Development of Pickering Emulsions Stabilized by Gliadin

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Development of Pickering Emulsions Stabilized by Gliadin/Proanthocyanidins Hybrid Particles (GPHPs) and the fate of Lipid Oxidation and Digestion Fu-Zhen Zhou, Li Yan, Shou-Wei Yin, Chuan-He Tang, and Xiao-Quan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05261 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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Journal of Agricultural and Food Chemistry

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Development of Pickering Emulsions Stabilized by Gliadin/Proanthocyanidins

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Hybrid Particles (GPHPs) and the fate of Lipid Oxidation and Digestion

4 Fu-Zhen Zhou †, Li Yan†, Shou-Wei Yin†‡*, Chuan-He Tang†‡, Xiao-Quan Yang †‡

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†

Research and Development Center of Food Proteins, School of Food Science and Engineering; ‡Guangdong Province

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Key Laboratory for Green Processing of Natural Products and Product Safety; South China University of Technology,

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Guangzhou 510640, PR China

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Running title: Pickering emulsions stabilized by GPHPs

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* Corresponding author:

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Phone: +86-2087114262.

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E-mail: [email protected] (Dr. Yin, S. W.)

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1 ACS Paragon Plus Environment


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ABSTRACT

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This work attempted to engineer emulsions’ interface using special affinity between proline-rich

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gliadin and proanthocyanidins (PA), to develop surfactant-free antioxidant Pickering emulsions

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with digestive-resistant properties. This binding interaction between gliadin and PA benefited the

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interfacial adsorption of the particles to corn oil droplets. Pickering droplets as building units

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assembled into interconnected three-dimensional network structure, giving the emulsions

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viscoelasticity and ultra-stability. Oxidative markers in Pickering emulsions were periodically

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monitored under thermally accelerated storage. Lipid digestion and oxidation fates were

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characterized using in vitro gastrointestinal (GI) models. Interfacial membrane constructed by

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antioxidant particles served as a valid barrier against lipid oxidation and digestion, in a PA

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dose-dependent manner. Briefly, lipid oxidation under storage and simulated GI tract was retarded.

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Free fatty acid (FFA) fraction released decreased by 55% from 87.9% (bulk oil) to 39.5%

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(Pickering emulsion), implying engineering interfacial architecture potentially benefited to fight

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obesity. This study opens a facile strategy to tune lipid oxidation and digestion profiles through the

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cooperation of Pickering principle and interfacial delivery of antioxidants.

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Key words: Pickering emulsions, gliadin/proanthocyanidins interaction, lipid digestion, lipid

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oxidation.

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ACS Paragon Plus Environment

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INTRODUCTION

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Emulsion is an important ingredient in foods, pharmaceuticals, and cosmetics.1 Lipid oxidation

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of emulsion is a major issue resulting in quality deterioration even carcinogen formation.2, 3 The

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reactive aldehydes formed are cytotoxic and genotoxic at very low concentrations.4 The oxidation

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of emulsified oil mainly occurs at the oil-water interface of emulsion system. Amphiphilic primary

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oxidant products (lipid hydroperoxides, LH) tend to adsorb at interface.5,6 Pro-oxidants, such as

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transition metals usually initiate lipid peroxidation by reducing LH into highly reactive radicals,

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promoting the chain reaction of lipid oxidation in emulsions.7 Nowadays, obesity cause a global

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crisis which brings health and social issues to humans. Lipid, one of high energy dense

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macronutrients, is often related to obesity.8 Delaying lipid lipolysis is a potential strategy to fight

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obesity. Previous studies confirmed that limited lipid digestion might activate the so-called ileal

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brake feedback mechanism (overexpression of hormones) lowering food or calorie intake while

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satiety don't lower.9,10 Both lipid oxidation and hydrolysis are prevalent at the interfacial region of

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emulsions. Hence, we aimed to design interfacial layer makeup by gliadin colloid particles using

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Pickering principle, to prevent lipid oxidation and lipolysis under storage and simulated GI tract.

