Gelatin Particle-Stabilized High-Internal Phase Emulsions for Use in

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Gelatin Particle-Stabilized High Internal Phase Emulsions for use in Oral Delivery Systems: Protection E#ect and in Vitro Digestion Study Huan Tan, Lifeng Zhao, Sisi Tian, Hui Wen, Xiaojun Gou, and To Ngai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04705 • Publication Date (Web): 08 Jan 2017 Downloaded from http://pubs.acs.org on January 19, 2017

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Journal of Agricultural and Food Chemistry

Gelatin Particle-Stabilized High Internal Phase Emulsions for use in Oral Delivery Systems: Protection Effect and in Vitro Digestion Study Huan Tan†, Lifeng Zhao†, Sisi Tian§, Hui Wen§, Xiaojun Gou*, †, and To Ngai*, ‡

† Key Laboratory of Medicinal and Edible Plants Resources Development of Sichuan Education Department, Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu, 610052, PR China § Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu, 610065, PR China ‡ Department of Chemistry, The Chinese University of Hong Kong, Shatin, N. T. Hong Kong

*Corresponding authors

*(Xiaojun Gou) Tel: +86-028-84216578; Fax: +86-028-84333218; E-mail: [email protected].

*(Ngai To) Tel: (+852) 39431222; Fax: (+852) 2603 5057; E-mail: [email protected]. ORCID Xiaojun Gou: 0000-0003-3211-3985 1

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ABSTRACT: The potential application of Pickering high internal phase emulsions (HIPEs) in the food

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and pharmaceutical industries has yet to be fully developed. Herein, we synthesized fairly

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monodispersed, non-toxic, auto-fluorescent gelatin particles for use as sole stabilizers to fabricate oil-in-

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water (O/W) HIPEs in an effort to improve the protection and bioaccessibility of entrapped β-carotene.

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Our results showed that the concentration of gelatin particles determined the formation, microstructure,

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droplet size distribution, and digestion profile of the HIPEs. For storage stability, the retention of β-

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carotene in HIPEs was significantly higher than in dispersion in bulk oil, even after 27 days of storage.

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In addition, in vitro digestion experiments indicated that the bioaccessibility of β-carotene was

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improved 5-fold in HIPEs. The present study will help establish a correlation between the

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physicochemical properties of gelatin particle-stabilized HIPEs with their applications in the oral

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delivery of bioactive nutraceuticals.

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KEYWORDS: gelatin particle, Pickering high internal phase emulsion, β-carotene, in vitro digestion,

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bioaccessibility

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INTRODUCTION

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In recent years, novel particle-stabilized emulsions (so called Pickering emulsions) have served as

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nutraceutical carriers for food and dietary supplements; these emulsions have been the subject of long-

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term research interests due to their outstanding stability, improved bioavailability, and palatability.1, 2 In

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particular, Pickering high internal phase emulsions (HIPEs), wherein the volume fraction of the internal

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phase (Φ) exceeds 0.74,3 have received growing attention for their uses in applications such as

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templates for scaffolds,4, 5 gels,6 and solid-like hydrophobic matrices.7 The advantage of utilizing HIPEs

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as nutraceutical carriers stems from their outstanding large Φ and tunable viscoelasticity properties,8

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where formulations with high nutraceutical loading and specific rheological behavior are desirable for

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flexible downstream applications. The thick interfacial particle layer around the droplets gives HIPEs

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advantages in the oxidation stability of the encapsulated compounds by greatly retarding the diffusion of

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pro-oxidants or free radicals. As delivery vehicles, HIPEs can also provide improved stability as well as

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controlled release in the gastrointestinal tract for encapsulated bioactive compounds. In addition, by

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virtue of their very low water content, oil-in-water (O/W) HIPEs can extend the shelf life of emulsions

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by preventing the growth of microorganisms.9 Although extensive research has addressed the

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application of HIPEs in the field of material science, much less research has been conducted that is

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aimed at understanding the oral delivery of nutraceuticals using HIPEs, especially Pickering HIPEs.

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To provide a physical barrier to prevent emulsion droplet flocculation and coalescence, the

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preparation of food emulsions generally involves the spontaneous accumulation of massive low

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molecular weight surfactants or biopolymers (5 ~ 50 vol%) at the oil-water interface to form dense

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packing layers.10 In contrast, particles with partial dual wettability are known to adsorb irreversibly at

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the oil-water interface to form rigid layers. These layers promote the stability of Pickering emulsions

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toward coalescence, creaming, and Ostwald ripening.8 To date, colloid particles suitable for O/W

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Pickering HIPE preparation are relatively limited in food science. Reported food-grade particles, such as

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soy protein nanoparticle aggregates,11 kafirin nanoparticles,12 and phytosterol colloidal particles,13 have

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been introduced in stabilizing Pickering emulsions. Even in these studies, few colloid particles meet the 3 ACS Paragon Plus Environment

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requirements for serving as effective stabilizers for HIPE preparation due to harsh intermediate

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wettability (contact angle θ approaches 90°), as well as insolubility in both liquid phases. Moreover, the

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employed particles are often highly polydispersed and poorly characterized. The colloidal particles

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coexisting with smaller soluble protein molecules in the aqueous phase obfuscate the real stabilizers that

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lower the interfacial tension during emulsification.14 In addition, information on the cytotoxicity of the

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synthesized particles is limited. Furthermore, the detection of colloid particles is rather complicated and

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always requires conjugation with an external fluorochrome. Thus, it is still a challenge to find effective

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Pickering stabilizers to formulate HIPEs for use in the food industry.

