Influence of Dairy Emulsifier Type and Lipid Droplet Size on

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Influence of Dairy Emulsifier Type and Lipid Droplet Size on Gastrointestinal Fate of Model Emulsions: In vitro Digestion Study Li Liang, Xiaoyun Zhang, Xing-Guo Wang, Qingzhe Jin, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02959 • Publication Date (Web): 02 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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Journal of Agricultural and Food Chemistry

Influence of Dairy Emulsifier Type and Lipid Droplet Size on Gastrointestinal Fate of Model Emulsions: In vitro Digestion Study

Li Liang a, b, Xiaoyun Zhang, b Xingguo Wang a, Qingzhe Jin a, *, David Julian McClements b,

*

a

School of Food Science and Technology, Collaborative Innovation Center of Food Safety

and Quality Control in Jiangsu Province, National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, Jiangsu 214122, China b

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

USA

* Corresponding authors E-mail addresses: [email protected] (Q. Jin), [email protected] (D.J. McClements). 1

1

ACS Paragon Plus Environment


2

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ABSTRACT

3

Human breast milk is a natural emulsion containing relatively large triacylglycerol

4

droplets coated by a distinct interfacial layer, known as the milk fat globule membrane

5

(MFGM).

6

milk in an infant’s gastrointestinal tract (GIT), but the membrane architecture is susceptible

7

to disruption by industrial processes. To formulate infant formula that simulates the

8

gastrointestinal behavior of breast milk, food manufacturers require knowledge of the impact

9

of the interfacial properties on the gastrointestinal fate of fat globules.

The unique properties of the MFGM impacts the release of nutrients from breast

In this study, a

10

simulated GIT was utilized to monitor the gastrointestinal fate of emulsified corn oil with

11

different dairy emulsifiers, including sodium caseinate, lactoferrin (LF), whey protein isolate

12

(WPI), and milk phospholipids (MPL) isolated from MFGM. The influence of droplet size

13

on the gastrointestinal fate of the MPL-stabilized emulsions was also examined. Our findings

14

provide valuable information for the optimization of infant formula and dairy-based

15

nutritional beverages.

16 17

Keywords: emulsifier type; droplet size; gastrointestinal fate; physicochemical properties; lipid digestion

2


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18

INTRODUCTION

19

Infant formula is designed to provide the full complement of macronutrients and

20

micronutrients to infants who do not have access to breast milk. Ideally, infant formula

21

should be created with a composition, structure, and digestibility as similar to human breast

22

milk as possible. Commercial infant formula is one of the earliest functional foods

23

developed by the food industry.

24

macronutrients (such as carbohydrates, proteins, and lipids), micronutrients (such as

25

vitamins and minerals), and other components (such as probiotics and prebiotics) similar

26

to those found in breast milk. 1, 2 In terms of milk fat, which typically provides around 40

27

to 50% of the calories to the infant, infant formula manufacturers aim to match the total fat

28

content and fatty acid profile of breast milk.

29

design the lipid droplets in infant formula so that they mimic the unique composition and

30

architecture of milk fat globules (MFGs) in breast milk and therefore behave in a similar

31

fashion in the infant digestive tract. 5

Manufacturers design these products to contain

3, 4

Moreover, manufacturers would like to

32

Developments in lipidomic analysis and spectroscopic imaging have led to rapid advances

33

in our understanding of the secretion, structure, and composition of MFGs and their

34

membranes (MFGM) over the past decade.

35

triglyceride cores coated by MFGM. The MFGM has a tri-layer structure that consists mainly

36

of phospholipids. Specifically, phospholipids are organized as a monolayer in contact with

6, 7

Structurally, native MFGs are composed of

3



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the triglyceride core and a bilayer isolating it from the aqueous environment, with a number

38

of proteins embedded inside it. The diameter of native MFGs ranges from around 0.1 to 15

39

µm with an average around 4 µm. At present, most commercial infant formula products

40

contain lipid droplets that have different interfacial compositions and dimensions than those

41

in MFGs.

