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.

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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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

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

306

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

307

effective vehicles for encapsulating active compounds.

308 309

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

310

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

311

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

312

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

313

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

314

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

315

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

316

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


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317

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

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

327

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

328

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

330

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

332

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

333

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

334

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

336

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

337

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

338

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

339

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

340

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

342

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

345

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

346

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

347

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

348

wt% gelatin particles.

349

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

350

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

351

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.,

354

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

355

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

356

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

357

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

358

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

359

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

360

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

361

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

362

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

363

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

365

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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369 370

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

372

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

374

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

375

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

376

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

377

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

378

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

379

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

380

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

381

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

382

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

383

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

384

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

385

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.

388 389

ACKNOWLEDGMENTS

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

391

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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Sci. 2010, 15, 40-49.

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Emulsions. J. Agric. Food Chem. 2013, 61, 8888-8898.

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Vehicles: Physicochemical Stability and in Vitro Digestion Profile. J. Agric. Food Chem. 2015, 63,

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(13) Liu, F.; Tang, C.-H., Phytosterol Colloidal Particles as Pickering Stabilizers for Emulsions. J.

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Agric. Food Chem. 2014, 62, 5133-5141.

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(14) Dickinson, E., Use of nanoparticles and microparticles in the formation and stabilization of food

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emulsions. Trends Food Sci. Technol. 2012, 24, 4-12.

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(15) Liang, R.; Shoemaker, C. F.; Yang, X.; Zhong, F.; Huang, Q., Stability and Bioaccessibility of β-

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Carotene in Nanoemulsions Stabilized by Modified Starches. J. Agric. Food Chem. 2013, 61, 1249-

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1257.

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(16) Xu, D.; Yuan, F.; Gao, Y.; Panya, A.; McClements, D. J.; Decker, E. A., Influence of whey

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

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digestion. Food Chem. 2014, 156, 374-379.

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(17) C. J. Coester, K. L. H. V. B. J. K., Gelatin nanoparticles by two step desolvation a new preparation

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method, surface modifications and cell uptake. J. Microencapsulation 2000, 17, 187-193.

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(18) Ax, K.; Mayer-Miebach, E.; Link, B.; Schuchmann, H.; Schubert, H., Stability of Lycopene in Oil-

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(19) Yuan, Y.; Gao, Y.; Zhao, J.; Mao, L., Characterization and stability evaluation of β-carotene

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nanoemulsions prepared by high pressure homogenization under various emulsifying conditions. Food

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changes occurring in emulsified lipids during in vitro digestion. Food Chem. 2009, 114, 253-262.

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carrier oil on β-carotene bioaccessibility. Food Chem. 2012, 135, 1440-1447. ACS Paragon Plus Environment

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(22) Kesavan S, A. B. S., Effect of surfactant on the release of ciprofloxacin from gelatin microspheres.

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Ars Pharm. 2010, 51, 1-16.

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Colloids Surf., A 2003, 217, 143-151.

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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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(28) Frelichowska, J.; Bolzinger, M.-A.; Chevalier, Y., Pickering emulsions with bare silica. Colloids

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Surf., A 2009, 343, 70-74.

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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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(31) Liang, R.; Shoemaker, C. F.; Yang, X.; Zhong, F.; Huang, Q., Stability and bioaccessibility of beta-

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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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Agric. Food Chem. 2008, 56, 8111-8120.

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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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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.

482 483

Notes

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The authors declare no competing financial interest.

485

Funding

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

487

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

488

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

489 490


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FIGURE CAPTIONS:

492

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

493

(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

495

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

496

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

497

scattering.

498

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

499

(right) laser (scale bar = 75 µm).

500

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

501

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

502

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

503

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

504

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

505

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

506

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

507

preparation.

508

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

509

2.0 wt% of the synthesized gelatin particles.

510

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

511

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

512

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

513

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

514

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


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515

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

516

HIPEs stabilized by different concentration of gelatin particles.


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Figure 1.


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Figure 2.


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Figure 3.


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Page 27 of 32


Figure 4.


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Figure 5．．


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Figure 6.


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Figure 7.


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Figure 8.


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Table of Contents (TOC) Graphic


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

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