Influence of Homogenization and Thermal Processing on the

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Influence of homogenization and thermal processing on the gastrointestinal fate of bovine milk fat: In vitro digestion study Li Liang, Ce Qi, Xing-Guo Wang, Qingzhe Jin, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04721 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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Journal of Agricultural and Food Chemistry

Influence of homogenization and thermal processing on the gastrointestinal fate of bovine milk fat: In vitro digestion study Li Liang a, b, Ce Qi a, 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 Tel: 413 545 2275; Fax: 413 545 1262; Email [email protected]

ACS Paragon Plus Environment


1

ABSTRACT

2

Dairy lipids are an important source of energy and nutrients for infants and adults. The

3

dimensions, aggregation state, and interfacial properties of fat globules in raw milk are

4

changed by dairy processing operations, such as homogenization and thermal processing.

5

These changes influence the behavior of fat globules within the human gastrointestinal

6

tract (GIT).

7

short time (HTST) pasteurized milk, and ultrahigh temperature (UHT) pasteurized milk

8

samples was therefore determined using a simulated GIT.

9

different regions of the GIT depended on the degree of milk processing. Homogenization

10

increased the initial lipid digestion rate, but did not influence the final digestion extent.

11

Thermal processing of homogenized milk decreased the initial rate and final extent of

12

lipid digestion, which was attributed to changes in interfacial structure. These results

13

provide insights into the impact of dairy processing on the gastrointestinal fate of milk

14

fat.

The gastrointestinal fate of raw milk, homogenized milk, high temperature

The properties of particles in

15 16

Keywords: milk fat globules; homogenization; thermal processing; gastrointestinal; lipid

17

digestion

18


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INTRODUCTION

20

Milk contains a range of macro- and micro-nutrients, such as lipids, proteins,

21

carbohydrates, vitamins, and minerals, which support the growth and development of

22

mammalian newborns 1 .

The lipids in human milk provide infants with about 40-50%

23

of their energy needs 1 .

Milk lipids are secreted in the form of fat globules, which are

24

natural colloidal particles that release energy-rich lipids (e.g., triacylglycerols) and

25

bioactive molecules (e.g., essential fatty acids, conjugated linoleic acid, phospholipids,

26

sphingolipids, cholesterol, carotenoids, and lipid soluble vitamins A, D, E and K) in the

27

gastrointestinal tract (GIT) 2. Structurally, milk fat globules consist of a triglyceride

28

core surrounded by a milk fat globule membrane.

29

a tri-layer structure that is comprised of phospholipids, sphingolipids, cholesterol,

30

glycoproteins, and enzymes 3.

31

phospholipids surrounded by a bilayer of phospholipids containing proteins. The

32

diameter of native milk fat globules ranges from around 100 nm to 15 µm with a mean

33

value around 4 µm 4.

34

relationship between the properties of milk fat globules and their nutritional attributes

35

5-7

36

and lipophilic fluorescent probes has greatly facilitated the study of the milk fat globule

37

membrane structure 8-10.

38 39

The milk fat globule membrane has

In particular, it is constituted by a monolayer of

A considerable research effort has focused on establishing the

. In recent years, the development of confocal laser scanning microscopy (CLSM)

Humans continue to consume fluid milk after infancy, although this typically comes from non-human sources, such as cows and goats.


Indeed, about 30% of the


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11

40

lipids ingested within the Western-style diet come from milk

41

microbiological risks associated with the possible presence of human pathogens (such

42

as Campylobacter, Salmonella spp. and Escherichia coli) 2, and the susceptibility of raw

43

milk to rapid creaming due to the large size of native milk fat globules 12, commercially

44

available milk typically undergoes a number of processing operations to improve its

45

safety and extend its shelf life, with thermal processing and homogenization being the

46

most common

47

structure of the milk fat globules considerably

48

droplet diameter is reduced to around 1 µm or less, and the nature of the interfacial

49

layer surrounding the fat globules changes dramatically. The disruption of the fat

50

globules during homogenization increases the interfacial area, and the newly formed

51

interfaces cannot be completely covered by the milk fat globule membrane.

52

Consequently, surface-active components in the aqueous phase (such as casein and

53

whey protein) adsorb to the fat globule surfaces and form a new interfacial coating 16.

