Triglyceride structure modulates gastrointestinal digestion fates of lipids

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Functional Structure/Activity Relationships

Triglyceride structure modulates gastrointestinal digestion fates of lipids: A comparative study between typical edible oils and triglycerides using fully designed in-vitro digestion model Zhan Ye, Chen Cao, Yuanfa Liu, Peirang Cao, and Qiu Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01577 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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Journal of Agricultural and Food Chemistry

Triglyceride structure modulates gastrointestinal digestion fates of lipids: A comparative study between typical edible oils and triglycerides using fully designed in-vitro digestion model Zhan Ye a; Chen Cao a, b; Yuanfa Liu a, b *; Peirang Cao a, b, Qiu Li c

Author Affiliations a. School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People’s Republic of China; b. State Key Laboratory of Food Science and Technology, National Engineering Laboratory for Cereal Fermentation Technology, National Engineering Research Center for Functional Food, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People’s Republic of China; c. Shandong LuHua group co., LTD, Laiyang 265200, Shandong, People’s Republic of China

* Corresponding Author Telephone: (086)510-85876799; Fax: (086)510-85876799; E-mail address: [email protected]

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ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry 1

Abstract:

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Three typical edible oils (Palm oil, PO; leaf lard oil, LO; rapeseed oil, RO) and triacylglycerols (TAGs)

3

(glycerol tripalmitate, GTP; glycerol tristearate, GTS; glycerol trioleate, GTO) were selected to

4

conduct digestion experiments using fully designed in-vitro digestion model. The evolution in mean

5

particle diameter, ζ-potential and micro-structural changes during different digestion stages were

6

investigated. FFA release extent and kinetics were monitored by pH-Stat method. The particle

7

characterization of different lipids during passage through the GIT depended on lipid type and the

8

micro-environment they encountered. Absorbed surface protein can hardly be the obstacle for pancreas

9

lipase to catalyze lipid hydrolysis after gastric digestion. The maximum FFA release level and apparent

10

rate constant in small intestine digestion stage of the three oils and TAGs were: PO>RO>LO,

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GTP>GTS>GTO, respectively. PO showed the highest FFA release level and rate mainly due to the

12

short chain length saturated palmitic acid (C16:0) specifically located in the Sn-1, 3 positions of TAG

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molecules in palm oil. While, the Sn-1, 3 positions of TAG molecules in RO and LO were mainly

14

mono- or poly-unsaturated fatty acids (C18:1 or C18:2), restricting the continuous hydrolysis reaction.

15

These findings can provide some basic understanding of the digestion differences of different lipids,

16

which may be useful for their nutritional and functional evaluation, and the applicability in food area.

17 18

Key words: Lipid digestion; Free fatty acid release; Chemical composition; In-vitro digestion;

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Triglyceride

20 21 22 23 24 25 26 27 2


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INTRUDUCTION

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Lipids are the unique macro-nutrients of human body, which encompasses not only dietary sources of

30

energy and the lipid constituents of cell and organelle membranes, but also the fat-soluble vitamins,

31

corticosteroid hormones, and certain mediators of electron transport, such as coenzyme Q 1. Digestible

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lipids derived from different sources have different TAG compositions, varying in their fatty acids

33

types (w-3 or w-6), locations (Sn-1, 2 or 3), fatty acid chain length (C14, C16 or C18) and unsaturation

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(saturated, mono- or poly-unsaturated) 2. The differences in lipid chemical or physical structures can

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contribute to appreciable differences in their digestion and absorption properties, which can further

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modulate lipid nutritional bioaccessibilities 3. Many researchers also showed that the initial type of the

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lipid phase within an ingested edible oil might influence its subsequent digestion and absorption fates

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within human body, which were mainly attributed to differences in fatty acid compositions and TAG

39

structures 1, 4.

40

Dietary lipids are consumed by humans in different types of O/W emulsions where the lipids are

41

embedded in form of droplets in an aqueous continuous medium 5. Lipid digestion is a complex

42

process which is involved in a series of physical chemical events in the GIT conditions 6-7. As shown in

43

Figure 1, lipid digestion contains three stages: oral processing, gastric digestion and intestinal

44

digestion. In the mouth, lipids are mixed with saliva, which contains various salivary proteins or

45

enzymes, such as mucins and proline-rich proteins. In the presence of the low molecular weight salts,

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lipids interact with these enzymes and proteins, and experience high shear effects between the oral

47

mucosa and teeth, which may alter the structural organization, physical state and interfacial properties,

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then forming coarse lipid emulsion. In the stomach, lipid emulsions are mixed with highly acidic

49

gastric fluids (pH 1-3), containing minerals, biopolymers, surface active lipids and gastric enzymes.

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With the peristaltic wave of the stomach, dietary lipids undergo coalescence or disruption due to

51

alteration of the emulsion droplet surface charge, the nature and composition of the lipid-water

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interface also impose significant effects on the gastric digestion process. Before reaching the small

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intestine, the transpyloric and retropulsive flow within the antrum produces shear forces to reduce the

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average particle size of emulsions down to ∼15-30 µm. This (re)emulsification of lipid can further 3



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facilitates efficient pancreatic lipid digestion by expanding the surface area available for lipase

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adsorption 8. In the small intestine, lipids are mixed with intestinal fluids (pH 6-7.5) that contain

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pancreatic lipase, colipase, proteases, bile salts, and phospholipids etc. The bile salts and phospholipids

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may adsorb to the surface of the lipid droplets through ‘orogenic displacement mechanism’. The

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lipase/colipase complex may then adsorb to the lipid-water interface and convert the core TAG into

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FFAs and monoglycerides (MAGs). Bile salts then combine with these digestion products and

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phospholipids to form the mixed micelles or vesicles, promoting continuous lipase hydrolysis reaction

62

7, 9-10

63

Many previous researchers did research with different type of lipids aimed at figuring out their

64

digestion differences when passing through the GIT tract using in-vitro digestion model. Qing G. et al.

