In Vitro Gastrointestinal Digestibility of Crystalline Oil-in-Water Emulsions

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In Vitro Gastrointestinal Digestibility of Crystalline Oilin-Water Emulsions: Influence of Fat Crystal Structure Wenjuan Jiao, Lin Li, Anling Yu, Di Zhao, Bulei Sheng, Medinu Aikelamu, Bing Li, and Xia Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04287 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 5, 2019

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Journal of Agricultural and Food Chemistry

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In Vitro Gastrointestinal Digestibility of Crystalline Oil-in-Water Emulsions:

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Influence of Fat Crystal Structure

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Wenjuan

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Zhang1, *

Jiao1,

Lin

Li1,2,

Anling Yu1, Di Zhao3, Bulei Sheng1, Medinu Aikelamu1, Bing Li1, *, Xia

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1 School of Food Science and Engineering, Guangdong Province Key Laboratory for Green

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Processing of Natural Products and Product Safety, South China University of Technology,

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Guangzhou 510640, China

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2 School of Chemical Engineering and Energy Technology, Dongguan University of Technology, College Road 1, Dongguan, 523808, China

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3 Key Laboratory of Meat Processing and Quality Control, MOE; Jiangsu Collaborative

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Innovation Center of Meat Production and Processing, Quality and Safety Control; Key

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Laboratory of Meat Products Processing, MOA; Nanjing Agricultural University; Nanjing,

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210095, P.R. China

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* Correspondence: [email protected] (B.L.); [email protected]; Tel.: +86-20-8711-3252 (B.L.)

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ACS Paragon Plus Environment


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ABSTRACT: To investigate how the fat crystal structure affects lipid in vitro digestibility, 30%

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palm stearin-in-water emulsions were prepared after storage at different temperatures (4, 25 and

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37°C) for 1 h, which consisted of different polymorphic forms, sizes and quantities of fat crystals.

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The variation of particle size (d4,3), zeta potential and microstructure during the gastrointestinal

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digestion, and the free fatty acid (FFA) released in small intestine phase were investigated. After

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oral and gastric digestion, all the emulsions underwent partial or complete coalescence and

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flocculation. During intestinal digestion, the d4,3 and zeta potentials did not notably affect lipid

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digestion. The FFA released assay results indicated that the lipid digestion extent decreased as the

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fat crystal size and content of the β polymorph increased, and there was no obvious relationship

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between FFA release and fat crystal quantity or solid fat content (SFC). This study highlighted the

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crucial roles of fat crystal size and polymorphic form in regulating the digestion behavior of

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lipid-based O/W emulsions.

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KEYWORDS: fat crystal structure; emulsion; in vitro digestion; polymorphic form


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INTRODUCTION

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Dietary lipids are essential nutrients and very important for human health1. The lipid

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digestibility within the gastrointestinal tract (GIT) directly determines their absorption and

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bioavailability2, 3. In the GIT, dietary lipids are exposed to numerous physicochemical and

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biochemical events4 and broken down into O/W emulsions because of mechanical stresses and

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various surface-active species5, 6. In the mouth, after mixing with saliva and subjecting to

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mechanical forces, the ingested food structure may be altered7-9. Then, the lipids in “bolus”

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undergo further structural changes with mechanical contractions, after blending with the highly

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acidic gastric juice in the stomach5,

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separation12, 13. Approximately 10-30% of the total lipid content is digested in the stomach where

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the lipid digestion process is initiated by the gastric lipase absorbing at the lipid-water interface5.

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Then the “chyme” containing partially hydrolyzed and emulsified lipids is exposed to small

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intestinal fluid mainly including various enzymes and bile salts and undergo a drastic pH change5,

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14, 15.

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absorbing at the oil droplets surface together with colipase and bile salts17. The hydrolysis

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products (eg., monoglycerides (MAGs) and free fatty acids (FFAs)) assemble themselves into

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mixed micelles, and diffuse to the brush border of the mucosa for absorption18.