54

The properties of these new interfaces may also be changed appreciably during thermal

55

processing due to alterations in the conformation and interactions of the different types

56

of proteins present 15, 17 .

3, 13

.

Due to

. These technological treatments change the composition and 14-15

. After homogenization, the mean

57

Previous studies have shown that the nature of the interfacial layer surrounding the

58

fat droplets in milk and milk-analogs has a significant impact on lipid digestibility 14,

59

17-18

60

digested more rapidly than homogenized lipid droplets (mainly surrounded by added

.

For example, human milk fat globules (surrounded by native membranes) were


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proteins) in infant formula 19.

62

lipase depends on the composition of the phospholipids in the coatings surrounding

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lipid droplets

64

(whey proteins or sodium caseinate) were digested faster than those coated by lecithin

65

using a simulated small intestine model 21.

66

have also been shown to may modulate adipose tissue development in mice feeding

67

studies

68

fate and digestibility of fat droplets also depends on their aggregation state, such as the

69

degree of flocculation or coalescence

70

impacts the interfacial structure or aggregation state of milk fat globules would be

71

expected to impact milk fat digestion 23. Recently, the effects of processing on milk fat

72

digestion has been studied using GIT models, which has provided some insight into the

73

major factors impacting the gastrointestinal fate of milk fat globules

74

studies monitored the change in lipid digestion under simulated gastric and small

75

intestine conditions, but did not include the oral phase in their GIT models.

76

phase is important because saliva contains mucin, a charged biopolymer, that can

77

interact with lipid droplets and alter their interfacial properties and aggregation state

78

e.g., due to depletion or bridging flocculation

79

through the mouth may impact the subsequent gastrointestinal fate of the milk fat

80

globules.

81

22

.

20

.

It has also been shown that the activity of human gastric

Other studies have shown that lipid droplets coated by proteins

The interfacial properties of lipid droplets

Studies with oil-in-water emulsions have shown that the gastrointestinal

23

. Therefore, any processing treatment that

27

.

24-26

.

These

The oral

As a result, the passage of milk

The objective of the present study was to determine the impact of homogenization



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and thermal processing on the physicochemical properties and digestion of milk fat

83

globules using a simulated GIT that included oral, gastric, and small intestine phases.

84

Our hypothesis was that the rate and extent of lipid digestion depends on the size and

85

interfacial properties of milk fat globules, which is effected by processing conditions.

86

The results of this study could be useful for manufacturers of dairy products and infant

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formula because it provides valuable information about the impact of milk fat globule

88

properties on their digestibility. In this study, the behavior of four bovine milk samples

89

was compared: native; homogenized (HM); high temperature short time (HTST); and,

90

ultrahigh temperature (UHT). The HTST and UHT samples were homogenized prior to

91

thermal processing to mimic commercial dairy products.

92

samples were passed through a simulated GIT model and the rate and extent of lipid

93

digestion was measured.

94

MATERIALS AND METHODS

After preparation, the

95

Bovine milk origin and preparation: Fresh raw whole bovine milk (“raw milk”) was

96

obtained from a local farm, and then stored at 4oC prior to use. Homogenized milk (HM)

97

was obtained by passing raw milk through a high-pressure homogenizer (M110L,

98

Microfluidics, Newton, MA, USA) with a 75 µm interaction chamber (F20Y) at a pressure

99

of 10,000 psi for 3 passes. The sample chamber was cooled using an ice-batch during HTST milk (72oC,

100

homogenization to prevent excessive heating of the milk samples.

101

15s) and UHT milk (138oC, 2s) were prepared by passing raw milk through a small-scale

102

sterilization processor, which also contains a homogenizer between the preheat and final


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heat sections (MicroThermics, Raleigh, NC, USA). The thermally processed milk

104

samples were stored at 4oC prior to use. The fat content of the initial milk was 3.5%

105

according to the supplier.

106

may experience difference sequences of homogenization and thermal processing than

107

used in this study, which may affect their subsequent gastrointestinal fate.