65

11

66

emulsions on their digestion, and showed that the rate and extent of lipid digestion decreased with

67

increasing solid fat content, which indicated that lipid physical state, or more specifically, lipid

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composition might modulate lipid hydrolysis process. Whereas, some other researchers showed lowest

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rates and extents of lipid digestion were observed for emulsified flavor oil, followed by emulsified krill

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oil. However, no appreciable differences were observed between the final amounts of FFA released for

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emulsified digestible oils, including corn oil, olive oil, sunflower oil and canola oil

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studies also explored the influences of lipid types on oil digestion fates in the form of O/W emulsions

73

using static in-vitro digestion model, and gave a general conclusion that there existed appreciable

74

differences in the digestion rate of different oils composed of different types of FFA: short chain >

75

long chain; saturated > unsaturated 13-14. However, apart from the lipid types, the positional distribution

76

of fatty acids within TAG molecules was also reported to affect lipid digestion. Nagata J. et al. 15 found

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that the in-vitro TAG hydrolysis rate of medium chain-linoleic-medium chain type lipids was 2-3 fold

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higher than linoleic-medium chain-linoleic types upon pancreatic lipases, and the corresponding serum

79

TAG levels higher in rats fed with medium chain-linoleic-medium chain type lipids versus

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linoleic-medium-linoleic types, which confirmed that the positional distribution of fatty acids within

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TAGs affected lipid digestion and absorption. However, the conclusions made from animal study

.

and Lucile B. et al. 12 explored the influences of physical state of the dispersed oil phase within O/W

4


10

. Some other

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didn’t show significant difference between soybean oil (mainly long-chain triglycerides, LCTs) and

83

coconut oil (mainly medium-chain triglycerides, MCTs)

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degree of unsaturation of TAGs did not appear to significantly affect lipid digestion. These suggested

85

that more work still need to be done in this area.

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Palm oil (PO), porcine leaf lard (LO) and rapeseed oil (RO) are the three of the most widely consumed

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edible oils in southeast Asia, especially in China

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digestive differences in-vitro or in vivo. As we know, the diversity of digestion fates of different lipids

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imposes significant effects on their absorption and nutritional value, therefore, in the present study, the

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commercial PO, LO and RO, and three purity TAGs (GTP, GTS and GTO) were selected to conduct

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in-vitro digestion experiment using fully designed single stage in-vitro model, which included the

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mouth, stomach and intestine digestion phase, in order to figure out their gastrointestinal digestion

93

differences in view of the FFA and TAG compositions. As we kwon, the GIT digestion system mainly

94

contains mouth, stomach and small intestine stages, thus, the fully designed in-vitro digestion model

95

displayed here is relative to partly designed in-vitro digestion model, which just includes one or two

96

stages of GIT digestion system (e.g. oral, stomach or small intestine stage). Our group investigated the

97

digestion differences of different lipids by analyzing the emulsion droplet characterizations, surface

98

protein changes, FFA release profiles and kinetic behaviors using purity TAGs as control tests. All of

99

our efforts are aimed at providing some basically understanding of digestion fates of different lipids

100

16

, besides, some studies showed that the

17

. However, few studies were focused on their

composed of different FFAs and TAGs when passing through the simulated GIT stages.

101 102

MATERIALS AND METHODS

103

Materials. Whey protein isolate (WPI) was obtained from Davisco Foods International Inc.

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(Davisco Foods International Inc., USA). Glycerol tripalmitate (CAS 555-44-2, >85%), glycerol

105

tristearate (CAS 555-43-1, >80%) and glycerol trioleate (CAS 122-32-7, >80%) were purchased from

106

J&K Scientific Ltd., (Shanghai, China). Fractionated palm oil (PO) and rapeseed oil (RO) were

107

purchased from Wilmar International Ltd (Shanghai, China), and porcine leaf lard (LO) purchased

108

from Jinen Food Co., Ltd. (Wenzhou, China). Mucin Type II from porcine stomach and lipase from 5



porcine pancreas Type II (100-500 units/mg) were purchased from Sigma Chemical Co. (St. Louis,

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USA). Pepsin from porcine gastric mucosa (USP grade, Valence 1: 3000) was purchased from Yuanye

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Biological Technology co., LTD (Shanghai, China) and bile salt was purchased from Xiya Chemical

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Industry Co., Ltd (Qingdao, China). Bovine Serum Albumin (BSA) was BR grade obtained from

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Macklin Biochemical Co., Ltd., (Shanghai, China). Nile red was purchased from J&K Scientific Ltd.,

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(Shanghai, China). All other chemicals of analytical grade purchased from Sinopharm Chemical

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Reagent Co. Ltd. (Shanghai, China) and Fisher Scientific (Shanghai, China). The water was purified

116

by a water purification system (Milli-Q Direct 8, Millipore, USA) before using.

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Fatty acid and triacylglycerol analysis. Fatty acid composition was analyzed using a gas

118

chromatography (GC) system (Shimadzu, Model GC-2010 PLUS) equipped with a capillary gas

119

chromatography column (TR-FAME 60 m × 0.25 mm i.d. × 0.25 µm) and a flame ionization detector

120

(FID) according to AOCS Official Method Ce 2-66 18 and our previously published paper 19.

121

Triacylglycerol profiles were analyzed by Ultra-Performance Liquid Chromatography Mass

122

Spectrometry (UPLC-MS). The UPLC system (Waters, Milford, Massachusetts, USA) was equipped

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with a BEH C18 column (i.d. 2.1 mm × 50 mm, 1.9 µm). TAGs of the samples were identified and

124

quantified using the quadrupole time of flight (Q-TOF) mass spectrometry (MS) instrument (Waters,

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Milford, Massachusetts, USA) with ESI probe. The analysis protocols and data processing were

126

detailedly summarized in the previous published paper developed by our lab 19.