10, 11,

which result in flocculation, coalescence and phase

Lipids hydrolysis primarily takes place in intestinal phase16, with the aid of pancreatic lipase

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The type, structure and position of fatty acids (FAs) in TAGs impact lipid digestion19-21. The

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structure and composition of the droplet surface and particle size in the emulsion substantially

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influence the digestion10, 22-24, as do the physical states of the lipid phase (e.g., crystalline, liquid

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crystalline, or liquid)25,

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digestion and absorption27-29. In the bulk system of a rat model, the degree of tristearin hydrolysis

26.

Lipids in the solid fat phase at body temperature showed lower



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(melting temperature of 73°C) was lower than that of triolein, which was completely liquid at

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37°C27. The intake of interesterified cocoa butter (37% solid fat content (SFC)) resulted in lower

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post prandial lipemia in humans compared with that associated with native cocoa butter (1% SFC)

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at 37°C28. Another study in guinea pigs demonstrated that the high-melting-temperature portions

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(42-44°C) in milk fat was less digested and absorbed than the low-melting-temperature portions

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(13-14°C)29. In the emulsion system, Guo et al.26 reported that the oil physical state largely

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influenced lipid digestion, and the digestion rate and extent decreased as the SFC increased. To

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exclude the influence of lipid composition, only one TAG composition (tripalmitin) was used in

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the study of the in vitro hydrolysis of emulsified lipids25, and that study showed that the

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digestibility of a liquid-state emulsion was higher than that of a solid-state emulsion.

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There are many types of high-fat food products, including butter and margarine, in which fat

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exists in a semisolid-state, and their physical properties, such as rheological, mechanical and

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sensory properties, depend on the underlying fat crystal network microstructure30. Moreover,

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products with the same SFC can vary in the fat crystal structure characteristics (e.g., fat crystal

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size, shape and polymorphic form), and factors that may affect the fat digestion behavior are

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poorly documented. However, to date, the differences in the digestion behaviors of foods with

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different fat crystal structures but the same lipid compositions remains unclear. Thus, further

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works are needed to explore the influences of fat crystal structure (SFC or fat crystal quantity, fat

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crystal size and polymorphic form) on lipid digestion behavior, which would help to modulate fat

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release and absorption.

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This work was aimed at characterizing the in vitro digestibility of crystalline O/W emulsions

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containing fats with different crystal structures but the same lipid compositions. We attempted to


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elucidate the relationship between the fat crystal structures (SFC or fat crystal quantity, fat crystal

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size and polymorphic form) and digestion. The focus was on investigating the key factors in

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determining the fat digestibility during the intestinal phase by primarily studying the changes of

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the physicochemical properties, microstructure and interfacial compositions in crystalline O/W

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emulsions.

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MATERIALS AND METHODS

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Materials. Sodium caseinate (C8654), mucin (M2378), bile extract (48305) and pancreatic

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lipase (P1750) were purchased from Sigma-Aldrich (Shanghai, China). Palm stearin (PS) (iodine

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value of 30.48±1.01 g I/100 g oil; acid value of 0.39±0.05 mg KOH/g FFA; melting point of

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49±0.2°C) was obtained from Cargill Inc., (Dongguan, China). And the other analytical grade

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chemicals were used in this case.

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Emulsion Preparation. To prepare an aqueous emulsifier solution, sodium caseinate powder

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(10 g) was dissolved into water (690 g) with stirring (MS-H-Pro+, DragonLab, China) for 2 h at

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60°C. Followed a period of heating (60°C, 30 min), 30 wt% of PS and 70 wt% of sodium

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caseinate solution were mixed to prepare the coarse emulsions using a high-speed shear

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emulsifying machine (T18, IKA, Germany) at 13000 rpm for 2 min, followed by using an

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ultrasonic device (VCX500, SONICS, USA) at 40% power (200 W) for 30 s (10 s sonication with

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a 10 s interval). The final emulsions were transferred into serum bottles and stored at different

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temperatures (4, 25 and 37°C) for 1 h, respectively. The emulsions after storage were used as the

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initial samples for digestion assays.