It should be noted that in commercial practice, milk samples

108

Pepsin from porcine gastric mucosa and lipase from porcine pancreas were purchased

109

from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA) and as reported by the

110

manufacturer their activity were 250 units/mg and 100-400 units/mg, respectively. Mucin

111

from porcine stomach, porcine bile extract, sodium chloride, calcium chloride, monobasic

112

phosphate and dibasic phosphate were obtained from either Sigma-Aldrich (Sigma

113

Chemical Co., St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). All solvents and

114

reagents were of analytical grade. Double distilled water from a water purification system

115

(Nanopure Infinity, Barnstaeas International, Dubuque, IA, USA) was used for

116

preparation of all solutions.

117

In vitro digestion model: The milk samples were passed through a static GIT model

118

that simulated the mouth, stomach, and small intestine phases so as to model the different

119

environments that milk may experience as it passes through the different sections of the

120

human GIT 28:

121

Initial phase: Milk was diluted with phosphate buffer solution (5 mM, pH 6.80) to

122

obtain a fat content of 2%, since this level of fat is usually appropriate to give full

123

digestion under the simulated GIT conditions used. 20 mL of milk was then placed into a



124

glass beaker in a thermally-controlled shaker (Innova Incubator Shaker, Model 4080,

125

New Brunswick Scientific, New Jersey) at 37oC.

126

Mouth phase: 20 mL of milk sample was mixed with 20 mL of artificial simulated

127

saliva fluid (SSF) containing 0.6 g mucin that was prepared according to previous studies

128

29

129

at 37oC with continuous stirring at 100 rpm to mimic the mouth phase. Although this time

130

is typically longer than a milk product spends in the human mouth, it was used to ensure

131

consistency between different samples.

132

model since physicochemical events occurring in the earlier stages of the upper

133

gastrointestinal tract may subsequently alter lipid digestion 30.

. After being adjusted to pH 6.80, the mixture was incubated in the water bath for 2 min

The mouth phase was included in the GIT

134

Stomach phase: Simulated gastric fluid stock solution (SGFSS) was prepared by

135

dissolving 2 g of NaCl and 7 mL of 12 N HCl in 1 L of double distilled water. Simulated

136

gastric fluid working solution (SGFWS) was prepared by mixing 20 mL of SGFSS with

137

pepsin (800 units/mL). 20 mL of the sample obtained from the mouth phase was added to

138

20 mL of SGFWS, which was preheated to 37oC before mixing. The mixture was then

139

adjusted to pH 2.50 and incubated at 37oC for 2 h with continuous agitation at 100 rpm.

140

Small intestine phase: 30 mL of the sample resulting from the stomach phase was

141

placed into a 100 mL glass beaker incubated in a water bath at 37oC and then adjusted to

142

pH 7.000. Then, 1.5 mL of CaCl2 (36.7 mg/mL) and NaCl (219.1 mg/mL) solution was

143

added, followed by 3.5 mL of bile salt solution with continuous stirring. The reaction

144

system was adjusted back to pH 7.000. Next, 2.5 mL of porcine pancreatic lipase (24


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mg/mL) dissolved in buffer solution was incorporated into the mixture (final lipase 64-

146

256 units/mL) 28. An automatic titration (pH-stat) device (835 Titrando, Metrohm USA

147

Inc., Riverview, FL) was then used to monitor the volume of 0.15 M NaOH solution

148

required to maintain the system at pH 7.000 throughout a 2 h incubation period at 37oC.

149 150

The amount of free fatty acids (FFAs) released within the small intestine phase can be calculated from the following formula: V ×mNaOH MFFA = NaOH Vmilk

151 152 153

Where, MFFA is the molar amount of FFAs released per milliliter of milk (µmol/mL),

154

VNaOH is the volume of alkaline solution consumed (mL), MNaOH is the molarity of the

155

alkaline solution (0.15 M), Vmilk is the volume of milk digested in the small intestine

156

phase (4.29 mL), which was calculated according to the dilution ratio used during the in

157

vitro digestion process.

158

It should be noted that the relatively simple in vitro GIT model used in this study

159

cannot simulate the complex physicochemical and physiological events occurring in vivo

160

within the human GIT.

161

samples and the identification of the key physicochemical phenomena occurring, and so

162

they are widely used for this purpose 31.

