

174

layer formed in the tubes after centrifugation was assumed to be the mixed micelle

175

phase, which contained the bioaccessible fraction of quercetin. The bioaccessibility

176

of quercetin was then determined by measuring its relative concentration in the

177

micelle phase compared to the initial amount using high performance liquid

178

chromatography (HPLC):

179

After the simulated

Bioaccessibility (%) = 100 × As/ Ainitial

180

Here AS is the amount of quercetin solubilized in the clear middle layer and Ainitial is

181

the initial amount of quercetin added into the system.

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Protein hydrolysis

183

The pepsin digest of the casein was collected at different periods during the

184

gastric phase and analyzed using SDS-PAGE based on a method described earlier 26.

185

The casein digest was treated with SDS loading buffer, heated at 95–100 °C for 10

186

min, and then centrifuged (3000 rpm) for 10 s. The obtained samples were cooled to

187

room temperature and loaded (10 µL) onto a gel system containing 5% stacking gel

188

and 12% separating gel (SDS-PAGE Gel Kit, Solarbio, Beijing, China). The gel was

189

run in a Bio-Rad Miniprotein unit (Bio-Rad Laboratories, Inc., Hercules, CA, USA)

190

at 200 V, then stained with Coomassie Brilliant Blue Fast Staining solution (Solarbio,

191

Beijing, China) and subsequently destained using pure water.

192

Quercetin analysis

193

The quercetin content of the samples was determined by HPLC (Agilent 1260, 10

194

Agilent Technologies, USA) as described previously

195

absolute ethanol to an appropriate concentration and then centrifuged (6,000 rpm) at

196

4 °C for 5 min.

197

nylon filter before further analysis. The chromatogram was recorded at 25 °C, and

198

area under the peaks was used to calculate the quercetin concentration from a

199

pre-established standard curve (0.5-20 µg/mL, R2 = 0.9991).

200

Statistical analysis

201

. Samples were diluted with

The supernatant was then collected and filtered through a 0.22 µm

All experiments were repeated three times and the results are expressed as the 11



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202

mean and standard deviations of these measurements. Data analysis was carried out

203

by one-way analysis of variance (ANOVA) using SPSS software (Version 17.0), and

204

values of P < 0.05 were considered statistically significant.

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RESULTS AND DISCUSSION

206

Impact of polysaccharide type on emulsion stability

207

Interactions between proteins and polysaccharides may lead to thermodynamic

208

incompatibility of protein-polysaccharide mixtures, promoting segregative phase

209

separation 36.

210

impact of polysaccharide type on emulsion stability. Casein-stabilized emulsions

211

were mixed with different levels of polysaccharides (0.1-0.3%) and then stored for

212

24 hours.

213

storage some of them separated into a droplet-rich cream layer at the top and a

214

droplet-depleted serum layer at the bottom (Figure 2). In particular, distinct phase

215

separation was observed in the emulsions containing pectin, carrageenan, and

216

xanthan gum.

217

to promote extensive depletion and/or bridging flocculation of the droplets in the

218

emulsions 37.

219

gum remained homogeneous after storage.

220

of gum arabic gives rise to compact molecules with a relatively small hydrodynamic

221

volume and as a consequence even 30% gum arabic solutions have a low viscosity 38.

222

Consequently, gellan gum was used in the remainder of the study to produce

Consequently, an initial experiment was carried out to ascertain the

Initially, all the emulsions had a uniform white appearance, but after

These effects can be attributed to the ability of these polysaccharides

On the other hand, the emulsions containing gum arabic and gellan However, the highly branched structure

12


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emulsions with a range of rheological properties.

224

Effect of gellan gum and acidification on emulsion rheology

225

In this section, a dynamic shear rheometer was used to characterize the impact

226

of gellan gum and acidification on the rheology of the emulsions. The initial

227

casein-stabilized oil-in-water emulsions had a low shear modulus and high damping

228

factor (tan δ > 1) (Figure 3a, b, c), which indicated that could be considered to be

229

viscous liquids. The elastic (G') and viscous (G'') components of the shear modulus

230

of these emulsions increased, while the damping factor decreased (tan δ < 1), as the

231

amount of gellan gum added increased, which indicated that the emulsions became

232

viscoelastic fluids after polysaccharide addition.

233

The gelled emulsions were formed by adding GDL to caseinate-stabilized

234

emulsions so as to reduce the pH (Figure 3d). As the pH decreased, the surface

235

potential (zeta-potential) of the casein-coated oil droplets decreased (Table 1),

236

which led to a reduction in the electrostatic repulsion between them.

237

three-dimensional network of aggregated droplets was formed that increased the

238

shear modulus of the emulsions (Figure 3a, b).

239

emulsions became predominantly elastic, i.e., G' > G'' (Figure 3c) and emulsion gels

240

were formed (Figure 4).