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Thermal Behavior. For analyzing the melting and crystallization behavior, approximately 20

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mg emulsion or 6 mg PS was placed in a high-pressure stainless-steel pan and characterized by a



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differential scanning calorimeter (DSC-8000, PerkinElmer, USA). To ensure the initial

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temperature was the same with the storage temperature, the emulsion sample or bulk fat in the

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aluminum pan was equilibrated at 4, 25 and 37°C for 2 min, followed by a heating process to

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80°C then a cooling process to 0°C at 10°C/min. The DSC software was used to determined to

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total enthalpy of melting (ΔHm), the melting and crystallization temperature (Tm and Tc). Three

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replicates were carried out for each determination.

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Solid Fat Content (SFC). After melting (80°C, 30 min), the completely melted bulk fat (PS)

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was stored at temperatures (4, 25 and 37°C) for 1 h. And the bulk fat SFC (SFCfat) was measured

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by a Bruker NMR SFC analyzer (mq-one, Bruker, Germany). Three replicates were carried out for

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each determination.

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The SFC of each emulsion (SFCemulsion) was determined using the following equation31:

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SFCemulsion  SFCfat

H m emulsion 100% H m  bulkfat

(1)

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where ΔHm-emulsion and ΔHm-bulk are determined by the DSC software, which represent the total

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melting enthalpy of the emulsion and bulk fat.

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X-ray Diffraction (XRD) Measurement. XRD (Smartlab, Rigaku, Japan) was applied into the

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polymorph analysis of fat crystals in the samples, working at 40 kV/40 mA, with Cu KR radiation

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and a Ni filter. And the scans (5-30°) with a scanning rate of 2.0°/min were kept at 4, 25 and

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37°C. The relative contents (%) of polymorphic forms (β′, β) crystals were calculated according to

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the method of Reshma et al.32

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Simulated Gastrointestinal Tract Model. All specimens were continuously incubated in the

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simulated mouth, stomach, and intestinal fluids with a magnetic stirring device at 100 rpm (RO


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15, IKA, Germany).

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Initial system: The emulsion samples after storage contained 30% (w/w) oil were transferred into

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glass beakers placing in a water bath.

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Mouth phase: The initial emulsions (2 mL) in the glass beakers were blended with simulated

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saliva fluid (SSF) (28 mL), which was prepared by a previous method of Sarkar et al.33. And the

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final mixture contained 2% (w/w) oil was adjusted to pH 6.8 then kept for 10 min at 37°C.

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Gastric phase: According to a previously reported method12, NaCl (2 g), HCl (37%, 7 mL) and

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water (1 L) were used to prepare simulated gastric fluid (SGF), and the SGF was adjusted to pH

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1.2 using HCl (1 M), then added to the ‘‘bolus’’ at a ratio of 1:1 (v/v). The final mixture contained

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1% (w/w) oil was adjusted to pH 2.5 with 1 M NaOH and stirred for 2 h at 37°C.

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Small intestinal phase: After adjusting pH to 7.0, 30 mL “chyme” was kept at 37°C. Next, the bile

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extract (4 mL, 46.87 mg/mL) and CaCl2 solution (1 mL, 110 mg/mL) were incorporated, followed

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by adding the pancreatic lipase suspension (2.5 mL, 24 mg/mL).

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Particle Size and Distribution Measurement. To determine the particle size (average

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volume-weighted mean diameter, d4,3) and distribution, a laser-diffraction-size analyzer

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(Mastersizer 3000, Malvern Instruments Ltd., UK) was used. And the refractive index of PS,

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1.478 was used for calculating. Five replicates were carried out for each determination.

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Zeta potential Determination. A Malvern Zetasizer Nano-ZS (MPT-2, Malvern Instruments

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Ltd., UK) was used to test the zeta potentials. A 1-mL sample was transferred into a DTS1070 cell

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with a balance time of 2 min. Each sample was determined in triplicate and the initial samples

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were diluted 100-fold with a casein sodium solution.