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Preparation of initial lipid digestion emulsions. Emulsifier solution was prepared by dispersing

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1.0 wt % WPI into 5 mM PBS (pH 7.0). The emulsifier solutions were then stored overnight at 4 °C to

129

ensure complete hydration. The pH of emulsifier solutions was adjusted back to pH 7.0 if required.

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The stock emulsions were prepared by homogenizing 10% (w/w) lipid phase with 90% (w/w)

131

emulsifier solution using a high-speed blender for 3 min at 12 000 r/min (T 18D S25, IKA, Germany).

132

The GTP and GTS emulsions were prepared by homogenizing the two phase in warm water bath at

133

~75 °C for melting the solid fat. The coarse emulsions were then passed three times through a

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two-stage valve ultra-high pressure homogenizer (AH 2010, ATS nano technology Co., Ltd., Suzhou,

135

China) operating at 80 bar and 350 bar in the first and second stages respectively. 6


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Determination of percentage of adsorbed protein (AP %). The AP% of freshly prepared 20

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emulsions were determined as described by Shao, Y.

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emulsion was centrifuged at 12 000 g for 30 min at 4 °C. Then, two phases could be observed: the

139

creamed oil droplets on the top and the aqueous phase at the bottom. The aqueous phase was then took

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out using a syringe, and filtered through a 0.22 µm filter (Millipore Corp.) for protein content

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determination. The protein concentration of the filtrate (Cf) was determined with the Lowry method

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using BSA as the standard. The initial digestion oil emulsion were also centrifuged at the same

143

conditions to allow determination of protein concentration (Cs) in the supernatant. The AP% was

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calculated as Equation (1).

AP% =

145

Cs − C f Co

with some modifications. In brief, 2.0 mL of

×100

(1)

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However, the AP% of emulsions before or after gastric digestion were measured according to Ye, A. 21

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with some modifications. The main steps were briefly summarized as follows: 2.0 mL of emulsion was

148

centrifuged at 12 000 g for 30 min at 4 °C. The up creamed oil droplets layer and a little volume of the

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down layer aqueous phase were carefully collected using a syringe to ensure droplets were completely

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gathered, then transferred to a small glass test tube. Then, 3 times of solvent (Acetone) was added in to

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extract the lipid, then vortexed (MS3 basic, IKA, Germany) for 3 minutes before using N2 blowing

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until the solvent and trace water volatilized, then repeat this step. The protein content was measured

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using Lowry method described as above. The AP% after gastric digestion (Cp, mg/mL) was calculated

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as the Equation (2).

AP% =

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Cp Co

×100

(2)

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In the equation (1) and (2), Co (mg/mL) was the WPI content applied for the every emulsion

157

preparation.

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Preparation of artificial digestion fluids. The artificial saliva (ASF), gastric (AGF) and small

159

intestine (AIF) fluids were prepared referred to the previous work 22-24. The chemical composition and

160

concentration were summarized in the Table 1.

161

Simulated gastrointestinal tract (GIT) model design. A fully designed single-stage in-vitro 7



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digestion model including mouth, stomach and small intestinal phases were promoted according to the

163

previous studies with some modifications

164

initial digestion oil emulsion contained 2% (wt %) lipid was obtained by diluting the stock emulsion

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with 5 mM PBS. Then was placed in a swirling water bath thermostat shaker (SHZ-82A, Jinda

166

instrument Manufacturing Co., Ltd. Jingtan, China) to incubate for 30 min under 37.8 °C for

167

completely mixing. 20 mL of ASF was preheated to 37.8 °C and then mixed with the initial emulsions.

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After being adjusted to pH 7.0, the mixture was incubated in 37.8 °C water bath with strong

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mechanical agitating (RW 20, IKA, Germany) under 240 r/min for 10 min to imitate the physical

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action in the mouth. After being preheated to 37.8 °C, 20 mL AGF was added to the above system,

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adjusting pH to 2.0. The mixture was incubated in the water bath incubator shaker for 120 min under

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37.8 °C to imitate the stomach digestion. The “chyme” from the stomach phase mixed with another 20

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mL AIF (37.8 °C), and adjusting pH to 7.0 to imitate small intestine digestion under 37.8 °C in water

174

bath incubator shaker (165 r/min) 28. During the 120 min small intestine digestion period, the released

175

FFA was monitored by pH-stat method using 0.1 M NaOH solution.

13, 25-27

, which was displayed in Figure 2. Briefly, 20 mL

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Free fatty acid (FFA) release and hydrolysis kinetics analysis. The released FFA was

177

monitored by a pH-stat automatic titration system (ZDJ-4A, INESA Scientific Instrument Co., Ltd,

178

Shanghai, China), and the volume of 0.1 M NaOH (in mL) to neutralize the FFA was recorded. During

179

this process, pancreatic lipase converted TAGs into a complex mixture of DAGs, MAGs and FFAs 1.

180

The released FFAs was expressed as a percentage which was calculated from the number of moles of

181

NaOH required to neutralize the FFA by using Equation (3) (Assuming 2 FFA produced per TAG

182

molecule) 29.

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FFA(%) = 100 ×

VNaOH × mNaOH × M Lipid WLipid × 2

(3)

184

Here VNaOH is the volume of NaOH solution required to neutralize the FFAs (in mL), mNaOH is the

185

molarity of the NaOH solution (in M), WLipid is the total weight of lipid initially present in the reaction

186

vessel, and MLipid is the (average) molecular weight of the experiment lipids (in g/mol).

187

During the intestine digestion process, the released FFA will gradually increase with digestion time, 8


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potentially attaining the total FFA release (Φmax). The kinetic parameters for the initial FFA release

189

were calculated using Equation (4) 30.

ln [ (φmax − φt ) / φmax ] = − kt + b

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(4) Here k is the first-order rate constant for FFA release (s ) and t is the digestion time (s). The total FFA

192

release level (Φmax, %) was obtained from the FFA released curves.