241

more rapid gelation and to a higher final gel strength, which may have been due to

242

an interaction between the casein and gellan gum molecules

243

solution conditions (pH 6.5), both the casein and gellan gum were negatively

As a result, a

Eventually, the rheology of the

The presence of gellan gum in the initial emulsions led to

39

.

Under the initial

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charged (Table 1).

245

between the protein and polysaccharide molecules, which prevented them from

246

aggregating.

247

negatively charged, excluded-volume effects caused by the presence of the

248

highly-extended polysaccharide molecules may have led to a higher local protein

249

concentration, which caused the casein molecules to aggregate more rapidly (see

250

later).

251

gellan gum were formed

252

However, when the pH was reduced further, the casein became positively charged,

253

and so there was an electrostatic attraction between cationic patches on the protein

254

surfaces and anionic groups on the polysaccharide chains.

255

of an intermediate level of gellan gum (0.1%) led to a stiffer gel being formed than

256

at higher levels (0.2% or 0.3%).

257

polysaccharide concentration on bridging flocculation 42.

258

there is insufficient polysaccharide present to interact with all the protein-coated

259

droplets.

260

cover the oil droplet surfaces and link numerous oil droplets together.

261

gellan gum levels, some of the oil droplets are completely coated by polysaccharide

262

molecules and are therefore are not involved in bridging flocculation.

263

Emulsion microstructure

264

Consequently, there would be an electrostatic repulsion

When the pH was reduced the casein molecules became less

What’s more, by lowering the pH, hydrogen bonds between the helices of 40, 41

, which may have enhanced emulsion gel rigidity.

Interestingly, addition

This effect may have been due to the impact of At low gellan gum levels,

At intermediate gellan gum levels, the polysaccharide molecules partially At high

The microstructure of fluid and gelled emulsions containing different levels of 14


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265

gellan gum were recorded using confocal laser scanning microscopy.

266

casein were stained using Nile red (red channel) and Nile Blue A (green channel),

267

respectively.

268

droplets (d = 233 nm) that were evenly dispersed throughout the sample (Figure 5).

269

After acidification for 12 h, some small protein aggregates were observed in this

270

emulsion but the fat droplets and proteins remained uniformly dispersed.

271

contrast, studies have shown that casein gels formed by heating have a more uneven

272

microstructure that became increasingly heterogeneous with increasing temperature

273

43

274

polysaccharide-rich zones (black regions) that appeared to form fibrous structures.

275

The nature of these structures depended on the level of gellan gum present, as well

276

as on acidification.

277

more extensive in the samples that had been acidified with GDL, which may be due

278

to the enhanced helix–helix associations between the junction zones of gellan at

279

lower pH values

280

compact structure: the higher the content of gellan gum, the more compact structure.

281

Additionally, after acidification for 12 h, some emulsion aggregates were

282

incorporated in black gellan gum zones in gelled emulsions with 0.2% or 0.3%

283

gellan (Figure 5), which may be attributed to electrostatic attraction between

284

positively charged protein and negatively charged polysaccharide (Table 1).

285

Oil and

Initially, the casein-stabilized emulsions contained relatively small

In

. In the samples containing gellan gum, there was evidence of distinct

40

.

In particular, the polysaccharide-rich zones appeared to be

Addition of gellan gum into gelled emulsions led to a more

We hypothesized that differences in the microstructures and rheology of the 15



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286

emulsions would lead to alterations in their gastrointestinal fate, and so we

287

investigated the digestion and bioaccessibility of the fluid and gelled emulsions

288

using a simulated GIT.

289

Protein digestibility

290

Commercial sodium caseinate is a mixture of different proteins, with the major

291

fractions being α-casein (23.5 kDa), β-casein (24.0 kDa) and κ-casein (19.0 kDa) at

292

levels of around 50%, 35% and 15%, respectively

293

that the sodium caseinate used in our study had two bands with fairly similar

294

molecular weights corresponding to α-casein and β-casein, and another band with a

295

lower molecular weight corresponding to κ-casein (Figure 6). Comparison with the

296

standard protein markers, suggested that all three protein bands had molecular

297

weights > 25 kDa, which is higher than expected, but has also been reported in other

298

studies

299

strongly bind sodium ions thereby increasing their molecular weight.

300

change in the position or intensity of the electrophoresis bands from the casein

301

fractions in the initial fluid or gelled samples tested, which suggests that protein

302

polymerization or fragmentation did not occur during emulsion formation.

45-47

.

44

. Gel electrophoresis showed

This effect may be due to the ability of the phosphate-rich caseins to There was no

303

The proteolysis of the casein fractions under simulated gastric conditions was

304

then investigated using SDS-PAGE (Figure 7). In the fluid emulsions, which were

305

not exposed to GDL treatment, the casein molecules were rapidly hydrolyzed into

306

peptides with the disappearance of all three casein bands after 2 min of digestion. 16


Page 17 of 41


307

As the digestion time increased, there was an increase of in the amount of smaller

308

peptide fragments that accumulated at the bottom of the acrylamide gel.