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Microstructure Measurements. Polarized light microscopy (PLM) (BX51, OLYMPUS,



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Japan) was used to perform the microstructural analysis of all samples and the fat crystal particles

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obtained by centrifuging the digestion juices at 12,000 rpm for 15 min. Specimens were placed on

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the microscope slides (7101, Sail Brand, China) with a coverslip (10212020C, Citoglas, China) at

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room temperature (25°C).

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Free Fatty Acid (FFA) Release. FFA released from the small intestine phase was measured by

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an automatic titrator (902, Metrohm, USA), where the pH was maintained at 7 with 0.1 M NaOH.

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And the content of FFA released was calculated according to the following equation:

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%FFA  100  (VNaOH  m NaOH  M Lipid ) / (WLipid  2)

(2)

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where VNaOH (mL) represents the consumed NaOH volume, mNaOH is the NaOH molarity (0.1 M),

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respectively. WLipid (g) and MLipid refer to the total lipids weight and the PS molecular (~ 850

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g/mol). Three replicates were carried out for each determination.

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Statistical Analysis. The experimental data are listed as the means ± standard deviations.

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SPSS 16.0 (SPSS Inc., Chicago, USA) and the Turkey’s test were used to evaluate the

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significance analysis. Significant differences existed in the data when p < 0.05.

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RESULTS AND DISCUSSION

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Polymorphism and Thermal Behavior of Initial Crystalline O/W Emulsions. The SFC, fat

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polymorphism and thermal behavior of the initial crystalline emulsions were determined to show

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the influence of storage temperature on the fat crystal properties, as these changes may result in

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different digestion behaviors1. The enthalpies (ΔHm) and SFCs of the bulk fats and emulsions are

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shown in Table 1. The enthalpies (ΔHm) and SFCs of the bulk fats and the emulsions showed

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obvious decrease with the increasing storage temperature (p < 0.05). For example, the SFCs of the

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emulsions stored at 4, 25 and 37°C were 26.57%, 15.20%, and 8.42%, respectively. DSC is


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commonly used to evaluate the thermal behaviors of fat in the bulk fats and the emulsions. The

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melting and crystallization profiles of the O/W emulsions are shown in Figure 1, and the peaks of

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the emulsions correspond to the crystal-to-liquid phase transition. The Tm values of the emulsions

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stored at 4, 25 and 37°C were 45.25, 46.22 and 47.71°C, respectively, indicating that all the

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emulsion samples contained fat crystals. The discrepancies in the Tm values were attributed to

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variations in the fat crystal structures. When the samples underwent the cooling process (80 to

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0°C), two crystallization peaks (peak 1 and peak 2) were observed in the cooling curve (Figure 1).

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In the crystallization curves of the emulsions stored at 4, 25 and 37°C, the Tc values of peak 1

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were 4.34, 4.31 and 4.29°C (p > 0.05), respectively, while Tc values of peak 2 were 26.30, 25.98

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and 25.73°C (p > 0.05), respectively. The Tc values of all the samples were similar (p > 0.05)

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because their TAG compositions from the PS were the same.

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The short and long spacings of fat crystals determined by XRD34 are shown in Figure 2. The

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position of the peaks in the short-spacing region is indicative of different polymorphic forms of

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the TAGs. The least stable polymorph α displays a single peak at 4.15 Å35; while the metastable

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polymorph β′ forms small crystals, with two obvious diffraction peaks at approximately 3.8 and

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4.2 Å36; and the most stable polymorph β (d-spacing at 4.6 Å) tends to form large, plate-like

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crystals37. The results of the short-spacing region indicated that both polymorph β′ and β were in

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the emulsions stored at 25 and 37°C, while the emulsion stored at 4°C only had β′ form (Figure 2).

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The emulsions stored at 25 and 37°C had 6.25% and 97.8% of the β form, respectively.