190

-1

193

Particle size and ζ-potential characterization. The particle size determined by static light

194

scattering using a particle analyzer (Nano Brook Omni, Brookhaven Instruments Corporation, US).

195

The sizes of emulsion droplets were reported as the surface weighted mean diameter d3, 2 (µm) and

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were calculated using the equation d3, 2 = Σnidi3/Σnidi2, where ni is the number of particles and di is the

197

diameter of emulsion droplets.

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The ζ-potential was measured using a Zetasizer Nano instrument (Zetasizer nano ZS, Malvern

199

Instruments Ltd., UK). Prior to analysis, one millilitre of sample was diluted to approximately 0.005

200

wt% droplet concentration by Milli-Q water to avoid multiple scattering effects, then proper volume of

201

the diluted solution was placed in a folded capillary cell (DTS 1070, Malvern Instruments Ltd., UK)

202

for ζ-potential measurements. All the particle size and zeta-potential measurements were carried out at

203

25 °C, and average of three readings were reported.

204

Fluorescence microscope. Fluorescence images of the emulsions in each digestion stage were

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captured using an upright fluorescence microscope (Leica DM2700 M, Germany) equipped with a

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Leica fluorescent generator (Leica EL6000, Germany). The 10× eyepiece lens and 20× objective lens

207

were selected. Prior to analysis, 1 mL samples were mixed with 20 µL Nile red solution (10 mg/mL in

208

ethanol), and vortex for 3 min, stained for 15 min and then covered with a cover slip. Avoid light and

209

stored at 4 °C before taking microscope images. The excitation wavelength of Nile red was 480 nm.

210

Statistic analysis. All experiments were conducted at least in triplicate for freshly prepared

211

samples. The results were expressed as mean ± standard deviation of replicated measurements.

212

Statistical analysis was performed with Spss 16.0 software (IBM SPSS software, USA) by One-way

213

ANOVA. Tukey adjustment was used to determine the significant difference between treatments.

214

Significant differences were declared at P< 0.05. 9



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215 216

RESULTS AND DISCUSSION

217

The fatty acid and TAG composition analysis. Fatty acid and TAG compositions were

218

summarized in Table 2 and Table 3. The major fatty acids of PO were the palmitic acid (C16:0), oleic

219

acid (C18:1) and stearic acid (C18:0) (81.03, 11.32 and 4.31%, respectively). In LO, the main fatty

220

acids were oleic acid (C18:1), palmitic acid (C16:0) and stearic acid (C18:0) (35.70, 29.76 and 17.59%,

221

respectively), while the top three fatty acids in RO were oleic acid (C18:1), linoleic acid (C18:2) and

222

linolenic acid (C18:3) (62.39, 20.42 and 7.51%, respectively) (Table 2). In comparison, the major fatty

223

acids in the three TAG samples were very simple, palmitic acid (C16:0), stearic acid (C18:0) and oleic

224

acid (C18:1) composed of over 90% of total fatty acids of GTP, GTS and GTO, respectively. The short

225

chain length saturated fatty acids (C16:0) were dominated in PO, and longer chain length unsaturated

226

fatty acids (C18:1, C18:2 and C18:3) were the major fatty acids in RO, whereas, LO contained both

227

saturated (C16:0 and C18:0) and unsaturated (C18:1 and C18:2) fatty acids.

228

The major TAGs in PO were PPP, OOP and POP, which were composed over 55% of all TAGs; the

229

main TAGs in LO were SOP, OPO and LPS (17.98, 16.92 and 8.69% respectively) and OOL, OLS and

230

LLO were the top three TAGs in RO, which accounted for over 40% of the total TAGs (Table 3). In the

231

TAG samples, the main TAGs in GTP, GTS and GTO were PPP, SSS and OOO, respectively, although

232

about 10% could not be identified. From Table 3, it could be concluded that the Sn-1, 3 positions of

233

TAG molecules in PO were mainly palmitic acid (C16:0), however, the major fatty acids in Sn-1, 3

234

positions within TAG molecules in RO were mono- or poly-unsaturated fatty acids (C18:1 and C18:2).

235

The fatty acids located in Sn-1, 3 positions in TAG molecules of LO were more complex, which

236

contained stearic (C18:0), oleic (C18:1) and linoleic acids (C18:2). The fatty acid and TAG

237

compositions of the lipid samples showed significant difference. And these differences might modulate

238

their digestion fates, which were also our overall hypothesizes in the present study.

239

Mean particle diameter analysis. The overall trend of the changes in mean particle diameter of

240

different emulsions when they passed though different GIT stages showed an ‘n’ model (Figure 3). The

241

particle diameter in mouth and gastric digestion phase was obviously larger than that in initial and 10


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Journal of Agricultural and Food Chemistry 31-32

242

small intestine phase, which were in consistent with previous studies

. Smaller mean particle

243

diameter can be observed for the three initial oil emulsions (0.213 to 0.330 µm) (Figure 3A). The mean

244

particle diameter of the TAG samples ranged from 0.220 to 0.670 µm, and that of GTS emulsion was

245

relatively larger than the GTP and GTO emulsions (Figure 3B). Although significant difference could

246

be observed among the initial emulsions (P < 0.05), they could hardly affect the digestion fates when

247

passing through different digestion stages due to the uniform particle size distribution (data not shown)

248

and small particle size

249

flocculation occurred for all the initial lipid emulsions (Figure 4).

32

. The fluorescence microscope images also showed that no droplet

250

After being incubated with ASF for 10 min, the average particle diameter increased, which could

251

be explained by the addition of a certain amount of porcine stomach mucin 33. Moreover, large amount

252

of salts (Na+, K+, PO43- etc.) addition also impose negative impact on the physical stabilities, thus

253

causing particle flocculation

254

droplet aggregation occurred in the mouth stage. Compared with LO, RO and GTS, GTO emulsions, it

255

could be obviously observed that the particle diameter of PO and GTP emulsion droplets was much

256

larger. As shown in Figure 1, droplet aggregation might be caused by either bridging or depletion

257

flocculation induced by mucin in the SIF during oral digestion, which was also detailedly reviewed in

258

a recent paper 35. However, it could be concluded that PO and GTP emulsion droplet were more prone

259

to interact with mucin molecules, thus leading to bridging flocculation occurred between lipid droplets.