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In our previous work, gelatin, one of the most versatile biopolymers in the food industry, was

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assembled into spherical nanoparticles to stabilize O/W HIPEs with hexane as the internal phase. The

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prepared HIPEs exhibited outstanding stability against coalescence and were inherently permeable,

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which promotes the long-term controlled release of encapsulated bioactive compounds. Inspired by

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these results, in the present study, we investigated the feasibility of gelatin particle-stabilized HIPEs to

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be functionalized as a novel encapsulation system for a nutraceutical, β-carotene, and as an oral delivery

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system. β-carotene is a well-known active phytochemical constituent that plays a major role in

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preventing certain chronic diseases caused by free radicals, such as cancer, heart disease, and colorectal

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adenomas.15,

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possesses poor water-solubility and bioaccessibility, which greatly restrict its use in food formulations.

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Herein, the purpose of our study was to further illustrate the preparation, auto-fluorescence, and

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cytotoxicity of gelatin particles, and then to introduce them as sole stabilizers to prepare O/W HIPEs

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with edible oil as the internal phase. The stability of the encapsulated β-carotene under normal storage

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conditions was investigated. The digestion profile of the HIPEs was also performed to mimic the

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digestion process in the gastrointestinal tract in order to determine the extent of lipolysis and the

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bioaccessibility of β-carotene.

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However, β-carotene is highly susceptible to thermal and oxidative degradation and

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MATERIALS AND METHODS

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Materials. Gelatin type B (~250 g bloom), β-carotene (95%), porcine pepsin, lipase from porcine

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pancreas, and bile salt were obtained from Aladdin (Shanghai, China). HEK293 (human embryonic

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kidney 293) and LO2 (human normal liver) cells were purchased from the American Type Culture

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Collection (ATCC). Methylthiazoleterazolium (MTT) was purchased from Sigma-Aldrich (St. Louis,

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MO, USA). Medium-chain triacylglycerol (MCT) oil with 60% octanoic acid and 40% decanoic acid

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was obtained from LIPO (USA). Sunflower oil (STANDARD FOODS, Shanghai, China) was used as

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received. All other chemicals were purchased from Kelong Chemical Reagent (Chengdu, China) and

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used without further purification. Water purified by a Milli-Q system was used for sample preparation.

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Preparation of Gelatin Particles. The gelatin particles were prepared using a modification of the

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procedure used by Coester and others.8, 17 In brief, 1.25 g of gelatin type B was dissolved in 25 mL of

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distilled water to prepare a gelatin solution under constant heating and stirring. Then, 25 mL of acetone

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was added to precipitate the high molecular weight (HMW) gelatin. After that, the HMW gelatin, i.e.,

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the precipitate, was collected and re-dissolved in 25 mL of distilled water with the pH of the solution

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adjusted to 12.0. A total of 75 mL of acetone was added drop-wise into the gelatin solution, followed by

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the addition of 125 µL of glutaraldehyde solution (25% aqueous solution) to form gelatin particles. The

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resulting mixture was then stirred at 50 °C for 3 or 14 h. At the end of the process, the dispersion was

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centrifuged at 10 000 g (Thermo ST16R, USA) for 40 min, and the particles were purified by three-fold

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centrifugation and redispersion in an aqueous acetone mixture (30 vol%, acetone). Finally, the particles

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were dispersed in distilled water, and the residual acetone was removed by slow vaporization. The

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resultant nanoparticles were stored at 4 °C for further experiments.

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Characterization of Gelatin Particles. Particle Size Measurement. The size of the gelatin particles

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was determined by photon correlation spectroscopy using a Zetasizer (Malvern Nano-ZS ZEN3600,

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England) at a detection angle of 90° at 25 °C. The concentration of the gelatin particles was 0.5 wt%.

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Atomic Force Microscopy (AFM). The size and morphology of the gelatin particles prepared under

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different cross-linking times were imaged by AFM (SPM-9600, Shimadzu, Japan). The gelatin particle

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solution was pre-prepared, and one drop of the dilute dispersion was placed on a freshly cleaved mica

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substrate and then dried in a desiccator for one day at room temperature. A tapping mode with a

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Dimension 3100 Nanoscope IV equipped with TAP 150 A1-G AFM probes was applied for the analysis

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of the samples in air.

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Confocal Laser Scanning Microscopy Observation. Gelatin particles cross-linked for 3 h were placed

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in a Petri dish and were observed using a TCS SP5 laser scanning confocal microscope (CLSM, Leica,

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Germany) with a 40 × objective (NA = 0.85). Two lasers at 488 and 543 nm were used to excite the

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gelatin particles, and a series of x/y layers were scanned.