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Considering the higher tendency of the β form to form large crystals, the fat crystal sizes in the

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emulsion stored at 37°C, with a higher β content, could be larger than those in the emulsion stored

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at 25°C, while the fat crystal sizes in the emulsion stored at 4°C were smallest. Because the Tm



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value of polymorph α is the lowest and that of polymorph β′ and β is intermediate and highest37,

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the XRD results (Figure 2) were in accordance with the DSC results (Figure 1), in which the

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temperature of the melting peak temperature increased with increasing storage temperature. The

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diffraction peaks of the long-spacing regions of the emulsions were 43.80 and 14.62 Å (the

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emulsion stored at 4°C), 43.08 and 14.45 Å (the emulsion stored at 25°C), 41.70 and 13.92 Å (the

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emulsion stored at 37°C), which were attributed to as the double chain length structure of TAGs34,

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38.

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Subsequently, each sample was subjected to the simulated GIT digestion. The variation of

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d4,3, zeta potential, microstructure and release of FFA were determined throughout the digestion to

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elucidate the potential influence of fat crystal structure on digestion behavior.

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Evolution of Droplet Size (d4,3) and Distribution. The particle size (d4,3) and distributions of

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lipid droplets after exposure to the GIT phases are shown in Figure 3. Initially, all emulsions

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showed relatively small lipid droplets, predominantly in the 0.01-10 μm range, and a small portion

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of large lipid droplets was also detected (10-100 μm). After oral digestion, a small fraction of

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large particles (> 100 μm) appeared in the emulsion stored at 4°C, while the distributions of

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particle size in the emulsions stored at 25 and 37°C were mainly in the 1-100 μm range. After

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gastric digestion, the particle size distribution in the emulsion stored at 37°C was mainly between

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10 and 100 μm, and an obvious peak at approximately 100 μm was found in the emulsion stored at

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4°C. These findings suggested that these droplets were unstable and that aggregation/flocculation

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or coalescence occurred during gastric digestion. The distributions of particle size in all samples

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were mainly at 10-100 μm after intestinal digestion (Figure 3-A, B, C).

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Figure 3-D shows the changes of the d4,3 values in all samples after the simulated GIT


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digestion. After oral and gastric digestion, a significant increase of the d4,3 values in all samples

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was observed (p < 0.05), while the d4,3 values in all samples decreased after being subjected to

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intestinal digestion phase (p < 0.05). After oral digestion, the obvious increase (p < 0.05) in d4,3

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values of three emulsions was attributed to the interaction between mucin and the droplet, causing

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bridging and/or depletion flocculation33, 39. Additionally, the d4,3 values for the emulsion stored at

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4°C were significantly larger than those of the emulsions stored at 25 and 37°C (p < 0.05). This

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result possibly occurred due to the weaker steric hinderance effect between emulsions, as they

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were more prone to aggregate with each other, leading to destabilization. The values of d4,3 in all

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samples had drastic increase after gastric digestion (p < 0.05), and the d4,3 values decreased

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significantly in the order of 4°C > 25°C > 37°C (p < 0.05). A previous study40 showed a similar

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result where the sodium caseinate-stabilized emulsions were highly unstable and formed

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aggregates during gastric digestion phase, owing to the weakening of electrostatic repulsion14, 41

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and

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undergo partial coalescence due to the fat crystals43. The smaller crystals provided better droplet

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coverage than the relatively larger crystals44, but the d4,3 values of the emulsion stored at 4°C was

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the highest, which can be explained by that the needle-shaped crystals of the β′ form being more

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likely to lead to destabilization37,

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decrease after intestinal digestion (p < 0.05), and they decreased obviously in the following

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sequence of 4°C > 37°C > 25°C (p < 0.05). This decrease in d4,3 may be explained by the

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dissociation of the flocculated droplets caused by emulsification of the bile salts or by TAG

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hydrolysis48. Moreover, the fat crystal structure and location can be changed by agitation and

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digestion, which may also influence the emulsion droplet size.

depletion or bridging flocculation caused by mucin33, 42. Additionally, all emulsions may

45-47.