260

Whereas depletion flocculation might also occur due to the increase in the osmotic attractive forces

261

between the droplets generated by non-adsorbed mucin molecules during this process. After passing

262

through the stomach digestion stage, mean particle diameter further increased, and extensive droplet

263

aggregation could be observed from the fluorescence microscope images (Figure 4). Under the

264

strongly acidic condition (pH 2.0), in the presence of pepsin, the protein-stabilized lipid emulsions

265

were prone to aggregation under gastric conditions due to hydrolysis of adsorbed proteins, which

266

weakened electrostatic repulsion and depletion or bridging flocculation induced by mucin

267

However, the increase magnitude in the particle diameter of LO and GTS emulsions was smaller than

268

the other emulsions. In fact, the mean particle diameter of PO, RO, GTP and GTO emulsions were all

34

. The fluorescence microscope images also showed that extensive

11


7, 10

.


exceeded 300 µm, and obvious lipid droplet aggregation could be observed for these four emulsions.

270

After the small intestinal digestion, the mean particle diameter were all decreased. This might be

271

attributed to the lipid hydrolysis upon pancreas lipase in the assistance of bile salts after the surface

272

WPI being hydrolyzed by porcine pepsin in the stomach digestion stage 36. The mean particle diameter

273

of the all lipid emulsions were about or under 1 µm. For the oil emulsions, it ranged from 0.678 to

274

1.010 µm, while, 0.481 to 1.071 µm for TAG emulsions. Compared with gastric or mouth digestion

275

phase, fewer particles and smaller particle size could be observed for all the lipid emulsions in the

276

same size vision field of the fluorescence microscope images in Figure 4.

277

It could be concluded that the mean particle diameter of lipid droplets in different digestion phase

278

were also different (Figure 3). For PO and GTP, they shared similar trends, higher particle diameter

279

appeared after mouth and stomach digestion stages. For RO and GTO, the highest particle diameter

280

occurred after stomach digestion stage. Obvious differences could be observed for LO and GTS, the

281

highest particle diameter for LO appeared after stomach digestion stage, while the particle diameter for

282

GTS were large in both mouth and stomach digestion stage. These differences might be ascribe to their

283

particular fatty acid and TAG compositions, which might further affect their interaction with digestion

284

enzymes and the lipid hydrolysis process.

285

Surface charge characterization. Changes in the electrical characteristics of different lipid

286

emulsions were measured as they passed through the different GIT digestion stages (Figure 3). For

287

both oil and TAG initial emulsions, they had a highly negative charge (from -39.733 to -49.167 mV for

288

oil emulsions; from -42.233 to 49.267 mV for TAG emulsions). The strong negative charge could

289

mainly be attributed to the presence of the WPI molecules at the droplet surfaces. WPI was above its

290

isoelectric point (pI= 5.1) at the initial solution conditions (pH 7.0), and therefore has a negative

291

charge

292

prepared using different oils, which might be attributed to the impurities present within the lipid phase.

293

For example, anionic or cationic impurities in the oil phase could adsorb to the oil-water interface and

294

contribute to the overall interfacial charge, e.g. FFAs, phospholipids, or trace mineral ions 34. While the

295

different electric charges of TAG emulsions droplets might be explained by the different fatty acids

10

. Nevertheless, there were differences in the electrical characteristics of emulsion droplets

12


Page 12 of 36

Page 13 of 36 296


compositions and TAG structures, e.g. fatty acid chain length or degree of unsaturation 37.

297

After passing through the mouth and stomach digestion stages, there was an appreciable decrease

298

in the magnitude of the negative charge on both of the oil and TAG emulsion droplets, especially for

299

the stomach phase, where the negative charge of different emulsion droplets almost reached zero. The

300

similar results were also reported in the previous studies

301

stage might be attributed to the electrostatic screening by mineral ions present within the simulated

302

saliva, however, the negative charge reduction after stomach digestion phase could be ascribed to the

303

strongly acidic and high ionic strength within the gastric digestion micro-environment, which could

304

result in the changes of the electrical properties of the lipid emulsion droplets

305

intestine digestion stage, the all samples had relatively high negative charges, which could be

306

attributed to the presence of anionic species in the various types of particles present (such as

307

undigested lipids, undigested proteins, micelles, vesicles and calcium salts). These anionic species

308

might come from the original emulsions or the gastrointestinal fluids (e.g. FFAs, bile salts or

309

phospholipids). Previous studies also reported that the presence of phospholipids, bile salts or FFAs

310

could decrease the ζ-potential of the lipid droplets in the form of W/O emulsions stabilized by WPI 10,

311

38

31

. The negative charge reduction in mouth

34

. After the small

.

312

As we know, the higher negative/positive charge indicated higher stability of the emulsion system.