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In Vitro Cytotoxicity against HEK293 and LO2 Cells. Briefly, HEK293 and LO2 cells (4 × 103 per

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wells) were added in a 96-well plate and cultured in a 5% CO2 humidified incubator at 37 °C for 24 h.

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Then, the cells were exposed to gelatin particles at concentrations of 78.125, 156.25, or 312.5 µg/mL for

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another 72 h incubation. At the end of the exposure period, the typical qualitative external morphology

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of the cells exposed to the 312.5 µg/mL gelatin particle solution was observed by Nikon optical

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microscopy. After that, 20 µL of MTT solution (5 mg/mL) was added per well and incubated for another

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2 h at 37 °C. Afterward, the supernatant fluid was removed, and 150 µL of DMSO was added per well

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for 15 ~ 20 min. The optical density of the mixtures at 570 nm was measured. Cell viability was

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determined as the percent absorbance relative to the control, which was obtained in the absence of

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gelatin particles under the same conditions. Each assay was carried out at least three times.

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Fabrication of β-carotene-encapsulated HIPEs Stabilized by Gelatin Particles. To determine the

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wettability of gelatin particles in the formation of HIPEs with edible oil as the internal phase, 20 vol%

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gelatin particle solution with different concentration (0.1, 0.3, 0.5, 1.0, 1.5, or 2.0 wt%) and 80 vol%

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sunflower oil were mechanically mixed with an Ultra Turrax T18 homogenizer (IKA, 10 mm head)

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operating at 12000 rpm for 30 s. ACS Paragon Plus Environment

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For the fabrication of β-carotene-encapsulated HIPEs, the oil phase was first prepared by dissolving

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β-carotene in sunflower oil or MCT oil (final concentration = 0.83 mg/mL) under continuous stirring in

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the dark until a homogeneous dispersion was produced. Next, 20 vol% gelatin particle solution with a

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concentration of 0.5, 1.0, or 1.5 wt% was mixed with 80 vol% oil phase by mechanically shearing with

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an Ultra Turrax T18 homogenizer (10 mm head) operating at 12000 rpm for 30 s.

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Characterization of Emulsions. The microstructure of the emulsions was characterized on an

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inverted CLSM (Nikon, Japan) using a 543 nm laser to excite the samples. The emulsion was directly

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placed on the slides, and a 60 × objective was used for observation. The average droplet size was

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determined by manually measuring over 100 droplets from the CLSM images using image analysis

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software (Nikon EZ-C1FreeViewer).

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Storage Stability of β-Carotene in the HIPEs. Typically, β-carotene-encapsulated HIPEs stabilized

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by 0.5, 1.0, or 1.5 wt% gelatin particles with sunflower oil as the internal phase were flushed with a

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nitrogen stream for 15 min to exclude the oxygen. Then, the glass vials were sealed and placed in a 37

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°C water bath for 27 days. The retention of β-carotene in HIPEs over time was determined following a

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modification of the method used by K. Ax and others.18,

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emulsion was removed and extracted with a certain volume of a mixture of ethanol and hexane (volume

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ratio = 2:3). After shaking well, the hexane phase was collected and appropriately diluted with hexane,

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followed by an absorbance measurement at 450 nm via a UV-visible spectrometer (Alpha-1, Shanghai

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Lab-spectrum Instruments Co., Ltd.). The β-carotene concentration was quantified by referencing a

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calibration curve of β-carotene under the same conditions. The retention of β-carotene in the HIPEs was

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expressed as a percentage of the relative β-carotene concentration: C(t)/C0, where C(t) is the β-carotene

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content in the HIPEs at time t, and C0 is the β-carotene content in the original HIPEs. A total of 12 mL

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of bulk sunflower oil with dispersed β-carotene (0.83 mg/mL) was treated in the same manner as the

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control.

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At certain time intervals, 0.5 g of the

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In Vitro Digestion. To determine the FFAs released from the emulsions, a dynamic in vitro digestion

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procedure with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was conducted with

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minor modifications to previously described methods.15, 16, 20 For the digestion of the Pickering emulsion,

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the thick particle interfacial layer around the droplets may hinder the diffusion of lipase to lipid subjects,

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leading to low lipolysis efficiency at a certain time. To reduce the impact of this adverse aspect, MCT

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oil was chosen as the carrier oil based on reports that lipids with medium chain triglycerides have

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increased digestion rates compared to long chain triglycerides.21 In this experiment, 1 L of SGF was

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freshly prepared by adding 2 g of NaCl and 3.2 g of pepsin, and then the pH was adjusted to 1.2 using

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1.0 M HCl. To initiate the gastric digestion, 0.5 g of β-carotene-encapsulated HIPEs with MCT oil as

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the internal phase was first mixed with 15 mL of phosphate buffer and incubated at 37 °C under

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magnetic stirring for 10 min. Then, 12 mL of SGF was added, and the pH of the mixture was adjusted to

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2.0 by adding 1.0 M HCl. The mixture was maintained at 37 °C with continuous shaking at 95 rpm/min

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for 2 h to mimic the conditions in the human stomach. After the digestion in stimulated SGF, the pH of