42

smaller with a mean particle diameter around 0.5 µm (range 0.1 to 1 µm) and an interfacial

43

layer comprised mainly of dairy proteins or surfactants (such as citrate glycerides,

44

monoglycerides, or diglycerides). 9

8

In particular, the lipid droplets in commercial products are typically much

45

Formula feeding could increase the incidence of obesity and metabolic syndrome in adult

46

life compared to breast feeding. 10, 11 Considering that the lipolysis rate of milk fat is likely

47

to impact its absorption and metabolism, 12, 13 the long-term health benefits of breastfeeding

48

may at least be partly related to the complex architecture of the MFGM. 14

49

therefore attempting to create lipid droplets in infant formula products that more closely

50

simulate the digestion and absorption of MFGs in the gastrointestinal tract.

51

Gallier et al. fabricated biomimetic fat globules by adsorption of bovine MFGM material to

52

the surfaces of relatively large lipid droplets (around 4 µm) in oil-in-water (O/W) emulsions.

53

15

54

accumulation in adult mice. 16-18

55

Researchers are

For instance,

The same group then showed that their biomimetic infant formula reduced excess body fat

In general, the rate and final extent of lipid digestion depends on the physicochemical 4


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characteristics of the interfacial layer, such as surface area, charge, and composition, and

57

therefore controlling these properties may lead to the design of more effective MFG-

58

analogs. 19-22

59

bovine milk due to industrial processing, such as homogenization and heat treatment, also

60

modified the kinetics of lipid digestion.

61

considered when developing biomimetic infant formula products.

62

applications in nutritional and pharmaceutical products,

63

commercially available in sufficient quantities and quality for use in infant formula and

64

other dairy-based beverage products. 25

Our previous study showed that changes in the interfacial properties of raw

23

Consequently, these factors should also be

24

Due to its potential

MFGM has recently become

65

In previous studies, researchers have shown that industrial processing of raw bovine

66

MFGs promoted disruption of the native interfacial membrane and adsorption of soluble

67

proteins, such as caseins and whey proteins, from the surrounding aqueous phase. 23, 26 In

68

this study, we investigated the influence of dairy emulsifier type and droplet size on the

69

gastrointestinal fate of model infant formula.

70

different dairy emulsifiers were prepared and characterized, including lactoferrin (LF),

71

sodium caseinate, whey protein isolate (WPI), and milk phospholipids (MPL) obtained

72

from MFGM. These emulsions then underwent a simulated gastrointestinal tract (GIT) and

73

the rate and final extent of lipid digestion was monitored.

74

influence of droplet size on the gastrointestinal fate of the MPL-stabilized emulsions to

Corn O/W emulsions surrounded by

5


In addition, we examined the


75

determine if their digestibility could be modulated using this approach. To the authors

76

knowledge, there have been no previous studies comparing the digestion of lipid droplets

77

coated with MPL and various other dairy proteins.

78

MATERIALS AND METHODS

79

Materials

80

The information about the powdered dairy proteins used in this study is listed in Table

81

1. MFGM phospholipid (MPL) was provided by Fonterra Co-operative Group Limited

82

(Rosemont, IL). The phospholipid concentrate (PC 700) was obtained from bovine milk

83

and its composition is shown in Table 2. MPL contained about 16.5% sphingomyelin,

84

which is not typically present in plant phospholipids. The unique composition of MPL

85

makes it possible to be applied in dairy and other functional food industry. All the

86

emulsifiers were used without further purification. All concentrations and composition data

87

cited were obtained from the manufacturers.

88

The fluorescent dye, including fluorescein isothiocyanate (FITC) and Nile red, and the

89

enzymes and extracts from digestive juices including mucin and pepsin from porcine stomach,

90

porcine bile extract, and porcine lipase (type II), were obtained from Sigma-Aldrich (Sigma

91

Chemical Co., St. Louis, MO). All other chemicals and reagents were analytical grade and

92

purchased from Fisher Scientific (Pittsburgh, PA). Corn oil was purchased from a local 6


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grocery store. All aqueous solutions were prepared with high purity water (Nanopure Infinity,

94

Barnstaeas International, Dubuque, IA).