313

Thus, it could be concluded that all of the initial lipid emulsions were more stable, while the emulsions

314

after stomach digestion were completely unstable, where the emulsion droplets were more prone to be

315

aggregated (Figure 4). This also suggested that the stability of the all lipid emulsions decreased from

316

initial to stomach digestion stage, while, increased after intestine digestion stage. These results were

317

highly in consistent with the mean particle diameter results showed in Figure 3. Significant differences

318

could be observed among groups between PO, LO and RO emulsions in initial and after stomach stage

319

(p olive oil >

380

corn oil ≈ soybean oil. However, Zhu et al. 14 reported that the order of total amount of FFAs released

381

level displayed as: milk fat > soya oil > fish oil, which were attributed to the different fatty acids

382

composition within the lipids. By analyzing the individual FFAs released profiles, it further suggested

383

that saturated fatty acids (C16:0 and C18:0) were released faster than unsaturated fatty acids (C18:1n9,

384

C18:2n6 and C18:3n3) from soybean oil emulsions; short chain fatty acids were released faster than

385

long chain fatty acids from milk fat emulsions; long chain polyunsaturated fatty acids (e.g. EPA and

386

DHA), were released more slowly than other fatty acids from fish oil emulsions. All these results

387

indicated that fatty acid chain length, unsaturation and oil physical state can strongly affect their

388

digestion fates. Actually, lipid digestion was a complex process which involves in a series of physical

389

chemical reactions occurred on the emulsion droplet surface. With the assistant of bile salts, the

390

pancreas lipase could bind with the lipid droplet on the oil-water interface, then hydrolyzed the lipid in

391

the hydrophobic core efficiently

392

occurred in intestine digestion stage 8, i.e. the ions type and concentration, pH conditions of the

393

digestion micro-environment and the secretion of co-lipase, among which, the most important one

394

might be the spcifities of fatty acid and TAG compositions within different lipids.

5, 30

. This enzymatic process was influenced by various of factors

395

FFA release kinetics analysis. To further illustrate the connections between lipid chemical

396

compositions and their digestion differences, the FFA release first-order kinetics were analyzed. The

397

linear relationships for FFA release over a period of 120 min intestine digestion time were obtained

398

using Equation (4). The maximum FFA released, apparent rate constants and regression coefficients of

399

different oils and TAGs were all displayed.

400

All the FFA release first-order kinetics curves showed good linear correlationship, and the linear

401

correlation coefficients were all over 0.9000 (RAdj2 ≈ 0.9000 for the all lipids). Differences could be

402

observed for the apparent rate constants of different digestible lipids, which displayed as the absolute

403

value of the slopes of the different FFA release first-order kinetics curves. For the PO, LO and RO, the 16


Page 16 of 36

Page 17 of 36


404

apparent rate constants were 0.0367, 0.0295 and 0.0311 s-1, respectively, which suggested that the PO

405

were digested more quickly than the other two. And the digestion rate of LO and RO seemed not

406

significantly different. However, the apparent rate constants for GTP and GTS (0.0418 and 0.0344 s-1)

407

were higher than GTO (0.0305 s-1) during the 120 min small intestine digestion process. From the

408

results of the maximum FFA release levels and the apparent rate constants of different digestible lipids,

409

it could be concluded that PO and GTP shared quite similar results in maximum FFA release level and

410

FFA release rate, and the maximum FFA release levels and apparent rate constants of RO and GTO

411

were almost the same, however, significant differences could be observed between the LO and GTS.

412

Overall, both of the maximum FFA release level and digestion rate of TAGs were higher than oils in

413

the intestine digestion stage.

414

As mentioned before, the most abundant fatty acid in PO was palmitic acids (81.03%) (Table 2),

415

most of which were located in Sn-1, 3 positions of TAG molecules (Table 3), especially for the PPP,

416

the content of which were over 35% in PO. As we know, pancreas lipase could specifically and

417

preferentially act upon the Sn-1 and Sn-3 positions of TAG molecules, and converted TAGs into Sn-1

418

(3) MAGs and FFAs 1. Meanwhile, many previous studies showed that the hydrolysis extent and rate

419

of lipids composed of short chain fatty acid were higher than lipids composed of long chain fatty acids

420

40-42

421

chain saturated fatty acids. However, the Sn-1 and Sn-3 positions of TAG molecules within LO and

422

RO were mostly long chain mono- or poly-unsaturated fatty acids (C18:1 or C18:2), and these TAGs

423

were mainly UUU (Unsaturated-Unsaturated-Unsaturated) types. Many previous studies investigated

424

the influences of fatty acid positional distribution and unsaturation within TAG molecules on the lipid

425

digestion fate, and confirmed that positional distribution of fatty acid within TAGs could affect lipid

426

digestion, especially for those long-chain fatty acids located in the Sn-1 or Sn-3 positions

427

However, the degree of unsaturation of FFAs in TAGs (i.e., poly- or mono-unsaturation) did not seem

428

to impose significant effects on lipid digestion10. Thus, it suggested that the FFA release rate constant

429

and maximum FFA release level of PO were higher than LO and RO, which were attributed to the

430

specific FFA and TAG compositions.

, especially for those whose Sn-1 and Sn-3 positions of TAGs molecules within lipids were short

17


9, 43

.


Page 18 of 36

431

The results of maximum FFA release level and apparent rate constants of GTP, GTS and GTO just

432

supported the above conclusions (Table 4). The TAG compositions of the GTP, GTS and GTO were

433

relatively simple, which were mainly composed of PPP, SSS and OOO (84.33, 77.78 and 84.21%,

434

respectively). The maximum FFA release level and FFA release apparent rate constant of GTP were

435

both the highest among the three, which was just because that the Sn-1 and Sn-3 position of the TAG

436

molecules of GTP were all palmitic acids (C16:0), and it favored the pancreas lipase hydrolysis

437

reaction upon TAG. The maximum FFA release level and FFA release apparent rate constant of GTS

438

were relatively lower than GTP. This might be due to the Sn-1 and Sn-3 position of GTS were steric

439

acid (C18:0), the chain length of which was longer than palmitic acid (C16:0). The similar results were

440

reported by Rong Liang, et al. 41. However, there seemed no difference between GTO and the PO, LO

441

and RO for maximum FFA release level and FFA release apparent rate constant, which might be

442

attributed to that the Sn-1 and 3 positions of GTO were all oleic acid (C18:1), which were similar with

443

RO. Previous studies reported that the release of pancreas lipase hydrolysis products (e.g. FFAs or

444

MAGs) and their incorporation into micelles were both dynamic processes when using olive oil to do