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the mixture was adjusted to 7.5 with 0.5 M NaOH to mimic digestion in the small intestine. Next, 4 g of

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bile salt solution (375 mg bile salts dissolved in 10 mM phosphate buffer, pH 7.0), 2.5 g of lipase

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suspension (60 mg lipase powder dispersed in 10 mM phosphate buffer, pH 7.0), and 7 g of 10 mM

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phosphate buffer were added. During the two hours of lipolysis, the pH was maintained at 7.5 by

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manually adding 0.25 M NaOH. The amount of NaOH consumed over time was recorded throughout

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the intestinal digestion. It was assumed that one molecule of MCT liberated two fatty acid molecules by

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consuming two molecules of NaOH.15 Therefore, the percentage of FFAs released from the system was

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determined by the following equation:

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FFAs released(%) =

VNaOH (t )CNaOHMw, lipid × 100 2mlipid

(1)

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where mlipid represents the total mass of oil present in the sample during digestion (g), Mw,lipid is the

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average molecular weight of the lipids (g/mol), CNaOH is the concentration of the NaOH (mol/L), and

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VNaOH (t) is the volume of NaOH used in the intestinal digestion at time t to neutralize the FFAs that

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could be produced from triacylglycerols.

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Bioaccessibility Determination. After SIF digestion, the aqueous fraction containing formulated β-

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carotene micelles was separated by centrifuging 5 mL of samples at 4 °C and 40000 rpm for 10 min

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(Thermo ST16R, USA). Then, 1 mL of the aqueous phase was collected for extracting the β-carotene

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with the mixture of ethanol and n-hexane (volume ratio = 2:3). The concentration of β-carotene was

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determined by the same method mentioned above, and the bioaccessibility (%) of β-carotene was

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calculated using following equation:

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bioaccessibility (%) =

amount of solubilized β -carotene in micelles amount of β -carotene in the formulations

(2)

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RESULTS AND DISCUSSION

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Characterization of Gelatin Particles. Visual and AFM Observation. The preparation of gelatin

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particles is a typical desolvation and crosslinking process. Desolvation leads to the shrinkage of

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hydrated gelatin chains, and the subsequent chemical cross-linking helps to precipitate fine particles.

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The gelatin particle solutions at concentrations of 5, 10, and 15 mg/mL are displayed in Figure 1A. The

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obtained gelatin particle solutions show no obvious precipitation, even after three months, indicating

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that these solutions are stable and well dispersed during the storage period. Note that glutaraldehyde is

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used as the cross-linking agent to form gelatin particles. It is well known that the reaction between

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glutaraldehyde and protein proceeds easily at room temperature and shows an obvious color change

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characteristic of the Schiff’s base linkage. The color of the gelatin particle solution is therefore ascribed

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to the establishment of aldimine linkages (CH = N) between the free amino groups of gelatin and the

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aldehyde groups of the glutaraldehyde.22 Moreover, the color of the solutions shows a clear tendency to

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be darker with increasing particle concentration.

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The influence of cross-linking time on size, microstructure, and morphology of the gelatin particles

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was further investigated by AFM (Figure 1B and 1C). Note that the aggregation of gelatin particles on ACS Paragon Plus Environment

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the macroscopic substrate is due to lateral capillary forces acting during the drying stage of sample

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preparation.23 Gelatin particles cross-linked for 3 h and 14 h are both fairly monodispersed and exhibit

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typical spherical morphology, which agrees well with our previous observations.8 Additionally, the

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images indicate that the size of the gelatin particles increased with increasing cross-linking time. This

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result is also confirmed by dynamic light scattering (DLS) data of the gelatin particles cross-linked for 3

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h and 14 h (Figure 1D) insofar as the average size of the gelatin particles is 235.9 ± 2.2 nm (PDI = 0.119)

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and 279.2 ± 0.5 nm (PDI = 0.128), respectively. Since gelatin particle formation is a complex process

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involving both inter- and intra-molecular electrostatic interactions,24 increasing the cross-linking time

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provides segments more time to overlap with each other or with the pre-formed aggregates in the

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desolvation process, which leads to the formation of larger particles. Moreover, the formation of gelatin

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particles is always accompanied by the volatilization of the desolvation agent (acetone). This

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volatilization may weaken the desolvation effect, thus leading to the rehydration of gelatin molecules

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and subsequent large particles formation. However, gelatin particles prepared in the two different cross-

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linking time periods exhibit little difference in the subsequent emulsion fabrication (data not displayed).

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To increase the efficiency and to save time, gelatin particles cross-linked for 3 h would be used in the

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following experiments.