309

amount of gellan gum present in the emulsions (0 to 0.2%) had little impact on the

310

rate or extent of protein digestion.

311

exposed to GDL treatment, the casein appeared to be much more resistant to pepsin

312

digestion.

313

bands corresponding to the three casein fractions after 10 min of digestion (Figure

314

7d, e, f).

315

the intensity of the bands corresponding to casein peptides increased, indicating that

316

proteolysis did occur.

317

casein molecules in the gelled emulsions after 120 min of proteolysis. Visual

318

observation of the samples after gastric digestion indicated that there were still some

319

relatively large gel fragments present in the emulsion gels (Figure 9b). These results

320

suggest that gelation of the emulsions reduced the extent of protein digestion,

321

presumably by inhibiting the ability of the pepsin molecules from reaching the

322

casein molecules.

323

enzyme, while in the gelled samples, the main casein was trapped in gel cubes and

324

the enzyme only had immediate access to the protein on the surface of the cubes.

325

Additionally, the presence of the gellan gum in the emulsion gels led to more

326

undigested protein fragments remaining at the end of the stomach phase (Figure 9b)

327

and fewer peptide fragments being formed (Figure 7d, e, f).

The

In the gelled emulsions, which had been

Indeed, there was little change in the intensity of the electrophoresis

As proteolysis continued, the intensity of these bands decreased, while

Interestingly, there was still evidence of some undigested

For the fluid emulsions, all the protein was accessible to the

This result suggests 17



Page 18 of 41

328

that the gellan gum further suppressed protein digestion, which may have been

329

because it limited the accessibility of pepsin to casein.

330

gels (Figure 3) that were more resistant to breakdown under simulated GIT

331

conditions, reducing the surface area of gel particles exposed to the enzyme.

332

Previous studies have shown that soft gels break down faster than hard gels, thereby

333

making it easier for pepsin to hydrolyze the proteins 48.

334

contributed to the formation of more compact structures (Figure 5) in the emulsion

335

gels, which may reduce the diffusion rate of pepsin through the gel network.

336

Therefore, the diffusion rate of enzymes into the gels appears to be a key rate

337

limiting factor of digestion, which may be controlled by the porosity of the gel

338

matrix.

339

Fat digestion

340

Gellan gum led to stronger

Additionally, gellan gum

The rate and extent of lipid digestion in the emulsions under simulated small

341

intestine conditions were measured using a pH stat method (Figure 8).

For

342

lipolysis analysis, all intestinal fluids had an initial corn oil content of 0.5 wt%.

343

The lipids in all the samples were hydrolyzed rapidly during the first 10 min with

344

around 50 to 73% of the FFA being released, followed by a more gradual hydrolysis

345

at later times until over 96% of the FFA was released after 120 min.

346

extent of lipid digestion was fairly similar in all the fluid emulsions, regardless of

347

the gellan gum level, which suggested that the presence of this polysaccharide did

348

not interfere with the ability of the lipase molecules to access the oil droplet

The rate and

18


Page 19 of 41


349

surfaces.

350

The rate and extent of lipid digestion was somewhat suppressed in the gelled

351

emulsions, with the effect increasing with increasing gellan gum level (Figure 8).

352

The rate of lipid digestion may have been decreased in the gelled emulsions because

353

they contained large gel fragments that inhibited the ability of the lipase molecules

354

to reach the surfaces of the oil droplets trapped inside.

355

reported that trapping oil droplets in protein gels can inhibit their lipolysis

356

When the gellan gum content was raised from 0.1% to 0.2%, the rate of lipolysis

357

gradually decreased, which may be attributed to oil droplets being incorporated in

358

more compact gel structures at higher polysaccharide concentrations.

359

some oil droplets were trapped in polysaccharide-rich zones for the systems

360

containing 0.2% gellan gum (Figure 5), which may also have slowed the access of

361

bile salts and/or lipase to the corn oil.

362

increased to 1.0 wt%, there were even more large undigested emulsion gel fragments

363

remaining by the end of the intestinal stage (Figure 9b).

364

fragments appeared to increase with increasing gellan gum concentration, which

365

may result into suppression of lipid digestion.

366

Quercetin bioaccessibility

367

Previous studies have also 33, 34

.

What’s more,

When the initial corn oil content was

The size of these

The digestion studies showed that the fats and proteins in the emulsions were

368

digested at different rates depending on their initial compositions and textures.

In

369

this section, we therefore examined the impact of emulsion properties on the 19



Page 20 of 41

370

bioaccessibility of quercetin after in vitro digestion.

The was no statistical

371

difference (Duncan, p ≥ 0.05) in the measured quercetin bioaccessibility (~ 51.0%)

372

in any of the fluid emulsions, regardless of the gellan gum level present (Figure 9a).