95

Emulsion Preparation

96

Emulsions (O/W) in this study consisted of 10% corn oil and 90% aqueous solution. 27

97

The aqueous solution contained 1% emulsifier dissolved in buffer solution (5 mM

98

phosphate, pH 7.0). First, a coarse emulsion was produced by blending the lipid and

99

aqueous phases together with a high-speed pre-mix device for 2 min (M133/1281-0,

100

Biospec Products, Inc., ESGC, Switzerland), and then a coarse emulsion was prepared

101

using a high-pressure microfluidizer (M110Y, Microfluidics, Newton, MA) at a pressure of

102

83 MPa (three passes). Unless otherwise specified, emulsions were prepared under this

103

condition, and the concentration in this study was percentage of mass unless otherwise

104

specified.

105

Emulsifier types. Aqueous phases with different emulsifiers were prepared (LF,

106

caseinate, WPI, MPL), and the total emulsifier concentration was 1%. As MPL was

107

difficult to uniformly disperse in the aqueous phase, sonication was used to prepare the

108

aqueous phase with this emulsifier before preparing the emulsions. As described previously,

109

the following sonication procedure was used: 70% amplitude with pulse length of 5 s on

110

and 3 s off for 20 cycles. 27

111

Droplet size. The minimum mean particle diameter (d3,2) that could be achieved with 1% 7



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112

MPL under standardized homogenization conditions was greater than 2 µm, which was

113

considerably bigger than the droplets obtained using the other emulsifiers.

114

reason, a higher level of MPL (2%) was utilized in this part of the study to obtain smaller

115

droplets. MPL stabilized emulsions with different mean particle diameters were fabricated

116

using different homogenization conditions: (i) only the high-speed pre-mix device was

117

used for 2 min (large droplet size); (ii) a high-speed pre-mix device and a microfluidizer at

118

69 MPa for three passes were used (medium droplet size); (iii) a high-speed pre-mix device

119

and a microfluidizer at 138 MPa for three passes were used (small droplet size).

120

In vitro Digestion Model

For this

121

All the samples were subjected to a simulation of human gastrointestinal conditions，

122

including the mouth, stomach, and small intestine. In the current study, we utilized a static

123

GIT model with intestinal fluid compositions based on the values established in our

124

previous studies

125

model proposed by Minekus and co-workers 29. Control emulsions were prepared using a

126

non-digestible oil (hexadecane) as the hydrophobic core rather than corn oil.

28

.

This model is fairly similar to the standardized in vitro digestion

127

Before conducting the in vitro digestion experiments, several simulated digestive fluids

128

were prepared. Artificial simulated saliva stock solution (sodium chloride 27.28 mM,

129

ammonium nitrate 4.10 mM, monopotassium phosphate 4.67 mM, potassium chloride 2.71

130

mM, potassium citrate 0.95 mM, uric acid sodium salt 0.11 mM, urea 3.30 mM, lactic acid 8


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sodium salt 1.30 mM), simulated gastric fluid (SGF) (sodium chloride 34.22 mM,

132

hydrochloric acid 226.11 mM) and simulated intestinal fluid (SIF) (sodium chloride 3.75

133

M and calcium chloride dihydrate 249.49 mM) were prepared. Simulated saliva fluid (SSF)

134

was prepared by dissolving 0.6 g porcine gastric mucin (type II) into 20 g of saliva stock

135

solution, and the fluid should be stirred overnight at 4 °C to make sure mucin dissolved

136

fully. Bile salts dissolved in buffer solution (pH 7.0, 53.57 g/L) was prepared 24h before

137

the small intestinal conditions, since the bile salts was hard to dissolve fully. All the

138

simulated digestion fluids should be preheated to 37 °C before each digestion phase. 30

139

Initial Phase: First, all the samples were diluted to obtain 2% lipid content, which was

140

the suitable concentration for the following digestion procedure as validated in our

141

previous study. 31 Then, 20 g of the sample was preheated to 37 °C in an incubator shaker

142

(Model 4080, New Brunswick Scientific, NJ).

143

Mouth Phase: First, 20 g of the emulsion was mixed 20 g of SSF. Second, the mixture was

144

kept in a water bath at 37 °C, and pH was adjusted to 6.8. Third, the mixture was swirled at

145

100 rpm for 2 min to mimic the mouth condition.