The d4,3 values of three samples showed significant



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Evolution of the Emulsion Zeta Potential. The composition at the TAG-water interface

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substantially influences the TAG hydrolysis efficiency1. The electrical characteristics of lipid

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droplets were measured during GIT digestion to elucidate the interfacial properties (Figure 4), and

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the evolution in zeta potential of all emulsions was similar. The initial emulsion droplets showed

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the strongest negative charge (from -31.8 to -34.5 mV), which may be closely related to the

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presence of sodium caseinate (pI~5) at neutral pH41. The zeta potentials of the initial emulsions

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showed no obvious difference (p > 0.05), implying that both the sodium caseinate content at the

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droplet surface and the components in emulsion water phase of the initial emulsions were similar,

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regardless of the different fat crystal structures (SFC, fat crystal size and polymorphic form). The

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decreases in the absolute values of zeta potential during the mouth phase were caused by pH and

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ionic strength changes48, the presence of mineral ions within simulated saliva49 and the mucin

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adsorption or displacement onto the oil droplet surfaces42. The pH of the simulated stomach juices

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(pH 2.5) was below the pI of sodium caseinate, and the droplets coated by protein would have a

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highly positive charge if no change occurred in the interfacial composition50. After gastric

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digestion, the absolute values of the zeta potential were further decreased, which was possibly

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attributed to the highly ionic strength and acidic gastric juice. Notably, the electrical

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characteristics suggested that the sodium caseinate were displaced from the interface, possibly by

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mucin48. The droplets surfaces in all samples showed a stronger negative charge (p < 0.05) after

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intestinal digestion, because of the displacement of different types of surface-active anionic

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particles (eg., phospholipids and bile salts) in the intestinal fluids48. In addition, the lipid

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hydrolysis may alter the interfacial composition, and some negatively charged particles (e.g.,

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surface active FFAs and MAGs) may assemble at their surfaces14. This trend was similar to those


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observed in previous studies51, where wheat gliadin, caseinate sodium and whey protein isolates

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were used as emulsifiers.

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Emulsion and Crystals Microstructure Changes. The changes in the microstructures of

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all emulsions and fat crystal particles throughout the GIT digestion are shown in Figures 5 and 6.

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The observed dimensions were generally in line with the d4,3 results. For the initial emulsions, the

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fat crystal particles in emulsion stored at 4°C were small in size, while relatively larger fat crystal

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particles appeared in the emulsions stored at 25 and 37°C. In addition, combined with the SFC

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results (Table 1) and the XRD results (Figure 2), the emulsion stored at 4°C with primarily the β′

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polymorph and small fat crystal sizes had the highest fat crystal quantity (or SFC), while the

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emulsion stored at 37°C had the lowest fat crystal quantity (or SFC), which contained 97.8% β

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polymorph and had a large fat crystal size. Oral processing led to flocculation induced by mucin in

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all emulsions33, and larger particles were found in the emulsion stored at 4 and 25°C. Gastric

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digestion resulted in droplet aggregation, which was consistent with the d4,3 results (Figure 3), and

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no obvious change was observed in the fat crystal particles (Figure 6). Some apparent large

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aggregates were found in the emulsion stored at 4 and 25°C, particularly in the emulsion stored at

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4°C, which primarily occurred due to coalescence, flocculation promoted by the acidic pH and

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mechanical shearing14, 42. In addition, the exposure of fat crystals could lead to further aggregation

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due to partial coalescence43. The fat crystal size in all samples after intestinal digestion increased

294

slightly, whlie some apparent larger fat crystals appeared in the sample stored at 37°C, which can

295

be attributed to the aggregation of fat crystals (Figure 6). In addition, the negatively charged bile

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salts adsorbed on the droplet surface and provided electrostatic repulsions between droplets,

297

leading to the breakdown of large aggregates52. The disruption of large aggregates was also due to



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the TAG hydrolysis or emulsification of bile salt.