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Fluorescence of Gelatin Particles. We have found herein that gelatin particles cross-linked with

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glutaraldehyde are auto-fluorescent in CLSM observation (Figure 2). Gelatin particles exhibit green and

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red fluorescence when excited by the commonly used 488 nm and 543 nm lasers, respectively. The

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green fluorescence is obviously stronger than the red fluorescence. In contrast, no fluorescence signal is

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detected for gelatin or glutaraldehyde solution (data not displayed). Therefore, the auto-fluorescence is

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presumably due to the establishment of linkages as a result of cross-linking. It is proved that the

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formation of CH = N bonds from Schiff’s base in conjugation with the C = C double bonds from the

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glutaraldehyde are responsible for the auto-fluorescence of the particles.25 Owing to the auto-

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fluorescence, it is easy to directly detect the gelatin particles at the O/W interfaces or in the continuous 10 ACS Paragon Plus Environment

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phase when utilizing them as Pickering stabilizers. In addition, this property gives these particles

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superiority in the food industry and is useful for researchers to study the system and design novel food

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emulsions, given its convenience and safety in detecting gelatin particles throughout the process without

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conjugation to any fluorescent agent.

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In Vitro Cytotoxicity of Gelatin Particles. To assess the safety of gelatin particles as starting food

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materials, MTT assays with HEK293 and LO2 cells were used to evaluate the cytotoxicity of gelatin

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particles. Figure 3A shows that exposure to gelatin particles for 72 h results in a very slight

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concentration-dependent decrease in cell viability. However, cell viability for all the applied gelatin

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particle solution is more than 95%, indicating that gelatin particles exhibit no toxicity at concentrations

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of 78.125 ~ 312.5 µg/mL. Meanwhile, there is no significant difference in cell viability between LO2

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and HEK293 cells exposed to gelatin particles. As a supplement, the general external morphology of

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cells exposed to 312.5 µg/mL gelatin particles and the control cells cultured in particle-free media were

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investigated by optical microscopy. The treated cells show no indication of morphology change when

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compared with the control cells (Figure 3B, C, D, and E), which further demonstrates that gelatin

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particles are biocompatible and non-toxic. Hence, they can be safely used as potential Pickering

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stabilizers in fabricating food emulsions.

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Gelatin Particle-Stabilized HIPEs. Visual Observation. Food colloids are multicomponent systems

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that contain dispersed biopolymers, particles, droplets, bubbles, and crystals. Lowering interfacial

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tension during the preparation of emulsions always refers to the competition to adsorb at the oil-water

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interface for all the ingredients.14 Consequently, the stabilizers that actually lower the interfacial tension

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during emulsification are not always obvious; therefore, the need to isolate the particles from multi-

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component ingredients or to specifically fabricate well-defined particles to be sole effective Pickering

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stabilizers is strongly required. To examine the emulsification ability of our formulated gelatin particles

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in food emulsion preparation, sunflower oil-in-water HIPEs solely stabilized by gelatin particles were 11 ACS Paragon Plus Environment

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prepared as a function of particle concentration. Figure 4 shows the photographs of the emulsions after

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three months of storage. For c = 0.1 wt%, a macroscopic oil phase is observed in the emulsion after the

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storage period, indicating that the amount of gelatin particles is not sufficient to form stable HIPEs. It is

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noted that stable O/W HIPEs (80 vol% internal phase) are formed when the gelatin particle

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concentration exceeds 0.3 wt%. The corresponding emulsions are strong gel-type materials, and they

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can hold their own weights even though the vessels are inverted when the gelatin particle concentration

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is more than 0.5 wt%. Furthermore, all of the HIPEs are completely stable against coalescence, as no

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release of oil is visible even after three months, highlighting the ability of the gelatin particles to

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stabilize the emulsions.

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Microstructure of HIPEs. The microstructure of the gelatin particle-stabilized HIPEs formed at

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different concentrations of gelatin particles was evaluated using CLSM. As mentioned above, gelatin

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particles display red fluorescence when excited by a 543 nm laser. Since the fluorescence intensity of

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HIPEs stabilized with the gelatin particles at low concentration (0.3 wt%) is too weak to detect, we

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present the CLSM images of HIPEs stabilized by gelatin particles at concentration of 0.5 ~ 2.0 wt% in

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Figure 5. These results reveal that most of gelatin particles seem to be irreversibly trapped at the oil-

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water interface and provide a physical barrier to emulsion droplet flocculation and coalescence.

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Increasing the particle concentration provides more stabilizers for emulsification, which leads to an

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increased number of smaller droplets and thereby increases droplet contact at a fixed volume fraction of

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the internal phase. In particular, there are also some large aggregates and agglomerates either at the oil-

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water interface or in the aqueous phase, and the phenomenon is more pronounced with the increase of

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particle concentration. Presumably, edible oil such as sunflower oil generally has the characteristic of

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high viscosity, which may affect the dispersion of gelatin particles, leading to the formation of large

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aggregates via hydrogen bonds among the particles. It seems that for the stabilization of O/W Pickering

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emulsions, the conditions in the aqueous phase that promote the particles to be in a state of weak

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aggregation could create the most stable emulsions. A possible explanation is that small aggregates of ACS Paragon Plus Environment

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particles can be transported to the interface of droplets more easily than single particles during high

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shear mixing due to their larger relative inertia for adsorption.26,

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enhanced stability of HIPEs when the number of gelatin particles increases. For the HIPEs stabilized

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with gelatin particle concentrations above 0.5 wt%, the structures that prevent droplet coalescence