However, in vitro models do enable the rapid screening of

163

Particle Characterization: The mean particle diameter and particle size distribution

164

of the emulsions was determined using a static light scattering instrument (Mastersizer

165

2000, Malvern Instruments, Westborough, MA, USA). Samples were diluted in aqueous



166

buffer solutions to avoid multiple scattering effects, and then stirred (1200 rpm) to ensure

167

homogeneity. The refractive indices of phosphate buffer solution and milk fat droplets

168

were taken to be 1.330 and 1.462, respectively. The mean size of each sample is

169

represented as surface-weighted mean diameter (d32), which were calculated from the full

170

particle size distribution 32.

171

The electrical surface potential (ζ-potential) of the particles was measured using a

172

particle electrophoresis instrument that uses light scattering for detection of particle

173

movement (Nano-ZS, Malvern Instruments, Westborough, MA, USA). Initial, mouth, and

174

small intestine samples were diluted with buﬀer solution (5 mM, pH 7.0) prior to

175

measurements to avoid multiple scattering eﬀects, while stomach samples were diluted

176

with pH 2.50-adjusted distilled water.

177

A confocal scanning laser microscope with a 60× objective lens (oil immersion) and

178

10× eyepiece (Nikon D-Eclipse C1 80i, Nikon, Melville, NY, USA.) was used to

179

determine the microstructure of the samples. Prior to analysis, Nile red (1 mg/mL in

180

ethanol) and fluorescein isothiocyanate (FITC, 10 mg/mL in dimethyl sulfoxide) were

181

both used to dye the oil and protein phases, respectively.

182

for 5 min before observation.

183

545 nm and 488nm, and emission wavelength were 605 nm and 515 nm, respectively.

184

All microstructure images for confocal microscopy were acquired and processed using

185

image analysis software (NIS-Elements, Nikon, Melville, NY).

186

The samples were incubated

The excitation wavelength for Nile red and FITC were

Statistical analysis: All analysis was performed on three fresh samples with two


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repeated measurements per sample. Results are reported as means and standard deviations

188

of these measurements. The means among treatments were analyzed by a one-way

189

ANOVA followed by post-hoc Duncan test. Statistical significance was set as p < 0.05

190

(SPSS, IBM Corporation, Armonk, NY, USA).

191 192

RESULTS AND DISCUSSION

193

Influence of processing on gastrointestinal fate of milk fat globules: The impact of

194

homogenization and thermal processing on the gastrointestinal fate of the bovine milk fat

195

globules was examined by passing them through the mouth, stomach, and small intestine

196

phases of the simulated GIT.

197

(PSD), microstructure, and particle charge (z-potential) of the samples was then measured

198

after exposure to each GIT stage.

The mean particle diameter, particle size distribution

199

Initial: Initially, the raw milk contained significantly larger fat globules (d32 = 3.55

200

µm) than any of the processed milks (d32 ≈ 0.45 µm), with no significant differences

201

among the HM, HTST, and UHT milks (p < 0.05).

202

homogenization is expected because the intense mechanical forces generated within the

203

microfluidizer breakdown larger droplets into smaller ones.

204

size was similar for all of the processed milk samples suggests that the different thermal

205

treatments did not alter fat globule size.

206

attributed to the fact that the electrostatic and steric repulsion between the fat globules

207

was sufficiently strong to inhibit their flocculation or coalescence during thermal

A reduction in fat globule size after

The fact that the particle

Based on previous studies, this effect can be



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processing 33.

209

(Fig. 2a).

210

diameters between 1 to 13 µm, while the homogenized milks contained fat globules with

211

diameters between 0.1 to 3.5 µm.

212

globules (red) were initially evenly dispersed throughout the aqueous phases in all of the

213

milk samples tested (Fig. 3).

214

All the milk samples initially had monomodal particle size distributions

The PSD results also showed that raw milk mainly contained fat globules with

The confocal micrographs indicated that the fat

The fat globules in raw milk are known to be coated by a structurally complex

215

membrane, which is rich in polar lipids, cholesterol, proteins, and glycoproteins 22.

216

confocal microscopy images indicated that there were significant levels of free protein

217

(presumably caseins and whey proteins) dispersed in the aqueous phase surrounding the

218

native fat globules.

219

were detected in both the aqueous phase and the interfacial layer (Fig. 3, white boxes).

220

This effect can be attributed to the fact that homogenization disrupted the native milk fat

221

globule membrane and increased the interfacial area, thereby resulting in the adsorption

222

of caseins and whey proteins to the fat globule surfaces 4, 14, 26.