146

Stomach Phase: First, 0.064 g of pepsin was dissolved in 20 g of SGF to get the gastric

147

working solution, and the solution was preheated to 37 °C. Second, 20 g of the bolus sample

148

taken from mouth phase was added to the preheated gastric working solution. Third, the pH

149

of the mixture was adjusted to 2.5 and swirled at 37 °C for 2 h with the agitation speed of 9



150

100 rpm. Lipase was not contained in this phase, because reliable sources of gastric lipase is

151

hard to obtain now. 32

152

Small Intestine Phase: First, 30 g of the chyme sample obtained from last stage was kept

153

at 37 °C water bath and then adjusted to pH 7.000. Second, 1.5 mL of SIF and 3.5 mL of bile

154

salt solution was added into the chyme sample with continuous stirring. Third, the pH of the

155

mixture was adjusted to 7.000 again, followed by 2.5 mL of porcine pancreatic lipase (24

156

mg/mL) with continuous stirring. During the 2h digestion period, a pH-stat automatic

157

titration unit (Metrohm USA Inc., Riverview, FL) was utilized to record the volume of NaOH

158

solution (0.15 M) consumed which would neutralize the released free fatty acids (FFA) from

159

oil phase at 37 °C.

160 161

The following equation was utilized to calculate the percentage of FFA released in small intestine phase:

162 163

FFA% =

VNaOH × mNaOH × Mlipid × 100 Wlipid × 2

164 165

Where, VNaOH is the volume of NaOH consumed (mL), mNaOH is the molarity of the NaOH

166

solution (0.15 M), Mlipid is the average molecular weight of the corn oil (824 g/mol), and

167

Wlipid is the mass of the oil in the small intestine phase (0.15g). 33

168

Determination of Particle Size, Charge and Microstructure 10


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The particle size distribution (PSD) and mean particle diameter of samples was

170

measured by a static light scattering instrument (Mastersizer 2000, Malvern Instruments,

171

Westborough, MA). Prior to the measurements, samples were diluted in appropriate

172

aqueous solutions to avoid multiple scattering effects and stirred at speed of 1200 rpm to

173

ensure system homogeneity. Specifically, pH 2.5-adjusted distilled water was used to dilute

174

samples from stomach. While phosphate buffer with pH 7.0 was used to dilute samples

175

from other digestion stages. The refractive indices were 1.330 and 1.470 for phosphate

176

buffer solution and corn oil, respectively. The particle size was reported as the surface-

177

weight mean diameters (d32), which were calculated from the full particle size distribution.

178

34

179

The ζ-potential of these samples was tested using a particle electrophoresis device based

180

on dynamic light scattering (Nano-ZS, Malvern Instruments, Westborough, MA). The buffer

181

solutions used to dilute samples from different digestion stages and refractive indices in this

182

part were the same as in particle size measurements.

183

The microstructures of the samples from different GIT stages were visualized by a laser

184

scanning confocal microscope (Nikon D-Eclipse C1 80i, Nikon, Melville, NY).

185

objective lens (60×, oil immersion) and eyepiece (10×) were selected to acquire the

186

magnified microstructure images. Prior to analysis, the protein and lipid phases were stained

187

by FITC (10 mg/mL dimethyl sulfoxide) and Nile red (1 mg/mL ethanol), respectively. The 11


The


188

excitation and emission spectrum were 488 nm and 515 nm for FITC, while they were 543

189

nm and 605 nm for Nile red. All micrographs were taken and exported by NIS-Elements

190

analysis software (Nikon, Melville, NY).

191

Statistical Analysis

192

In this study, emulsions were prepared freshly in triplicate, and measurements were

193

conducted twice per sample. The means and standard deviations were calculated from these

194

results. The statistical significance was analyzed by a one-way ANOVA and tested by post-

195

hoc Duncan multiple range test (p < 0.05) (SPSS 19.0, IBM Corporation, Armonk, NY).

196

RESULTS AND DISCUSSION

197

Influence of Emulsifier Type and Droplet Size on Gastrointestinal Fate.

198

The impact of emulsifier type and droplet size on the gastrointestinal fate of the model

199

infant formula were examined using a simulated GIT model. The mean droplet diameter,

200

PSD, ζ-potential and microstructure of the emulsions was conducted after each GIT phase.