373

This suggested that the gellan gum did not interfere with the release and

374

solubilization of the nutraceutical from these emulsions.

375

expected because the rate and extent of fat and protein digestion was similar in all of

376

the fluid emulsions.

377

that the addition of another anionic polysaccharide (alginate) to protein-stabilized

378

fluid emulsions did not impact the lipolysis rate or curcumin bioaccessibility

379

Conversely, addition of a cationic polysaccharide (chitosan) to protein-stabilized

380

fluid emulsions inhibited lipolysis and decreased carotenoid bioaccessibility, which

381

was attributed to its ability to bind to anionic species such as bile salts and free fatty

382

acids 50.

This effect might be

These results are in agreement with other studies that reported

49

.

383

In contrast to the fluid emulsions, the bioaccessibility of quercetin in the gelled

384

emulsions decreased significantly with increasing gellan gum level (Figure 9a).

385

This decrease in bioaccessibility may be attributed to the fact that some undigested

386

fat remained in the gelled emulsions after exposure to the simulated GIT (Figure

387

9b).

388

phase.

389

contributing to solubilization of quercetin in micellar phase.

390

experiments showed that the amount of FFA produced during lipid digestion is

As a result, some of the quercetin remained trapped in the undigested oil What’s more, released quercetin could interact with mixed micelles

51

,

FFA release

20


Page 21 of 41


391

actually very similar between all the samples at the end of digestion, approaching

392

100% (Figure 8), therefore the concentration of mixed micelles must also be quite

393

similar.

394

is severely limited, that mixed micelles were also trapped in the network.

395

the digestion conditions between the lipid digestion and bioaccessibility experiments

396

were different, as evidenced by the remaining gel particles observed at the end of the

397

bioaccessibility study (Figure 8, 9b).

398

released from these emulsion gels, which may be related to micellization 52, which

399

ultimately determines the solubilization and bioaccessibility of the released

400

quercetin.

401

the bioaccessibility of nutraceuticals, such as β-carotene in fat droplet-loaded

402

carrageenan gels

403

can increase nutraceutical bioaccessibility.

404

have a higher bioaccessibility in starch-based emulsion gels than in simple

405

emulsions, which was attributed to the ability of the starch matrix to inhibit fat

406

droplet aggregation, thereby leading to a larger exposed lipid surface area 53.

407

impact of gelation on bioaccessibility therefore appears to be highly system

408

dependent and is closely linked to the impact of gelation on lipid digestion.

409

Typically, the greater the amount that lipid digestion is inhibited, the lower the

410

bioaccessibility of any hydrophobic nutraceuticals.

411

It is possible that, considering that diffusion of the enzymes into the matrix However,

There was a different amount of FFAs

Some previous studies have also reported that emulsion gelation reduces

27

.

However, other studies have reported that emulsion gelation For instance, β-carotene was found to

The

In summary, we have shown that nutraceutical-loaded emulsions with different 21



Page 22 of 41

412

rheological properties can be produced by polysaccharide addition and acidification.

413

A simulated gastrointestinal tract study showed that fluid emulsions were digested

414

more rapidly than gelled emulsions, which led to a higher bioaccessibility of

415

quercetin.

416

digestion could be suppressed by increasing the level of gellan gum present.

417

effect was attributed to the formation of gels that were more resistant to

418

fragmentation under simulated GIT conditions.

419

for digestive enzymes (proteases or lipases) to reach their substrates (proteins or

420

lipids) inside the gel fragments.

421

bioaccessibility of quercetin, which was attributed to the fact that some of the

422

quercetin remained in the undigested oil phase and there were less mixed micelles

423

available to solubilize the quercetin.

424

suitable for enhancing nutraceutical bioavailability while gelled emulsions may be

425

more suitable for inhibiting lipid digestion and controlling body weight.

426

results show that the gastrointestinal fate of emulsions can be modulated by altering

427

their composition, structure, and textural properties, which may be important for

428

optimizing the design of functional foods.

429

References

430 431 432 433 434 435

(1)

For the gelled emulsions, the rate and extent of lipid and protein This

As a result, it was more difficult

Inhibition of lipid digestion reduced the

Hence, fluid emulsions appear to be most

These

McClements, D. J., Enhanced delivery of lipophilic bioactives using emulsions: a review of

major factors affecting vitamin, nutraceutical, and lipid bioaccessibility. Food & Function 2018, 9, 22-41. (2)

Aronson, J. K., Defining 'nutraceuticals': neither nutritious nor pharmaceutical. Br. J. Clin.