223

The

For the homogenized milk samples, significant levels of protein

Initially, the fat globules in the raw milk had a z-potential that was negative (-26.4±

224

0.9 mV).

The processed milk samples all had fairly similar electrical characteristics,

225

with z-potential values of -32.9±1.2 mV, -31.0±0.4 mV and -31.2±0.5 mV for HM,

226

HTST and UHT, respectively (Fig. 4).

227

different from that of the raw milk (p < 0.05).

228

raw and homogenized milks can be attributed to the disruption of the milk fat globule

The z-potential of the processed milks was The difference in z-potential between the


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membrane and the adsorption of surface-active proteins from the aqueous phase, mainly

230

caseins

231

appear to change the z-potential appreciably, which suggests that the overall interfacial

232

charge properties did not change much.

233

milk does promote the denaturation of adsorbed whey protein molecules, as well as

234

changes in the interactions between the casein and whey protein molecules in the

235

interfacial layer

236

some alterations in interfacial composition, but it is difficult to determine precisely what

237

the molecular origins of these changes are in terms of differences in interfacial

238

composition and structure from electrophoresis measurements.

17, 34-35

.

The two thermal treatments applied to the homogenized milk did not

15, 36-37

.

Nevertheless, it is known that heat treatment of

Thus, the z-potential measurements suggest that there were

239

Mouth: There was a large increase in the mean diameter of the particles in all of the

240

milk samples after they were exposed to simulated mouth conditions (Fig. 1), indicating

241

that droplet aggregation occurred.

242

previously, where it was attributed to electrostatic screening by mineral ions in the

243

simulated saliva, as well as bridging or depletion flocculation induced by the presence of

244

mucin

245

coagulated structures (Fig. 1b).

246

increase in the number of large particles in the milk samples exposed to mouth conditions

247

(Fig. 2), which also indicated that extensive aggregation had occurred.

248 249

37-39

This type of aggregation has been reported

. Visual observations of these samples indicated that they contained large The PSD measurements indicated that there was an

The aggregates present in the sediment formed at the bottom of the test tubes after exposure to the mouth phase were too large to examine using confocal microscopy.


It


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250

was postulated that they contained a relatively high amount of undigested materials that

251

were denser than water, such as proteins, mucin, and mineral ions.

252

examine the structure of the particles within the aqueous phase above the sediment using

253

confocal fluorescence microscopy (Fig. 3).

254

extensive milk fat globule flocculation in the processed milk samples after exposure to

255

the mouth phase, especially for the heat-treated ones.

256

change in the structure of the fat globules in the raw milk.

257

the microscopy images of the milk samples appeared to decrease after exposure to the

258

mouth, which may have been due to dilution effects or because some fat globules were

259

trapped in the protein aggregates at the bottom of the test tubes.

260

samples all exhibited fat globule flocculation, with the thermally processed samples

261

(HTST and UHT) containing the largest aggregates.

262

flocculation observed in the processed milk samples can be attributed to changes in the

263

interfacial composition and structure caused by homogenization and thermal processing.

264

The fat globules in the raw milk would be surrounded by milk fat globule membranes,

265

whereas those in the homogenized milk would be surrounded by a mixture of caseins and

266

whey proteins

267

adsorbed whey proteins, which could increase the extent of fat globule aggregation due

268

to increased hydrophobic and disulfide interactions 40.

269 270

28

It was possible to

These images indicated that there was

Conversely, there was little The level of fat globules in

The homogenized milk

The higher degree of fat globule

. Thermal processing is known to promoted thermal denaturation of

After exposure to the mouth phase, there was an appreciable decrease in the magnitude of the z-potential for the three processed milk samples (p>0.05).


Conversely, the

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surface potential of the particles in the raw milk remained relatively constant.

The

272

surface potentials of all the samples were fairly similar (-25.1 to -26.5 mV) after the

273

mouth phase, with no statistical difference (p>0.05) among them (Fig. 4).

274

in electrical characteristics of the milk fat globules in the processed samples may have

275

been due to electrostatic screening effects by mineral ions in the simulated saliva 41 and/or

276

due to the association of mucin molecules with the fat globule surfaces 33.