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include not only the packed particle layer around the droplets but also the rigid disordered layers or

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networks formed in the continuous phase. The latter structures are probably contiguous with the

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adsorbed layer around the droplets. Therefore, these structures hold whole oil droplets together into a

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3D network, which in turn forms a physical barrier preventing the coalescence of droplets and

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enhancing the stability of HIPEs.1, 26

27

This is one explanation for the

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Emulsion Size. The CLSM images show a clear tendency that an increase in particle concentration

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results in a decrease in the droplet size. We further evaluated the average droplet size distribution of the

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gelatin particle-stabilized HIPEs at particle concentrations of 0.5 ~ 2.0 wt% using the incorporated

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image software in CLSM. The gelatin particle content sets the amount of available stabilized interface at

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a fixed volume fraction of the oil phase. It means that more particles have more opportunity to migrate

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onto the interface to stabilize the emulsions, which then leads to the production of small oil droplets.28, 29

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As expected, Figure 6 shows that the approximately calculated droplet size decreases with increasing

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particle concentrations. Moreover, the presence of oil droplets with sizes less than 10 µm is apparently

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obvious when the particle concentration exceeds 1.5 wt%. Furthermore, it is interesting to see that the

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droplet size distribution is quite narrow, especially when a high particle concentration is incorporated in

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the emulsion stabilization. This is significantly important in practice because the droplet size as well as

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the loading capacity of active compounds in each droplet can be tuned with the particle concentration.

288 289

Protective Effect of Gelatin Particle-stabilized HIPEs on β-Carotene. β-carotene is a highly

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polyunsaturated nutraceutical that is very susceptible to thermal and oxidative degradation during

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emulsification, processing, and storage.30 The retention of β-carotene during 27 days of storage was ACS Paragon Plus Environment

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periodically measured to investigate its stability when encapsulated in HIPEs. As shown in Figure 7, the

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retention of β-carotene in the control (bulk oil) decreases steadily with time, and only 8% β-carotene is

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left at the end of the storage period. In contrast, β-carotene encapsulated in gelatin particle-stabilized

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HIPEs exhibits improved stability and high retention. More surprisingly, the final retention is nearly

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90%, meaning that there is little loss of β-carotene throughout the process. The high retention of β-

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carotene in HIPEs benefits from the dense and thick particle layer around the oil droplets, which can

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retard the diffusion of pro-oxidants or free radicals. In other words, the thick coverage of the interface

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can provide sufficient screens to protect β-carotene from degradation and oxidation. Interestingly, there

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is almost no difference in the retention of β-carotene among the HIPEs stabilized with different

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concentrations of gelatin particles. This implies that in addition to the thickly packed particle layers, the

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concentrated dispersed phase also plays a role in the protection of β-carotene, as observed, which may

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dominate the decisive role in the gelatin particle-stabilized HIPE encapsulation. Compared to other

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emulsions stabilized by Tween,19 starch,31 soybean soluble polysaccharides and chitosan,32 the retention

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of β-carotene in the gelatin particle stabilized HIPEs is greater after 27 days of storage under roughly

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equal storage conditions. Therefore, it can be expected that gelatin particle-stabilized HIPEs are highly

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effective vehicles for encapsulating active compounds.

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In Vitro Digestion Profile of β-carotene-encapsulated HIPEs. The in vitro digestion of β-carotene-

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encapsulated HIPEs was examined to study their potential as delivery systems for lipid-soluble

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nutraceuticals. Herein, the effect of particle concentration on the lipid digestion as well as

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bioaccessibility of β-carotene were assayed in two steps, including treatment with SGF and SIF. As

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presented in Figure 8A, the release profiles of FFAs generally follow a similar trend, which includes an

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initial rapid increase of FFAs followed by a more gradual increase with digestion time. Before reaching

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the plateau value, HIPEs stabilized with 0.5 wt% gelatin particles exhibit a sharp release of FFAs during

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the first 7 min, while the other two HIPEs show slightly slower release kinetics during the first 15 min.

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Previous studies suggest that the rate of lipid digestion has a tendency to increase with decreasing

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droplet size because of the enhanced surface area of the lipid phase exposed to the aqueous phase.33

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According to the results in the CLSM observation, the droplet size of the HIPEs decreases in the order

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of 0.5 wt% > 1.0 wt% > 1.5 wt%. However, the final percentage of FFAs produced during the digestion

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is in the following order: 0.5 wt% (43.76%) > 1.0 wt% (37.01%) > 1.5 wt% (35.79%), which is not in

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accordance with the reported hypothesis. The reason that we do not see a similar tendency likely reflects

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the fact that most previous studies focus on nanoemulsions or Pickering emulsions with a volume

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fraction of the internal phase below 74%. These emulsions are of low viscosity and are easily dispersed

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in SGF and SIF. In contrast, HIPEs are normally highly concentrated emulsions; thus, increasing the

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concentration of gelatin particles always leads to the formation of thick interfacial layers and large

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aggregates, which will consequently reduce the ability of the lipase to access the lipids and retard the

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release of FFAs.34 In addition, gelatin particles are negatively charged in neutral dispersions.8 Increasing

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the concentration of gelatin particles, therefore, makes the HIPEs more negatively charged, which may

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decrease the attachment of negatively charged pancreatic lipase due to electrostatic repulsion.16 Hence,

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the extent of lipid digestion of gelatin particle-stabilized HIPEs is slightly lower than that for

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conventional emulsions or Pickering emulsions with a volume fraction of the internal phase below

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74%.12, 15 The microscopy images in Figure 8A (inset) show the changes in the microstructure of typical

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HIPE stabilized by 1.0 wt% gelatin particles before and after digestion.