445

intestine digestion experiment, meanwhile, the kinetic results indicated that the release of hydrolysis

446

products was much faster than their incorporation into micells, however, the FFA release rate and

447

extent were highly related with the fatty acid chain length

448

discussed the lipolysis rate constants predicted by an established mathematical model to assess the

449

fatty acids release rate of lipids composed of different TAGs, and gave the following order: C8:0,

450

C10:0 >> C18:1 (n-9) >> C12:0 > C14:0 > C16:0 ≈ C16:1 > C22:6 (n-3), which were not only in close

451

agreement with the available literature on the substrate specificity of pancreatic lipase, but also with

452

our present results. Two main factors were reported to be responsible for these results. Firstly,

453

short-chain triglycerides with a relatively high water solubility was more accessible to contact and

454

hydrolysis by pancreatic lipase in water system

455

produced from PO and GTP diffuse more easily into the surrounding aqueous phase than the long

456

chain FFA digestion products arising from other four lipids which further decrease the contact of lipase

457

to oil droplets in the emulsions 47.

44

. Research from T.M. Giang et al.

45

46

. Secondly, shorter chain FFA digestion products

18


Page 19 of 36


458

However, from these results, traces of evidence could be observed that the degree of fatty acid

459

unsaturation and TAG types (UUU and SSS (Saturated-Saturated-Saturated); OOO and LLL) affect

460

lipid digestion. Besides, the bile salts play an important role during lipid hydrolysis process as reported

461

in some previous research

462

rate constants of different lipids might also be affected by their different interactions with bile salts. So

463

further work still need to be proceeded to illustrate these intriguing findings. Besides, previous studies

464

showed that the gastric pre-digestion facilitated lipid digestion in the small intestine in normal

465

physiological conditions. The formation of hydrolysis products (e.g. Sn-1, 2 DAG and MAG) from

466

gastric digestion could increase the solubilization of TAGs, the binding of co-lipase, and by the release

467

of fatty acids that stimulate the release of cholecystokinin from the stomach

468

digestion studies as the present work, the lipid digestion rate and extent in small intestine might

469

probably be lower than in the real physiological conditions, or in the presence of gastric and lingual

470

lipase conditions. So the influences of the hydrolysis products produced from gastric digestion in the

471

presence of gastric and lingual lipase should be further considered.

5, 8, 30

, and the differences in FFA release extent and FFA release apparent

48

. So, during in-vitro

472

In summary, in the present study, a fully-designed single stage in-vitro digestion model including

473

mouth, stomach and small intestine digestion phase was promoted to investigate the digestion

474

difference of PO, LO and RO in comparison with GTP, GTS and GTO, so as to illustrate the influences

475

of lipid compositions on the gastrointestinal digestion fates. Results showed that the mean particle

476

diameter and charge characterization of different lipid emulsions throughout different digestion stages

477

were significantly different. During stomach digestion stage, the negative charge of the all lipid

478

emulsions reached nearly zero, and the lipid droplets greatly aggregated, which were not only

479

attributed to the low pH and high ionic strength in gastric conditions, but also the displacement of the

480

surface protein coated on the lipid droplet surface. Although, the surface protein coated on the oil

481

emulsion droplet was higher than TAG emulsions, after gastric digestion, the surface protein sharply

482

decreased, which could hardly be the obstacle for pancreas lipase to catalyze lipid hydrolysis. PO

483

showed higher maximum FFA release level than LO and RO, which might probably be ascribed to that

484

the Sn-1, 3 positions within TAG molecules of PO were mainly shorter chain saturated fatty acids 19



(C16:0), which favored the hydrolysis reaction of pancreas lipase upon TAGs. While, the Sn-1, 3

486

positions of TAG molecules in LO and RO were mainly mono- or poly-unsaturated fatty acid (C18:1

487

or C18:2), which restricted the continuous hydrolysis process during intestine digestion. Moreover, the

488

FFA release profiles of GTP, GTS and GTO, which composed of PPP, SSS and OOO, respectively, just

489

strongly supported these conclusions. The present work can provide some basically understanding of

490

the digestion fates of different lipids, and may give some references for the nutritional and functional

491

evaluation.

492 493

ACKNOWLEDGEMENT

494

This work was supported by the Natural Science Foundation of China (31701528 and 31671786),

495

National Key R&D Program of China (2016YFD0401404), Northern Jiangsu province science and

496

technology projects (BN2016137), and the Fundamental Research Funds for the Central Universities

497

(JUSRP51501).

498 499

AUTHOR INFORMATION

500

Corresponding Author

501

*Telephone: 0510-85876799; Fax (086)510-85876799; E-mail: [email protected].

502

ORCID

503

Yuanfa Liu: 0000-0002-8259-8426

504 505

NOTE

506

The authors declare no competing financial interest.

507 508

ABBREVIATIONS USED

509

AGF, gastric fluid; AIF, small intestine fluid; AP, adsorbed proteins; ASF, artificial saliva fluid; FFA,

510

Free fatty acid; GIT, gastrointestinal tract; GTP, glycerol tripalmitate; GTS, glycerol tristearate; GTO, 20


Page 20 of 36

Page 21 of 36


511

glycerol trioleate; LCT, long-chain triglycerides; LO, Leaf lard oil; MAG, monoglycerides; MCT,

512

medium-chain triglycerides; PO, Palm oil; RO, Rapeseed oil; TAG, triacylglycerol; WPI, Whey

513

protein isolate.