Pharmacol. 2017, 83, 8-19. (3)

Flores, F. P.; Kong, F., In Vitro Release Kinetics of Microencapsulated Materials and the Effect 22


Page 23 of 41


436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

of the Food Matrix. Annu. Rev. Food Sci. Technol. 2017, 8, 237-259. (4)

McClements, D. J.; Xiao, H., Designing food structure and composition to enhance

nutraceutical bioactivity to support cancer inhibition. Semin. Cancer Biol. 2017, 46, 215-226. (5)

McClements, D. J.; Saliva-Trujillo, L.; Zhang, R.; Zhang, Z.; Zou, L.; Yao, M.; Xiao, H.,

Boosting the bioavailability of hydrophobic nutrients, vitamins, and nutraceuticals in natural products using excipient emulsions. Food Res. Int. 2016, 88, 140-152. (6)

Zou, L.; Liu, W.; Liu, C.; Xiao, H.; McClements, D., Julian, Utilizing food matrix effects to

enhance nutraceutical bioavailability: increase of curcumin bioaccessibility using excipient emulsions. J. Agric. Food Chem. 2015, 63, 2052-2062. (7)

Zou, L.; Zheng, B.; Liu, W.; Liu, C.; Xiao, H.; McClements, D. J., Enhancing nutraceutical

bioavailability using excipient emulsions: Influence of lipid droplet size on solubility and bioaccessibility of powdered curcumin. J. Funct. Foods 2015, 15, 72-83. (8)

Salvia-Trujillo, L.; McClements, D. J., Enhancement of lycopene bioaccessibility from tomato

juice using excipient emulsions: Influence of lipid droplet size. Food Chem. 2016, 210, 295-304. (9)

Salvia-Trujillo, L.; McClements, D. J., Improvement of beta-Carotene Bioaccessibility from

Dietary Supplements Using Excipient Nanoemulsions. J. Agric. Food Chem. 2016, 64, 4639-4647. (10) Chen, X.; Zou, L.; Liu, W.; McClements, D. J., Potential of Excipient Emulsions for Improving Quercetin Bioaccessibility and Antioxidant Activity: An in Vitro Study. Journal of agricultural and food chemistry 2016, 64, 3653-3660. (11) Chen, X.; McClements, D. J.; Zhu, Y.; Chen, Y.; Zou, L.; Liu, W.; Cheng, C.; Fu, D.; Liu, C., Enhancement of the solubility, stability and bioaccessibility of quercetin using protein-based excipient emulsions. Food Res. Int. 2018, 114, 30-37. (12) Conquer, J. A.; Maiani, G.; Azzini, E.; Raguzzini, A.; Holub, B. J., Supplementation with Quercetin Markedly Increases Plasma Quercetin Concentration without Effect on Selected Risk Factors for Heart Disease in Healthy Subjects. J. Nutr. 1998, 128, 593-597. (13) Boots, A. W.; Haenen, G. R.; Bast, A., Health effects of quercetin: from antioxidant to nutraceutical. Eur. J. Pharmacol. 2008, 585, 325-337. (14) Panchal, S. K.; Poudyal, H.; Brown, L., Quercetin ameliorates cardiovascular, hepatic, and metabolic changes in diet-induced metabolic syndrome in rats. J. Nutr. 2012, 142, 1026-1032. (15) Egert, S.; Bosy-Westphal, A.; Seiberl, J.; Kürbitz, C.; Settler, U.; Plachta-Danielzik, S.; Wagner, A. E.; Frank, J.; Schrezenmeir, J.; Rimbach, G.; Wolffram, S.; Müller, M. J., Quercetin reduces systolic blood pressure and plasma oxidised low-density lipoprotein concentrations in overweight subjects with a high-cardiovascular disease risk phenotype: a double-blinded, placebo-controlled cross-over study. Br. J. Nutr. 2009, 102, 1065-1074. (16) Murakami, A.; Ashida, H.; Terao, J., Multitargeted cancer prevention by quercetin. Cancer Lett. 2008, 269, 315-325. (17) Kawabata, K.; Mukai, R.; Ishisaka, A., Quercetin and related polyphenols: new insights and implications for their bioactivity and bioavailability. Food Funct. 2015, 6, 1399-1417. (18) Wang, W.; Sun, C.; Mao, L.; Ma, P.; Liu, F.; Yang, J.; Gao, Y., The biological activities, chemical stability, metabolism and delivery systems of quercetin: A review. Trends Food Sci. Technol. 2016, 56, 21-38. (19) Nabavi, S. F.; Russo, G. L.; Daglia, M.; Nabavi, S. M., Role of quercetin as an alternative for 23