277

behavior of the fat globules in the raw and processed milks may have been because the

278

affinity of mucin molecules for the fat globule surfaces was different.

279

mucin may have adsorbed more readily to the protein-coated fat globules in the processed

280

milks than to the milk fat globule membrane coated fat globules in the raw milk.

281

Nevertheless, further studies are needed to confirm this hypothesis.

The change

The different

For example, the

282

Stomach: For all the milk samples, the particle size decreased appreciably after passing

283

through the stomach phase (Fig. 1), which suggests that some of the large aggregates

284

formed in the mouth phase were dissociated.

285

could clearly be seen by visual observation of the samples, with much less sediment being

286

observed (Fig. 1b).

287

conditions, but they contained larger particles than in the initial samples (Fig. 2).

288

are a number of possible reasons for the disruption of the large aggregates in the stomach

289

phase.

290

molecules 42.

291

electrostatic interactions in the system.

Indeed, the breakdown of the aggregates

All of the samples had monomodal PSDs after exposure to stomach There

First, the stomach contains gastric pepsin, which partly hydrolyzes protein Second, the change in pH from neutral to acidic alters the nature of the Third, the samples were diluted (1:1) when they



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292

moved from the mouth to the stomach phase, which may have promoted dissociation of

293

some aggregates.

294

The confocal microscopy images indicated that the fat globules also changed

295

appreciably after exposure to stomach conditions (Fig. 3).

The breakdown of the protein

296

aggregates appears to have promoted the release of trapped fat globules.

297

globules in the raw milk did not appear to undergo appreciable flocculation in the

298

simulated stomach fluids, although there was still an increase in the size of the individual

299

fat globules, which suggests that some coalescence occurred.

300

suggested that the membranes surrounding the fat globules in raw milk can protect them

301

from flocculation within gastric fluids 42.

302

globules occurred in all of the processed milks.

303

the fat globules in these systems were coated by a mixture of proteins (caseins and whey

304

proteins).

305

aggregation under stomach conditions due to the acidic pH, high ionic strength, and

306

enzyme activity of the simulated gastric fluids 42-43.

The fat

Other researchers have

In contrast, appreciable flocculation of the fat Again, this may have occurred because

It is known that protein-coated fat droplets are highly susceptible to

307

After exposure to the stomach phase, the surface potentials of all the milk samples

308

were relatively low and fairly similar (+2.5 to +3.2 mV), with no statistical difference

309

(p > 0.05) among them (Fig. 4).

310

2.50), and this pH is well below the isoelectric point of the milk proteins.

311

would have expected that the protein-coated droplets would have had a much higher

312

positive z-potential then the measured values.

The simulated gastric fluids are highly acidic (pH Thus, one

This effect can mainly be attributed to


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the influence of the mucin molecules arising from the simulated saliva.

314

anionic biopolymer, whose negative charge decreases in magnitude as the pH is reduced

315

from 7 to 2

316

protein-coated droplets in the gastric fluids, thereby partly neutralizing their charge 43.

317

In addition, the relatively high ionic strength of the simulated gastric fluids would also

318

reduce the magnitude of the z-potential due to electrostatic screening and ion binding

319

effects 29.

44

.

Mucin is an

The anionic mucin molecules bind to the surfaces of the cationic

320

Small intestine: After exposure to the small intestine phase, the mean particle

321

diameters (d32) of all the processed milks decreased steeply (Fig. 1), which indicated that

322

many of the large particles present in the stomach phase dissociated.

323

was still a small number of large particles present in these systems, as seen in the PSD

324

data (Fig. 2).

325

intestine phase indicated that they did not contain any visibly large aggregates or

326

sediments.

327

to the small intestine phase (Figs. 1 and 2), however, the nature of the particles was very

328

different from those present in the stomach phase (Fig. 3).

329

for all the processed milk samples, and so only the photograph for the HTST is shown in

330

Fig. 1b.

However, there

Indeed, visual observation of the processed milk samples after the small

The raw milk sample still contained relatively large particles after exposure

Similar results were obtained

331

The confocal microscopy images indicated that there were few aggregates remaining

332

in the homogenized and HTST samples after exposure to the small intestinal fluids (Fig.

333

3).

On the other hand, the UHT samples contained a number of lipid-rich aggregates



334

dispersed throughout the images (Fig. 3).