514 515

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516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558

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23



Figure captions Figure 1. The microscopic changes of dietary lipids in the different GIT sites. Figure 2. Diagram of fully designed in-vitro digestion model for lipid samples in the present study Figure 3. Mean particle diameter (A, B) and Zeta-potential (C, D) of WPI-stabilized oil and TAG emulsions in different digestion phase. Error bars represent the standard deviation calculated from three independent experiments (P= 0.05). Significant differences (P < 0.05) are indicated with different letters above the bars. Capital letters indicate significant difference between groups; while lowercase letters indicate significant difference within a group. Figure 4. Microstructure images of emulsions with different lipid types after they were exposed to different regions of the simulated GIT, (A) for PO, LO and RO, (B) for GTP, GTS and GTO, respectively. Figure 5. Changes of percentage of adsorbed proteins (AP %) in initial emulsions and gastric digested samples. Capital letters indicate significant difference between groups; while lowercase letters indicate significant difference within a group. Figure 6. Levels of total FFAs released from different lipid emulsions within 120 min digestion time in small digestion phase, A, B and C for PO, LO and RO respectively; D, E and F for GTP, GTS and GTO, respectively. The curves y=×× displayed in each figure were represent the maximum FFAs released (as %) during the digestion process. Figure 7. Corresponding FFA release data plotted as a first-order kinetics reaction as a function of 120 min lipolysis time as calculated from Equation (4). A, B and C for PO, LO and RO respectively; D, E and F for GTP, GTS and GTO, respectively.

24


Page 24 of 36

Page 25 of 36


Figure 1.

25



Figure 2.

26


Page 26 of 36

Page 27 of 36


Figure 3.

B

A

D

C

27



Figure 4.

Initial

Mouth

Stomach

(A)

PO

LO

RO

28


Page 28 of 36

Intestine

Page 29 of 36


Initial

Stomach

Mouth

(B)

GTP

GTS

GTO

29


Intestine


Figure 5.

30


Page 30 of 36

Page 31 of 36


Figure 6.

A

B

C

D

E

F

31



Page 32 of 36

Figure 7.

A

B

C

D

E

F

32


Page 33 of 36


Table 1. Chemical Composition of Artificial Digestion Fluids in Each Stage of GIT Model. Names

Regents or chemicals

Chemical formula

ASF

Sodium chloride Ammonium nitrate Potassium phosphate Potassium chloride Potassium citrate Uric acid sodium salt Urea Lactic acid sodium salt Porcine gastric Mucin Type II Sodium choloride Hydrochloric acid Pepsin Sodium choloride Calcium chloride Bile salt (porcine bile extract) Porcine pancreas lipase

NaCl NH4NO3 KH2PO4 KCl K3C6H5O7.H2O C5H3N4O3Na H2NCONH2 C3H5O3Na -NaCl HCl -NaCl CaCl2 ---

AGF

AIF

33


Concentration (g/L for solid; mL/L for liquid chemicals) 1.594 0.328 0.638 0.202 0.308 0.021 0.198 0.146 30.0 2.0 7.0 3.2 6.574 1.1 5.0 1.6


Page 34 of 36

Table 2. Fatty Acid Composition (%) of Oil and TAG Samples. Fatty acid C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3(n-3) a Others a

PO

LO

RO

GTP

GTS

GTO

0.68 ± 0.00 81.03 ± 0.79 4.31 ± 0.21 11.32 ± 0.25 1.90 ± 0.07 0.54 ± 0.00

1.26 ± 0.07 29.76 ± 0.18 1.25 ± 0.02 17.59 ± 0.11 35.70 ± 0.56 10.43 ± 0.17 0.39 ± 0.01 1.88 ± 0.03

3.77 ± 0.02 0.16 ± 0.00 1.58 ± 0.01 62.39 ± 0.86 20.42 ± 0.53 7.51 ± 0.35 2.45 ± 0.04

0.19 ± 0.00 98.95 ± 0.81 0.20 ± 0.00 0.07 ± 0.00 0.43 ± 0.00

0.68 ± 0.00 98.27 ± 0.75 1.05 ± 0.01

0.47 ± 0.00 2.12 ± 0.02 90.72 ± 0.83 3.15 ± 0.02 1.71 ± 0.01

Others represent the minor content FFAs which were not listed in the table.

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Page 35 of 36


Table 3. Triglyceride Profiles of the Experimental Lipids. a

PO

LO

Triglycerides Profiles (%) RO GTP

PPP 35.90 ± 0.26 SPO 17.98 ± 0.14 OOL 19.01 ± 0.15 PPP 84.33 ± 0.21 OOP 11.84 ± 0.11 OPO 16.92 ± 0.12 OLS 11.88 ± 0.10 POP 8.73 ± 0.05 LPS 8.69 ± 0.08 LLO 9.88 ± 0.10 PSP 8.20 ± 0.03 SPoO 4.94 ± 0.03 SOO 8.40 ± 0.07 PPO 5.93 ± 0.03 SPS 4.62 ± 0.02 OOO 8.18 ± 0.04 PPM 5.54 ± 0.02 PPO 4.14 ± 0.03 LLL 5.84 ± 0.05 POS 4.39 ± 0.02 OPEi 4.10 ± 0.01 OLO 4.35 ± 0.04 OPL 4.10 ± 0.02 SSP 3.84 ± 0.02 LLP 4.24 ± 0.00 PPS 2.91 ± 0.00 PPS 3.39 ± 0.00 LLPo 3.80 ± 0.00 POM 1.40 ± 0.00 OPoL 3.34 ± 0.00 LLLn 3.52 ± 0.01 b Others 11.05 ± 0.14 Others 28.03 ± 0.15 Others 20.89 ± 0.17 Others 15.67 ± 0.13 a P, palmitic; S, Stearic; O, Oleic; L, Linoleic, M, Myristic; Ln, Linolenic; Po, Palm oleic; Ei, Eicosenoic; b

Others represent other TAGs not detected or in minor content are not listed in.

35


SSS SSP

GTS 77.78 ± 0.28 5.86 ± 0.02

OOO OOS

GTO 84.21 ± 0.25 6.68 ± 0.02

Others

16.37 ± 0.12

Others

9.11 ± 0.14


Graphic for table of contents

36


Page 36 of 36

Triglyceride structure modulates gastrointestinal digestion fates of lipids

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