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Free Fatty Acid (FFA) Release. Lipolysis is initiated by pancreatic lipase/colipase

300

complex with the aid of surface-active bile salts during intestinal digestion phase. As shown in

301

Figure 7, the content of FFA release increased with the increasing digestion time. An initial rapid

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FFA release was observed in the first 10 min of intestinal digestion, 14.33% of the FFA was

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released from the emulsion stored at 4°C, while 13.44% and 12.55% of the FFAs were released

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from the emulsions stored at 25 and 37°C, respectively. However, in 10 min to 120 min of

305

intestinal digestion, the content of FFA release from the emulsion stored at 4°C was significantly

306

higher than those of the emulsions stored at 25 and 37 °C. The initial rapid FFA release from the

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emulsion stored at 4°C was followed by a more gradual release after 60 min of intestinal

308

digestion, while more gradual changes existed in the FFAs released from the emulsions stored at

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25 and 37°C after 10 min of intestinal digestion. This phenomenon could be explained by that the

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lipolysis products (eg., MAGs and FFAs) accumulated at the droplet interface, which restricted the

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access of pancreatic lipase/colipase complex to the TAG core52. The contents of FFAs showed the

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following order of 4°C > 25°C > 37°C after 2 h of intestinal digestion, and the contents of FFAs

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released from emulsions stored at 4, 25, and 37°C were 82.28%, 66.63%, and 59.00%,

314

respectively.

315

The mean droplet diameter and the composition at the TAG/water interface substantially

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influence TAG digestion in the emulsion1, 24. The interface composition can alter the surface area

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exposed to the lipases, and the digestibility of TAG decreased along with increasing droplet

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diameter48, 53. The particle sizes of emulsion after gastric digestion significantly influenced the

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digestion rate and extent during intestinal digestion54. After gastric digestion, the values of d4,3


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decreased in the following order of 4°C > 25°C > 37°C and showed obvious differences (p < 0.05)

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(Figure 3). The values of FFAs released determined herein were inconsistent with a previous

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report that the small lipid droplets had highest digestion rate and extent24, 55. Moreover, the zeta

323

potentials of all emulsions after gastric digestion showed no obvious differences (p > 0.05) (Figure

324

4), but the digestion extents were obviously different. Therefore, in our investigation, the

325

interfacial composition and mean droplet diameter were not the most important parameters

326

influencing the digestion extent. In a previous report, all samples had different lipid

327

compositions26, and the digestion extent decreased with increasing SFC. In our investigation, all

328

samples had the same lipid compositions; the emulsion stored at 4°C, which had the highest SFC

329

(Table 1), showed highest digestion extent (Figure 7), while the lowest digestion extent (Figure 7)

330

was observed in the emulsion stored at 37°C, which had the lowest SFC (Table 1). Therefore, in

331

this study, SFC was not the determining factor influencing the extent of lipid digestion. Fat

332

crystals can lead to lipid droplet aggregation by partial coalescence and flocculation, altering the

333

accessibility of pancreatic lipase/colipase complex to hydrolyze the lipid droplets56. However, the

334

emulsion stored at 4°C with high quantities of fat crystals exhibited the highest digestibility, while

335

the emulsion stored at 37°C with low quantities of fat crystals had the lowest digestibility. This

336

finding illustrated that the droplet aggregation resulting from fat crystals was not the dominant

337

factor influencing the digestion extent, which was consistent with the d4,3 results described above.

338

Additionally, fat crystal structure may be determinants affecting lipid digestion. The larger fat

339

crystals may have greater spatial resistance to prevent subsequent adsorption of pancreatic

340

lipase/colipase complex. In addition, the densest β polymorph has a triclinic-parallel subcell in

341

which adjacent chains pack tightly together57, 58, further influencing the TAG hydrolysis. In this



342

study, the emulsion stored at 37°C, which had a high proportion of large fat crystals in the β form,

343

had the lowest digestibility, while the emulsion stored at 4°C, which only had small fat crystals in

344

the β′ form, showed the highest digestibility. Thus, lipid digestion in the crystalline O/W

345

emulsions was heavily influenced by the fat crystal structure (size and polymorphic form) when

346

the lipid composition within crystalline O/W emulsions was constant.

347

In summary, the digestion behavior of sodium caseinate-stabilized crystalline O/W emulsions

348

with different fat crystal structures was investigated, and the lipid compositions among samples

349

were the same in this study. The digestion extent decreased as fat crystal size and the β polymorph

350

content increased, and there was no obvious relationship between the FFA release and the fat

351

crystal quantity or SFC. Large fat crystals can prevent the subsequent adsorption of pancreatic

352

lipase/colipase complex to the droplet interface, and β polymorph is the densest polymorphic form,

353

thus further influencing the access of lipase to the TAG. The results in this work gain further

354

understanding of digestibility of the crystalline emulsions and facilitate the rational design of

355

crystalline emulsion-based vehicles that can modulate FFA bioavailability and lipid metabolism.