The presence of these aggregates would

335

account for the lower extent of lipolysis observed in the UHT samples (see next section).

336

The confocal microscopy images also suggested that the raw milk samples contained a

337

number of large lipid-rich structures.

338

large lipid droplets that had been only partially disrupted by digestion (Fig. 3, white box).

339

The other lipid-rich structures observed in this sample may have been aggregated lipid

340

droplets, or micelles and vesicles assembled from bile salts, phospholipids, free fatty acids,

341

and monoacylglycerols 45-46.

Interestingly, some of these structures looked like

342

Particle charge: After exposure to the simulated small intestine conditions, all the

343

samples had similar relatively high negative z-potentials (-42.6 to -43.7 mV) (Fig. 3).

344

This effect can be attributed to the fact that all the digested samples would have contained

345

a mixture of anionic bile salts, phospholipids, free fatty acids, and peptides that assembled

346

into colloidal particles, such as micelles, vesicles, or undigested fat globules 24, 47.

347

Influence of processing on milk fat globule digestion: The rate and extent of

348

digestion of the milk fat globules within the small intestine phase was monitored by

349

measuring the free fatty acids (FFAs) released over time using an automatic titration unit

350

(Fig. 5). In general, the digestion profiles were fairly similar for the four different milk

351

samples.

352

during the first 5 min, followed by a more gradual increase at longer times, until

353

eventually a relatively constant value was reached. The rapid initial rate of lipolysis

354

suggests that the lipase molecules quickly adsorbed to the fat globule surfaces and readily

There was a rapid increase in the level of FFAs released from the fat globules


Page 18 of 35

Page 19 of 35


355

accessed the emulsified triacylglycerol molecules.

356

longer times due to a reduction in the amount of substrate remaining, and because of the

357

accumulation of long-chain FFAs at the fat globule surfaces, as has been reported

358

previously

359

triacylglycerol core, which inhibits the ability of the lipase to operate.

360

48

The rate of lipolysis slowed down at

. These FFAs may form a liquid crystalline shell around the remaining

The initial rate of fat globule digestion was calculated as the linear slope of the FFAs

361

released versus time profiles during the first 5 minutes of lipolysis (correlation coefficient >

362

0.86) (Fig. 6). There were significant differences in the kinetics of lipid digestion among

363

the samples, with the initial rate decreasing in the following order: HM > HTST > Raw >

364

UHT. Homogenization of the raw milk increased the initial digestion rate (i.e., HM >

365

Raw), which can be attributed to the increase in fat globule surface area exposed to the

366

lipase in the surrounding aqueous phase.

367

homogenized milk appeared to decrease the initial rate of lipid digestion, with the effect

368

being more pronounced for the milk sample that had underwent the more severe thermal

369

treatment (i.e., HM > HTST > UHT).

370

of the interfacial coating surrounding the fat globules (e.g., due to protein denaturation

371

and cross-linking) 49, thereby making it more difficult for the lipase molecules to adsorb.

372

Alternatively, thermal treatment may have promoted a greater extent of fat globule

373

flocculation, which would again limit the ability of lipase to adsorb to the fat globule

374

surfaces 23.

375

However, thermal processing of the

Thermal treatment may have altered the structure

The final amount of FFAs released at the end of the small intestine phase also



Page 20 of 35

376

depended on the processing method used, decreasing in the following order: Raw ≈ HM >

377

HTST > UHT (Fig. 5).

378

the final extent of milk fat digestion (Raw ≈ HM), but heat treatment appeared to decrease

379

the overall extent of lipid digestion of the homogenized samples, with the effect becoming

380

more pronounced as the processing temperature increased (HM > HTST > UHT).

381

results suggest that even though the initial rate of lipid digestion was faster for the

382

homogenized milk than for the raw milk, there was still sufficient time for full digestion

383

to occur by the end of the small intestine phase.

384

the interfacial structure in the homogenized milks caused by thermal processing were

385

sufficient to inhibit lipid digestion throughout the duration of the small intestine phase.

386

The presence of some fat globule aggregates in the confocal microscopy images of the

387

UHT samples after small intestine digestion (Fig. 3) may therefore have been due to the

388

fact that lipid digestion was not complete.