478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519

Page 24 of 41

obesity treatment: you are what you eat! Food Chem. 2015, 179, 305-310. (20) Cai, X.; Fang, Z.; Dou, J.; Yu, A.; Zhai, G., Bioavailability of quercetin: Problems and promises. Curr. Med. Chem. 2013, 20, 2572-2582. (21) Buldo, P.; Benfeldt, C.; Carey, J. P.; Folkenberg, D. M.; Jensen, H. B.; Sieuwerts, S.; Vlachvei, K.; Ipsen, R., Interactions of milk proteins with low and high acyl gellan: Effect on microstructure and textural properties of acidified milk. Food Hydrocolloid. 2016, 60, 225-231. (22) Prajapati, V. D.; Jani, G. K.; Zala, B. S.; Khutliwala, T. A., An insight into the emerging exopolysaccharide gellan gum as a novel polymer. Carbohydr Polym 2013, 93, 670-678. (23) López, D. N.; Galante, M.; Alvarez, E. M.; Risso, P. H.; Boeris, V., Effect of the espina corona gum on caseinate acid-induced gels. LWT-Food Sci. Technol. 2017, 85, 121-128. (24) Kim, H. J.; White, P. J., Impact of the Molecular Weight, Viscosity, and Solubility of β-Glucan on in Vitro Oat Starch Digestibility. J. Agric. Food Chem. 2013, 61, 3270-3277. (25) Bai, Y.; Wu, P.; Wang, K.; Li, C.; Li, E.; Gilbert, R. G., Effects of pectin on molecular structural changes in starch during digestion. Food Hydrocolloid. 2017, 69, 10-18. (26) Hu, B.; Chen, Q.; Cai, Q.; Fan, Y.; Wilde, P. J.; Rong, Z.; Zeng, X., Gelation of soybean protein and polysaccharides delays digestion. Food Chem. 2017, 221, 1598-1605. (27) Soukoulis, C.; Tsevdou, M.; Andre, C. M.; Cambier, S.; Yonekura, L.; Taoukis, P. S.; Hoffmann, L., Modulation of chemical stability and in vitro bioaccessibility of beta-carotene loaded in kappa-carrageenan oil-in-gel emulsions. Food Chem. 2017, 220, 208-218. (28) Al-Yafeai, A.; Bohm, V., In Vitro Bioaccessibility of Carotenoids and Vitamin E in Rosehip Products and Tomato Paste As Affected by Pectin Contents and Food Processing. J. Agric. Food Chem. 2018, 66, 3801-3809. (29) Li, Z.; Dai, L.; Wang, D.; Mao, L.; Gao, Y., Stabilization and Rheology of Concentrated Emulsions Using the Natural Emulsifiers Quillaja Saponins and Rhamnolipids. J. Agric. Food Chem. 2018, 66, 3922-3929. (30) Matia-Merino, L.; Lau, K.; Dickinson, E., Effects of low-methoxyl amidated pectin and ionic calcium on rheology and microstructure of acid-induced sodium caseinate gels. Food Hydrocolloid. 2004, 18, 271-281. (31) Makkhun, S.; Khosla, A.; Foster, T.; McClements, D. J.; Grundy, M. M.; Gray, D. A., Impact of extraneous proteins on the gastrointestinal fate of sunflower seed (Helianthus annuus) oil bodies: a simulated gastrointestinal tract study. Food Funct. 2015, 6, 125-134. (32) Chen, X.; McClements, D. J.; Wang, J.; Zou, L.; Deng, S.; Liu, W.; Yan, C.; Zhu, Y.; Cheng, C.; Liu, C., Coencapsulation of (−)-Epigallocatechin-3-gallate and Quercetin in Particle-Stabilized W/O/W Emulsion Gels: Controlled Release and Bioaccessibility. J. Agric. Food Chem. 2018, 66, 3691-3699. (33) Guo, Q.; Bellissimo, N.; Rousseau, D., Role of gel structure in controlling in vitro intestinal lipid digestion in whey protein emulsion gels. Food Hydrocolloid. 2017, 69, 264-272. (34) McIntyre, I.; M, O. S.; D, O. R., Altering the level of calcium changes the physical properties and digestibility of casein-based emulsion gels. Food Funct. 2017, 8, 1641-1651. (35) Li, Y.; McClements, D. J., New mathematical model for interpreting pH-stat digestion profiles: impact of lipid droplet characteristics on in vitro digestibility. J. Agric. Food Chem. 2010, 58, 8085-8092. 24