201

Initial Phase

202

Particle Stability: Initially, the emulsion coated by 1% MPL contained significantly larger

203

lipid droplets (d32 = 2.48 µm) compared with those coated by the three dairy proteins. The

204

emulsions stabilized by lactoferrin contained somewhat larger droplets (d32 = 0.29 µm) than 12


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those containing either sodium caseinate or WPI (d32 = 0.15 µm) (Figure 1a). The proteins

206

therefore seem to be more effective at promoting droplet disruption and inhibiting droplet

207

coalescence during homogenization than the phospholipids. The larger droplets in the LF-

208

stabilized emulsions may be because it has an appreciably higher molecular weight than the

209

other two dairy proteins and so required a higher amount to saturate the droplet surfaces. 35

210

Moreover, some droplet flocculation may have occurred in this emulsion because the initial

211

pH was close to the isoelectric point of lactoferrin (pI ~ 8) and because absorption of anionic

212

phosphate ions (PO4-) from the buffer solution onto the cationic oil droplet surfaces may have

213

caused some charge neutralization. 36

214

An attempt was made to produce MPL-stabilized emulsions containing smaller droplets

215

so as to facilitate the comparison between different droplet size.

216

level of MPL (2%) was used to produce these emulsions.

217

conditions, we were able to produce MPL-stabilized emulsions with mean droplets diameters

218

(d32) of 10.70 µm (large), 2.70 µm (medium), and 0.69 µm (small), respectively (Figure 1b).

219

Emulsions coated by sodium caseinate, WPI and MPL initially had monomodal PSD

220

(Figure 2a). While the LF-stabilized emulsions had broad bimodal distributions (Figure 2a).

221

The confocal microscopic images showed that the oil droplets (red) were spread in the

222

aqueous phases evenly in all the emulsions (Figure 3).

223

For this reason, a higher

By altering the homogenization

Particle Charge: The sign and magnitude of the droplets surface charge account for their 13



224

stability and interaction with other components. We therefore measured the electrical charge

225

at the surface of the lipid droplets before and after passing through the simulated GIT model.

226

Initially, all the emulsions had negative charges, and the magnitude of the ζ-potential

227

increased in the order: LF < caseinate < WPI ≈ MPL (Figure 4a). The fact that LF-stabilized

228

emulsion has negative charge (﹣9.6 mV) at pH 7.0 had been attributed to the adsorption of

229

PO4- from the buffer solution in previous study. 36 The relatively high negative ζ-potential of

230

the caseinate or WPI stabilized emulsions was due to the pH of the initial solution (pH 7.0)

231

was obviously higher than their isoelectric point (pI ~ 5), and there were no appreciably

232

differences among the two emulsions. The negative ζ-potential of the emulsions coated by

233

MPL was presumably due to the presence of anionic phospholipids from the MPL that had

234

adsorbed to the oil droplet surfaces. The initial electrical charge of the MPL-stabilized

235

emulsions containing different droplet sizes also showed relatively similar high negative

236

charges (Figure 4b).

237

Mouth Phase

238

Particle Stability: The mean particle diameter of the LF-stabilized emulsions remained

239

constant after passage through the mouth stage, whereas there was a slight increase for all

240

the other emulsions (Figure 1a). Only for the MPL-stabilized emulsions, this change was

241

statistically significant (p < 0.05). The PSD of the emulsions had broad and multimodal

242

distribution after exposure to the mouth phase, with a fraction of particles with similar 14


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dimensions as those in the initial emulsions and another fraction with much larger

244

dimensions, which suggests that part of the droplets flocculated under the mouth conditions

245

(Figure 2b). Confocal microscopy also indicated that droplet aggregation occurred,

246

especially for the MPL-stabilized emulsions (Figure 3a). The visible appearance of the

247

emulsions also suggested that droplet flocculation had occurred because faster creaming

248

occurred (Figure 1c). This effect has already been proposed previously, where it was due

249

to depletion or bridging flocculation effects of mucin in the SSF. 37

250

For the 2% MPL stabilized emulsions with different droplet dimensions, the mean

251

diameter of the droplets increased appreciably after passage through the mouth phase (p
10 µm for the protein-coated emulsions (p < 0.05),