389

previous studies that have indicated that thermally processed milk is more resistant to

390

digestion than raw milk

391

processing (HTST or UHT) may actually increase the extent of lipid digestion under

392

simulated small intestine conditions 25.

393

the nature of the milk samples, processing conditions, or simulated GIT models used in

394

the different studies.

395

included mouth, stomach and small intestine phases, whereas in the previous study only

396

the stomach and small intestine phases were included.

Thus, homogenization appeared to have no significant effect on

23

.

These

They also suggest that the changes in

These results are consistent with some

However, other studies have reported that thermal

These differences may be due to differences in

For instance, in our study a static in vitro model was used that


Page 21 of 35

397


In summary, this study has demonstrated that the initial size and interfacial properties

398

of milk fat globules influence their hydrolysis under simulated GIT conditions.

Raw

399

milk is initially digested slower than homogenized milk because of its larger fat globule

400

size (lower surface area).

401

of the lipid phase in the raw milk to occur within the small intestine.

402

of homogenized milk reduced the rate and extent of fat globule digestion, which was

403

mainly attributed to changes in the interfacial properties induced by high temperatures.

404

In particular, heating milk above the thermal denaturation temperature of the adsorbed

405

whey proteins is known to promote their unfolding and interfacial cross-linking, which

406

could lead to appreciable changes in interfacial structure and fat globule aggregation.

407

As a result, the adsorption of lipase molecules to the fat globule surfaces may have been

408

inhibited, which slowed down digestion.

409

development of dairy products with tailored lipid digestion profiles.

410

be useful to carry out further studies in animal or human models.

However, there was still sufficient time for complete digestion Thermal processing

These results may be useful for the However, it would

411 412

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FUNDING STATEMENT This material was partly based upon work supported by the National Institute of Food and Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00491) and USDA, AFRI Grants (2014-67021 and 2016-25147).

The research work was partly

funded by BINC Nutrition and Care of Maternal and Child Research Funding Program (2017BINCMCF34), and National Key R&D Program (2017YFD0400600). Li Liang thanks the Chinese Scholarship Council for support (No. 201506790027).


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Page 27 of 35


FIGURE CAPTIONS Figure 1. Influence of GIT stage and processing on the mean particle diameter of milk samples. (a) Surface-weighted mean particle diameter (d3, 2). (b) Photos of digestion product. Different lowercase letters (a, b, c) represent significant differences (Duncan, p < 0.05) between different GIT stages (same processing). Different uppercase letters (A, B, C) represent significant differences (Duncan, p < 0.05) between different processing treatments (same GIT stage). Figure 2.

Particle size distributions of milk samples with different degrees of

processing after exposure to different stages of the simulated GIT. Figure 3.

Influence of processing on the microstructure of milk samples after

exposure to different simulated GIT stages.

Lipids located in the core of the fat

droplets were labelled using Nile red (red), while milk proteins were labelled with FITC (green). Scale bar = 20 µm. Particles in white boxes are 5 times magnified. (For interpretation of the references to color in this figure legend, the reader is referred to the web version.) Figure 4.

Electrical characteristics (z-potential) of milk samples with different

degrees of processing after exposure to different stages of the simulated GIT. Different lowercase letters (a, b, c) represent significant differences (Duncan, p < 0.05) between different GIT stages (same processing). Different uppercase letters (A, B, C) represent significant differences (Duncan, p < 0.05) between different processing treatments (same GIT stage).



Figure 5.

Amount of free fatty acids (FFAs) released from milk samples with different

degrees of processing under simulated small intestine conditions. Different lowercase letters (a, b, c) represent significant differences of final FFAs released (Duncan, p < 0.05) between different processing treatments. Figure 6.

Initial rate of FFAs released from milk samples with different degrees of

processing measured using a pH-stat under simulated small intestine conditions. Different lowercase letters (a, b, c) represent significant differences (Duncan, p < 0.05) between different treatment.


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Page 29 of 35


FIGURES

Figure 1



Figure 2


Page 30 of 35

Page 31 of 35


Figure 3



Figure 4


Page 32 of 35

Page 33 of 35


Figure 5



Figure 6


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GRAPHICAL FOR TABLE CONTENTS

Homogenization

Raw Milk Fat Globule

Homogenization + Heat


Influence of Homogenization and Thermal Processing on the

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