Page 25 of 41


520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561

(36) Le, X. T.; Rioux, L. E.; Turgeon, S. L., Formation and functional properties of protein-polysaccharide electrostatic hydrogels in comparison to protein or polysaccharide hydrogels. Adv. Colloid Interface Sci. 2017, 239, 127-135. (37) McClements, D. J., Food Emulsions: Principles, Practices, and Techniques. 3rd ed.; CRC Press: Boca Raton, FL, 2015. (38) Williams, P. A.; Phillips, G. O., Gum arabic In Handbook of Hydrocolloids (Second Edition), Phillips, G. O.; Williams, P. A., Eds. Woodhead Publishing: Cambridge, U.K., 2009; pp 252-273. (39) Nag, A.; Han, K.-S.; Singh, H., Microencapsulation of probiotic bacteria using pH-induced gelation of sodium caseinate and gellan gum. Int. Dairy J. 2011, 21, 247-253. (40) Yamamoto, F.; Cunha, R. L., Acid gelation of gellan: Effect of final pH and heat treatment conditions. Carbohyd. Polym. 2007, 68, 517-527. (41) Picone, C. S. F.; da Cunha, R. L., Interactions between milk proteins and gellan gum in acidified gels. Food Hydrocolloid. 2010, 24, 502-511. (42) Dickinson, E., Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloid. 2003, 17, 25-39. (43) Thomar, P.; Nicolai, T., Heat-induced gelation of casein micelles in aqueous suspensions at different pH. Colloids Surf. B. Biointerfaces 2016, 146, 801-807. (44) Siew, D. C. W.; Heilmann, C.; Easteal, A. J.; Cooney, R. P., Solution and Film Properties of Sodium Caseinate/Glycerol and Sodium Caseinate/Polyethylene Glycol Edible Coating Systems. J. Agric. Food Chem. 1999, 47, 3432-3440. (45) Chevallier, M.; Riaublanc, A.; Lopez, C.; Hamon, P.; Rousseau, F.; Thevenot, J.; Croguennec, T., Increasing the heat stability of whey protein-rich emulsions by combining the functional role of WPM and caseins. Food Hydrocolloid. 2018, 76, 164-172. (46) Luo, Y.; Pan, K.; Zhong, Q., Casein/pectin nanocomplexes as potential oral delivery vehicles. Int. J. Pharm. 2015, 486, 59-68. (47) Chen, Y. C.; Chen, C. C.; Chen, S. T.; Hsieh, J. F., Proteomic profiling of the coagulation of milk proteins induced by glucono-delta-lactone. Food Hydrocolloid. 2016, 52, 137-143. (48) Guo, Q.; Ye, A.; Lad, M.; Dalgleish, D.; Singh, H., Effect of gel structure on the gastric digestion of whey protein emulsion gels. Soft Matter 2014, 10, 1214-1223. (49) Pinheiro, A. C.; Coimbra, M. A.; Vicente, A. A., In vitro behaviour of curcumin nanoemulsions stabilized by biopolymer emulsifiers – Effect of interfacial composition. Food Hydrocolloid. 2016, 52, 460-467. (50) Zhang, C.; Xu, W.; Jin, W.; Shah, B. R.; Li, Y.; Li, B., Influence of anionic alginate and cationic chitosan

on

physicochemical

stability

and

carotenoids

bioaccessibility

of

soy

protein

isolate-stabilized emulsions. Food Res. Int. 2015, 77, 419-425. (51) Buchweitz, M.; Kroon, P. A.; Rich, G. T.; Wilde, P. J., Quercetin solubilisation in bile salts: A comparison with sodium dodecyl sulphate. Food Chem. 2016, 211, 356-364. (52) Ortega, N.; Reguant, J.; Romero, M.-P.; Macia, A.; Motilva, M.-J., Effect of fat content on the digestibility and bioaccessibility of cocoa polyphenol by an in vitro digestion model. J. Agric. Food Chem. 2009, 57, 5743-5749. (53) Mun, S.; Kim, Y. R.; McClements, D. J., Control of beta-carotene bioaccessibility using starch-based filled hydrogels. Food Chem. 2015, 173, 454-461. 25



Page 26 of 41

562

26


Page 27 of 41


563

Funding

564

The authors are grateful for the financial support of this study by the National

565

Natural Science Foundation of China (31601468 and 21766018), Major Discipline

566

Academic

567

(20162BCB22009), the Key Project of Natural Science Foundation of Jiangxi

568

Province, China (20171ACB20005), Open Project Program of State Key Laboratory

569

of Food Science and Technology, Nanchang University (SKLF-ZZB-201717), the

570

Postgraduate Innovation Fund of Jiangxi Province (YC2017-B010) and the

571

Postgraduate Innovation Fund of Nanchang University (cx2017127).

572

Notes

573

Technical Leader

Training

Plan

Project

of

Jiangxi

Province

The authors declare no conflict of interest.

574

27



Page 28 of 41

575

FIGURE CAPTIONS

576

Figure 1. Illustration of the experimental setup for the production of fluid and gelled

577

nutraceutical emulsions. GDL = glucono-delta-lactone.

578

Figure 2. Compatibility of natural polysaccharides with emulsions under neutral

579

conditions. Gel = gellan gum, Ara = arabic gum, Pec = pectin, Car = carrageenan,

580

Xan = xanthan gum.

581

Figure 3. (a) Elastic modulus, (b) Loss modulus, (C) Loss factor and (d) pH value of

582

casein-stabilized emulsions with different gellan gum content (0% - 0.3%) change as

583

a function of time during acidification process (glucono-delta-lactone was added

584

into emulsion samples at 0 h) .

585

Figure 4. Visual images of fluid and gelled nutraceutical emulsions (after

586

acidification for 12 h) with different gellan gum content (0% - 0.3%).