356

■ AUTHOR INFORMATION

357

Corresponding Author

358

* Bing Li: E-mail: [email protected]: +86 13650736070.

359

* Xia Zhang: E-mail: [email protected]. Tel: +86 20 87113252.

360

ORCID

361

Wenjuan Jiao: 0000-0003-0599-5249

362

Bing Li: 0000-0002-4407-2132

363

Funding


Page 16 of 33

Page 17 of 33


364

This work is supported by the National Natural Science Foundation of China (NSFC)-Guangdong

365

Joint Foundation Key Project (No. U1501214), the National Natural Science Foundation of China

366

(No. 31871758, 31401660), the Pearl River S&T Nova Program of Guangzhou (No.

367

201806010144) and the Fundamental Research Funds for the Central Universities, SCUT (No.

368

2017MS093).

369

Notes

370

The authors declare no competing financial interest.

371

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Figure Captions

Figure 1 Melting profiles of crystalline O/W emulsions stabilized by sodium caseinate after 1 h of

storage at 4, 25, and 37°C.

Figure 2 XRD spectra of crystalline O/W emulsions stabilized by sodium caseinate after 1 h of

storage at 4, 25, and 37°C.

Figure 3 Particle size (d4,3) and distribution of crystalline O/W emulsions stabilized by sodium

caseinate following in vitro oral, gastric and intestinal digestion. Particle size distribution: 4°C

(A), 25°C (B), 37°C (C) and d4,3 changes (D). Different capital letters indicate significant

differences (p < 0.05) in the d4,3 values from different storage temperatures within the same

digestion phase. Different lowercase letters indicate significant differences (p < 0.05) in the d4,3

values from the same storage temperature during different digestion phases.

Figure 4 Changes in the zeta potentials of crystalline O/W emulsions stabilized by sodium

caseinate following in vitro oral, gastric and intestinal digestion. Different capital letters indicate

significant differences (p < 0.05) in the zeta potentials from different storage temperatures within

the same digestion phase. Different lowercase letters indicate significant differences (p < 0.05) in

the zeta potentials from same storage temperature during different digestion phases.



Figure 5 Microstructure of crystalline O/W emulsions stabilized by sodium caseinate during oral,

gastric and intestinal digestion. Both optical (left column) and polarized light microscopy (right

column) were conducted at the indicated temperature. The scale bar is 20 μm.

Figure 6 PLM of fat crystal particles in crystalline O/W emulsions stabilized by sodium caseinate

during GIT after centrifugation. The scale bar is 20 μm.

Figure 7 Fatty acids release profiles of crystalline O/W emulsions stabilized by sodium caseinate

during intestinal digestion.

Table 1 The enthalpies (ΔHm) and SFCs of sodium caseinate-stabilized emulsions and bulk fats

stored at 4, 25, and 37°C.


Page 24 of 33

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Figure 1



Figure 2


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Figure 3



Figure 4


Page 28 of 33

Page 29 of 33


Figure 5



Figure 6


Page 30 of 33

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Figure 7



Page 32 of 33

Table 1

Bulk

Emulsion

Temperature ΔHm (J/g)

SFC (%)

ΔHm (J/g)

SFC (%)

4°C

31.81±2.03a

79.15±1.66a

10.55±1.49a

26.57±0.02a

25°C

16.53±2.34b

37.93±0.21b

6.61±0.94a

15.20±0.09b

37°C

6.08±0.69c

25.73±1.18c

2.02±0.29b

8.42±0.49c

Different lowercase letters within a column indicate significant differences (p < 0.05).


Page 33 of 33


Table of Contents/Abstract Graphic


In Vitro Gastrointestinal Digestibility of Crystalline Oil-in-Water Emulsions

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