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Generally, lipid digestion is an interfacial phenomenon, and larger surface areas always benefit the

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interaction with lipases, thus producing faster reaction velocities. Many previous studies have found that

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the maximum FFAs release of emulsion-based vehicles is appreciably higher than that of bulk oil.12, 15, 35

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In the present study, the sharp release of FFAs of bulk oil is delayed to the first 30 min. Nevertheless, it

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exhibits a higher constant release than 1.0 and 1.5 wt% gelatin particle-stabilized HIPEs in the

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subsequent digestion process and shows 38.08% released FFAs at the end of the digestion. For

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Pickering HIPEs, the lipid droplets are trapped within a gel-like matrix. The lipid digestion thus

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includes the necessary disruption of the matrix and the alternative penetration or diffusion of lipase ACS Paragon Plus Environment

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through the particle interfacial layer so as to react with the lipid substrate.35 Consequently, the

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advantage of a large surface area is suppressed by the influence of thick interfacial layers, as well as the

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high viscosity property of HIPEs when the gelatin particle concentration is above 1.0 wt%. Accordingly,

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the extent of lipid digestion is inhibited, and the maximum FFAs release is slightly decreased. Based on

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these results, the lipolysis efficiency for MCT oil is desirable when entrapped in HIPE stabilized by 0.5

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wt% gelatin particles.

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At the end of the digestion process in the simulated small intestine, we investigated the influence of

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the gelatin particle concentration on the bioaccessibility of β-carotene. As reported, the process by

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which β-carotene to be bioaccessible is related to a micellization process during the digestion.

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Concretely, the surface-active substances come from food itself (e.g., proteins, surfactants,

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phospholipids), or hydrolysis products generated as a result of the digestion process (e.g.,

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diacylglycerols, monoglycerols, FFAs), or gastrointestinal tract (e.g., bile salts, phospholipids, or

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proteins) can be adsorbed to the oil droplet surfaces and displace any existing emulsifiers to form

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micelles, through which the originally dissolved β-carotene in the lipids will be solubilized in the

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micelle cores.35 Figure 8B shows that the bioaccessibility of β-carotene in the HIPEs is in the order of

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0.5 wt% (11.36%) ≈ 1.0 wt% (11.59%) < 1.5 wt% (13.53%), while the value for bulk MCT oil is only

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2.74%. Interestingly, our results are not in agreement with the principle reported by others who suggest

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that the extent of lipolysis is positively correlated with the bioaccessibility. These previous studies

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presume that increasing the extent of lipolysis can improve the content of released FFAs, which are one

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of surface-active substances in the micellization process that could improve the bioaccessibility of the

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encapsulated substance.12, 15, 36 However, the residual gelatin particles can also be emulsifiers in the

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micellization process. The residual particles may compete with the surface-active substances already

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present at the oil-water interface and potentially play a crucial role in solubilizing β-carotene by

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carrying it to the epithelium cells for absorption.37 Thus, increasing the gelatin particle concentration in

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HIPEs exhibits a pronounced effect on promoting the bioaccessibility of β-carotene. Nevertheless,

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further research is clearly required in order to interpret this phenomenon. ACS Paragon Plus Environment

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In summary, stable O/W Pickering HIPEs are fabricated using auto-fluorescent gelatin particles as

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Pickering stabilizers. During processing, particle concentration has a profound effect on the formation

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and droplet size distribution of the resulting HIPEs. As bioactive compound carriers, gelatin particle-

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stabilized HIPEs exhibit an outstanding protective effect upon the chemical stability of the encapsulated

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β-carotene, and there is little loss throughout the storage period. The digestion of HIPEs in vitro

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demonstrates that the amount of gelatin particles can alter the physical properties of the emulsion

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droplets as well as the dispersed phase, leading to different digestion profiles of the carrier oil and of the

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bioaccessibility of β-carotene. Typically, increasing the concentration of gelatin particles could help

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form a thick and dense layer over the oil droplets, which therefore limits the access of lipase to lipids in

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the emulsion droplet core and decreases the release of FFAs accordingly. Nonetheless, the

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bioaccessibility of β-carotene is found to be improved 5-fold after encapsulation in HIPEs. We assumed

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that in addition to the FFAs, gelatin particles could be another emulsifier in the micellization process

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that exhibit a pronounced effect on promoting the bioaccessibility of β-carotene. Further work is still

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needed to clarify the correlation between the FFAs release profile and the bioaccessibility of the

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encapsulated bioactive compounds in gelatin particle-stabilized HIPEs. Overall, the current work will

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broaden the knowledge of Pickering HIPEs in the development of new strategies to improve the oral

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bioaccessibility of bioactive nutraceuticals in food science and dietary supplements of various forms,

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such as mayonnaise, neutral creams, and cheese-type products.