278

while it was from around 3 µm to 6 µm for the MPL-stabilized emulsions (p > 0.05). These

279

results suggest that some form of droplet aggregation occurred within the gastric fluids. The

280

confocal microscope images suggest that the increase in droplet size was mainly due to 16


This

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droplet flocculation (Figure 3a). In contrast, significant droplet aggregation was not exhibited

282

in the MPL-stabilized emulsion. In line with our previous study, 23 the fat globules coated by

283

original membrane, including MPL and some membrane proteins, in raw milk also did not

284

present appreciable flocculation under simulated gastric conditions. Thus, MPL processes the

285

ability to stabilize droplets encapsulated in SGF. In contrast, several reasons contributed to

286

the fact that dairy proteins stabilized oil droplets are quite susceptible to aggregation under

287

gastric conditions. Reduced steric and electrostatic repulsion caused by pepsin hydrolyzation,

288

electrostatic screening resulted from relatively high ionic strength and bridging flocculation

289

arising from mucin molecules all contributed to the aggregation effect. 40, 41

290

For the 2% MPL stabilized emulsions with different droplets sizes, the mean diameter of

291

the particles in the small and medium size emulsions increased after passage through the

292

stomach phase (p < 0.05), but there was no change for the large size emulsions. Again, the

293

relative diameter of the droplets in these emulsions followed a similar trend to that in the

294

initial samples (Figure 1b). The PSD data clearly indicated an increased fraction of larger

295

particles in those samples that aggregated (Figure 2e). The confocal microscopy images

296

confirmed the particle size results, with appreciable aggregation only being observed in the

297

medium and small size emulsions (Figure 3b).

298

Particle Charge: After exposure to the region of stomach, the surface potentials of all the

299

samples were slightly positive (from + 0.9 to + 2.3 mV). There was no significant difference 17



300

among the different protein-stabilized emulsions (p < 0.05). The pH of the acidic gastric

301

fluids (pH 2.5) is fairly below the isoelectric point of all the dairy proteins, and so a strong

302

positive ζ-potential would be expected. 42 The fact that the ζ-potential measured was close to

303

neutral can be explained by charge neutralization resulting from the absorption of anionic

304

mucin molecules to the surfaces of the cationic protein-stabilized droplets. 41 The relatively

305

low magnitude of the charge on the lipid droplets would be insufficient to create a strong

306

electrostatic repulsion between them, which would partly explain the fact that extensive

307

droplet aggregation occurred. Furthermore, electrostatic screening and ion binding effects

308

resulted from the high ion levels in the gastric fluids would also have reduced the electrostatic

309

repulsion between the particles. The ζ-potential of all the MPL-stabilized emulsions was also

310

close to zero (Figure 4a), which can be attributed to similar reasons as for the protein-

311

stabilized emulsions.

312

Small Intestine Phase

313

Particle Stability: After incubation in the small intestinal conditions, the mean droplet size

314

of all the samples decreased appreciably (p < 0.05) (Figure 1a), with the effect being

315

especially pronounced for the MPL-stabilized emulsions (d32 = 0.32 µm). The PSD of the

316

protein-stabilized emulsions was monomodal, while that of the MPL-stabilized emulsion

317

became broad and bimodal (Figure 2d). The increase in the fraction of smaller particles in

318

the PSD (Figure 2d) and the microscopy images suggested that most of the large particles 18


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319

from the stomach stage have been dissociated in the small intestine stage (Figure 3a),

320

presumably due to the action of the digestive enzymes.

321

For the emulsions coated by 2% MPL, the mean particle diameters decreased steeply to

322

around 0.27 µm to 0.44 µm (p < 0.05) (Figure 1b). The PSD of all three of these emulsions

323

changed from monomodal under stomach conditions to bimodal under small intestinal

324

conditions (Figure 2e). The confocal microscopy images showed that most of the oil droplets

325

had been hydrolyzed after incubation in small intestine phase for 2h (Figure 3b).