587

Figure 5. Confocal fluorescence microscopy observations of fluid and gelled

588

nutraceutical emulsions (after acidification for 12 h) with different gellan gum

589

content (0% - 0.3%). (a) Oil stain (excitation at 488 nm); (b) Protein stain (excitation

590

at 633 nm); (c) Combined image of a and b.

591

Figure 6. SDS-PAGE profile of sodium caseinate (lane 1, standard marker; lane 2,

592

sodium caseinate; lane 3, fluid nutraceutical emulsions with 0% gellan gum; lane 4,

593

fluid nutraceutical emulsions with 0.1% gellan gum; lane 5, fluid nutraceutical

594

emulsions with 0.2% gellan gum; lane 6, gelled nutraceutical emulsions with 0%

595

gellan gum; lane 7, gelled nutraceutical emulsions with 0.1% gellan gum; lane 8, 28


Page 29 of 41


596

gelled nutraceutical emulsions with 0.2% gellan gum).

597

Figure 7. SDS-PAGE profile of the in vitro gastric digestion of sodium caseinate: (a)

598

fluid nutraceutical emulsions with 0% gellan gum; (b) fluid nutraceutical emulsions

599

with 0.1% gellan gum; (c) fluid nutraceutical emulsions with 0.2% gellan gum; (d)

600

gelled nutraceutical emulsions with 0% gellan gum; (e) gelled nutraceutical

601

emulsions with 0.1% gellan gum; (f) gelled nutraceutical emulsions with 0.2%

602

gellan gum (lane 1, standard marker; lane 2, sodium caseinate; lane 3-8, digested for

603

2, 5, 10, 30, 60, and 120 min).

604

Figure 8. Free fatty acids (FFA) release profile for fluid and gelled nutraceutical

605

emulsions (F-0%, fluid nutraceutical emulsions with 0% gellan gum; F-0.1%, fluid

606

nutraceutical emulsions with 0.1% gellan gum; F-0.2%, fluid nutraceutical

607

emulsions with 0.2% gellan gum; G-0%, Gelled nutraceutical emulsions with 0%

608

gellan gum; G-0.1%, Gelled nutraceutical emulsions with 0.1% gellan gum; G-0.2%,

609

Gelled nutraceutical emulsions with 0.2% gellan gum).

610

Figure 9. (a) Bioaccessibility of quercetin (QT) in fluid and gelled nutraceutical

611

emulsions; (b) Visual images of digesta from different gastrointestinal stage (F-0%,

612

fluid nutraceutical emulsions with 0% gellan gum; F-0.1%, fluid nutraceutical

613

emulsions with 0.1% gellan gum; F-0.2%, fluid nutraceutical emulsions with 0.2%

614

gellan gum; G-0%, Gelled nutraceutical emulsions with 0% gellan gum; G-0.1%,

615

Gelled nutraceutical emulsions with 0.1% gellan gum; G-0.2%, Gelled nutraceutical

616

emulsions with 0.2% gellan gum). Samples designated with different lowercase 29



617

Page 30 of 41

superscripts (a, b, c…) mean significantly different (Duncan, p < 0.05).

618

30


Page 31 of 41


Tables Table 1. Impact of pH on the Zeta-potential of Gellan Gum and Fluid Emulsions with Different Gellan Gum Contents (0% - 0.3 %). α Zeta potential (mV) pH

α

0%

0.1%

0.2%

0.3%

gellan gum

2

23.5±0.8 e A

24.1±0.3 e A

24.2±0.6 d A

25.7±1.0 d A

-3.0±0.6 c

3

35.9±0.6 f A

36.5±0.5 f A

41.7±1.3 e B

40.5±2.4 e B

-33.5±1.0 b

4

16.4±0.3 d B

13.5±1.2 d A

23.3±0.5 d C

23.4±0.4 d C

-47.6±4.6 a

5

-16.4±0.3 c A

-24.9±1.1 c C

-21.7±0.3 c B

-21.1±0.4 c B

-48.0±4.7 a

6

-33.4±0.6 b A

-39.8±1.2 b B

-40.6±1.0 b B

-44.4±4.9 a B

-41.1±4.9 a

7

-45.3±1.6 a B

-49.3±1.6 a C

-46.1±1.6 a B

-36.4±0.9 b A

-43.3±5.0 a

Different lowercase letters (a, b, c) in the same column or different capital letters

(A, B, C) in the same row for each parameter were significantly different (Duncan, p < 0.05).

31



Page 32 of 41

Figure 1

32


Page 33 of 41


Figure 2

33



Page 34 of 41

Figure 3

34


Page 35 of 41


Figure 4

35



Page 36 of 41

Figure 5

36


Page 37 of 41


Figure 6

37



Page 38 of 41

Figure 7

38


Page 39 of 41


Figure 8

39



Page 40 of 41

Figure 9

40


Page 41 of 41


Table of Contents Graphic

41


Gastrointestinal Fate of Fluid and Gelled Nutraceutical Emulsions

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