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ACKNOWLEDGMENTS

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We thank Liangyin Chu from the Department of Pharmaceutical and Bioengineering, Sichuan

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University, for insightful suggestions during CLSM experimentation. Additionally, we highly appreciate

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the grammar-checking, polishing, and proofreading work done by Zonglin YI of Prof Ngai To’s group,

393

the Chinese University of Hong Kong.

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protein–beet pectin conjugate on the properties and digestibility of β-carotene emulsion during in vitro

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drug encapsulation efficiency of gelatin nano-particles. Colloids Surf., B 2005, 45, 42-48.

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Application of Novel Microspheres Possessing Autofluorescent Properties. Adv. Funct. Mater. 2007, 17,

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microgels. Curr. Opin. Food Sci. 2015, 3, 94-109.

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Interfaces, Binks, B. P., Horozov, T.S., Ed. Cambridge University Press: UK, 2006.

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Aspects of Emulsification. Langmuir 2004, 20, 1130-1137.

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(30) Hou, Z.; Gao, Y.; Yuan, F.; Liu, Y.; Li, C.; Xu, D., Investigation into the Physicochemical Stability

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and Rheological Properties of β-Carotene Emulsion Stabilized by Soybean Soluble Polysaccharides and

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Chitosan. J. Agric. Food Chem. 2010, 58, 8604-8611.

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carotene in nanoemulsions stabilized by modified starches. J. Agric. Food Chem. 2013, 61, 1249-57.

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(33) Ahmed, K.; Li, Y.; McClements, D. J.; Xiao, H., Nanoemulsion- and emulsion-based delivery

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systems for curcumin: Encapsulation and release properties. Food Chem. 2012, 132, 799-807.

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(34) Siew, C. K.; Williams, P. A.; Cui, S. W.; Wang, Q., Characterization of the Surface-Active

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(35) McClements, D. J.; Li, Y., Review of in vitro digestion models for rapid screening of emulsion-

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based systems. Food Funct. 2010, 1, 32-59.

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bioaccessibility and loading of curcuminoids. Food Chem. 2012, 131, 48-54.

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Interfaces: Unique Interfacial Properties as Globular Proteins. Langmuir 2008, 24, 6812-6819.

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Notes

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The authors declare no competing financial interest.

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Funding

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This work was financially supported by Key Laboratory of Medicinal and Edible Plants Resources

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Development of Sichuan Education Department (10Y201612) and Antibiotics Research and Re-

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evaluation Key Laboratory of Sichuan Province (ARRLKF16-01).

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FIGURE CAPTIONS:

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Figure 1. (A) Visual observation of gelatin particle solution at concentration of 5, 10, and 15 mg/mL

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(from left to right). The photographs were taken after the samples were stored at rest under 4 °C for

494

three months. (B) 5 µm × 5 µm AFM topography images of gelatin particles cross-linked for 3 h. (C) 5

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µm × 5 µm AFM topography images of gelatin particles cross-linked for 14 h. (D) Size distribution of

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gelatin particles cross-linked for 3 h (red line) and 14 h (green line) as measured by dynamic light

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scattering.

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Figure 2. The CLSM images of gelatin particles excited by commonly used 488 nm (left) and 543 nm

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(right) laser (scale bar = 75 µm).

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Figure 3. (A) Cytotoxicity evaluations test of HEK293 and LO2 cells with different concentrations of

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gelatin particles. Optical microphotographs of HEK293 cells with gelatin particle treatment (B) and

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cells without any particle treatment (C). Optical microphotographs of LO2 cells with gelatin particle

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treatment (D) and cells without any particle treatment (E). All the samples were incubated at 37 °C for

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72 h, and the pH of cell culture medium is around 7.2 ~ 7.4. The scale bar is 25 µm.

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Figure 4. Photographs of HIPEs stabilized with different solid concentrations (0.1-2.0 wt%) of gelatin

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particles with sunflower oil as internal phase. The photographs were taken three months after the

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preparation.

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Figure 5. The CLSM images of HIPEs stabilized by (A) 0.5 wt%, (B) 1.0 wt%, (C) 1.5 wt%, and (D)

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2.0 wt% of the synthesized gelatin particles.

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Figure 6. Droplet size distributions of HIPEs stabilized by different concentration of gelatin particles.

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Figure 7. Retention of β-carotene in gelatin particle-stabilized HIPEs over 27 days of storage period.

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Figure 8. (A) FFAs release profiles from β-carotene−encapsulated HIPEs stabilized by different

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concentration of gelatin particles as a function of small intestine digestion time. (Insets) Typical

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microstructure observation of HIPE stabilized by 1.0 wt% gelatin particles before and after the digestion

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under 10 × magnification. (B) Bioaccessibility of β-carotene after in vitro digestion of MCT oil and

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HIPEs stabilized by different concentration of gelatin particles.

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Table of Contents (TOC) Graphic

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