326

Particle Charge: After passage through the small intestine stage, the surface potential of

327

all the samples became highly negative (-39.7 to -45.0 mV) (Figure 4), which can be caused

328

by the presence of various types of anionic species, such as bile salts, free fatty acids, peptides,

329

and phospholipids. 43

330

Influence of Dairy Emulsifier Type and Droplet Size on Lipid Digestion

331

The rate and final extent of lipid digestion of model infant formula containing oil droplets

332

surrounded by either dairy proteins or MPL was determined by measuring the FFA released

333

with an automatic titration device over small intestinal period. All the emulsions exhibited

334

fairly similar overall lipid digestion profiles (Figure 5). There was a rapid release of FFA in

335

the first 10 min followed by a gentle increment at later times until a relatively steady value

336

was obtained.

19



337

338

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Final Digestion Extent

There was no significant difference among emulsions coated by different emulsifiers in

339

terms of final digestion extent, with almost all of the lipids being digested (Figure 5a).

For

340

the emulsions coated by 2% MPL, the initial digestion rate and final digestion extent

341

depended on the diameter of the droplets, decreasing with increasing droplet size. The final

342

digestion extent of the large size emulsion was significantly lower than that of other two

343

emulsions (p < 0.05) (Figure 5b), which can be due to the reduction in the surface area of

344

lipid droplets for the lipase to access to.

345

Initial Digestion Rate

346

Although all the lipids in the samples were fully hydrolyzed by the end of the small

347

intestine phase, there were some differences in their initial digestion rates. More than half of

348

the lipid digestion happened in the first 10 min, thus the FFA released in this period was

349

examined. There was a short lag period in all the emulsions containing protein-coated

350

droplets, with the effect being most pronounced for the caseinate-stabilized emulsions

351

(Figure 6). This effect may have occurred because the protein-coated droplets were

352

flocculated extensively in the stomach phase and the flocs had to breakdown before the lipase

353

could readily access the lipid droplet surfaces.

354

the MPL-stabilized emulsions (Figures 5b and 6), which may have been because they did not

Conversely, a lag phase was not present in

20


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355


undergo extensive flocculation in the stomach.

356

In summary, the impact of dairy emulsifier type and oil droplet size on the

357

gastrointestinal fate of lipid droplets was studied under simulated GIT conditions. Our

358

results demonstrated that it is possible to modulate the lipid digestion profile through

359

changing the interfacial composition or surface area. Therefore, these results provide

360

valuable insights that may facilitate the development of functional foods with dairy

361

emulsifiers.

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nutritional requirements of particular populations, such as infants, the elderly, and patients

363

with metabolic diseases.

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layer on the performance of the emulsions.

365

of the lipid phase composition, as this is known to impact the digestion and solubilization

366

of both macronutrients (such as fatty acids) and micronutrients (such as vitamins and

367

nutraceuticals).

368

These functional foods could be manufactured to satisfy the specific

In this study, we mainly focused on the role of the interfacial In future studies, we will examine the impact

It should be noted that the simulated GIT utilized in this study is representative of the

369

healthy human adult gut.

Specialized in vitro digestion models are required to obtain a

370

more realistic understanding of the impact of interfacial properties on lipid digestion in

371

infants, the aged, or populations with particular disease states.

372

important to carry out in vivo studies, using animals or humans, to obtain a more detailed

373

understanding of the impact of interfacial properties on emulsion behavior. 21


Moreover, it will be

Nevertheless,


374

our study does provide some valuable insights into the role of interfacial properties on lipid

375

digestion in dairy-based emulsions that may be useful for optimizing their performance.

376

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Funding

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

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

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USDA, AFRI Grants (2016-08782). The research work was partly funded by the National

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First-class Discipline Program of Food Science and Technology (JUFSTR20180202). Li

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Liang thanks the Chinese Scholarship Council for support (No. 201506790027).

25



508

FIGURE CAPTIONS

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Figure 1. Influence of emulsifier type or droplet size on the mean particle diameter of corn oil

510

emulsions after passing through different GIT stages. (a) Emulsifier type. (b) Droplet size. (c)

511

Visual appearance of different emulsifier types. Different lowercase letters (a, b, c) represent

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significant differences (Duncan, p < 0.05) between different GIT stages (same emulsifier type or

513

droplet size). Different uppercase letters (A, B, C) represent significant differences (Duncan, p

Influence of Dairy Emulsifier Type and Lipid Droplet Size on

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