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Droplet interfacial characteristics (composition, thickness, and charge) play an important role in

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lipid oxidation and digestion fate of emulsions. Engineering interfacial structures by Pickering

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particle is a potent strategy to manipulate lipid oxidation and digestion profiles of emulsified oils.

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Pickering emulsion is a kind of solid particle stabilized emulsions where interfacial absorption of

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particles is nearly irreversible. The high resistance to coalescence is a major benefit of Pickering

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emulsion.11,12 Pickering emulsions stabilized by sustainable solid particles especially food-grade

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ones have attracted increasing interests for their unique characteristic and potential applications,

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such as reducing caloric intake, delivering functional substances.13 Typical food-grade particles for 3



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Pickering emulsion development included cellulose or chitin nanocrystals,14,15 phytosterol,16

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modified starch,17 zein and gliadin colloid particles.18,

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advantages relative to conventional emulsions, i.e., eminent stability (thicker interfaces), controlled

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delivery (responsive and tunable interfaces), and safety (surfactant-free). However, those works

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referred mainly to the physical performances of Pickering emulsions, little was known about the

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oxidant and digestive fate of them. Actually, it is a feasible strategy to tune interfacial

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characteristics via Pickering principle for controlling the performance of emulsified oil including

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oxidative and digestive fate.

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Pickering emulsions possess many

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Gliadins, one of prolamine-type proteins in cereals, are proline-rich proteins with

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amphiphilicity.20 Gliadins are capable of self-assembly to yield various forms of colloidal structures,

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e.g., nano/micro-scale particles. Previous works reported that pristine gliadins colloid particle

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(GCPs) were used as solid emulsifier to develop Pickering emulsions.21, 22 In our laboratory, we

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showed that GCPs are too hydrophilic to adsorb at the surface of corn oil droplets and tend to rest in

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the continuous phase at pH 2.9, and the Pickering emulsions developed were susceptible to

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creaming and/or coalescence. The ion strength increase and pH shift, and the association were

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utilized to manipulate the surface property of GCPs to fabricate stable emulsions. However, those

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emulsions developed still were labile to destabilize in highly acidic conditions, for example

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simulated gastric fluids (SGF). This behavior limited to some degree the application of Pickering

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emulsions as oral delivery. Accordingly, some pertinent solutions must be adopted to tune the

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colloidal and interface properties of GCPs to produce stable Pickering emulsions with robust

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interfacial architecture, for example, the unique interactions between proline-rich gliadins and

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active polyphenols.

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Grape seed proanthocyanidins (PA) belong to flavonoids that exhibit interesting biological 4


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characteristics with potent application in pharmaceuticals.23 Although PA possess superior

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antioxidant activities as revealed or shown by in vitro screening test, the polarity inhibits them from

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partitioning into oil phase and thus reduces to a certain degree the antioxidant effects.24 The special

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(unique) interaction between proline-rich proteins and PA is well known, mainly by hydrogen

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bond.25 PA usually consist of oligomeric and polymeric forms. Polymeric forms have a stronger

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affinity to proline-rich proteins while oligomeric forms receive attentions in view of the biological

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activity, e.g., antioxidant activity.26, 27 Though, gliadin and PA was used as stabilizer and natural

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antioxidants, respectively, no information was available on the development of gliadin/PA colloidal

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particles. Moreover, no study on the combination (or co-assembly) of gliadin and PA for preventing

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lipid oxidation and digestion in Pickering system was reported.

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Herein, we reported firstly the application of the special affinity between proline-rich gliadin

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and PA to construct duel-function protein colloid particles, aiming to control the self-assembly

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nucleation process of gliadin, and to promote interfacial adsorption and regulate interfacial

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architecture, so as to manipulate the lipid oxidation and digestion fate of emulsified oils. Confocal

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laser scanning microscopy (CLSM) and optical microscopy were used to characterize the

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microstructure of Pickering emulsions developed, and rheological characteristics of them were

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investigated by dynamic oscillatory measurements. The thermally accelerated oxidation of lipid in

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the Pickering emulsions was analyzed by detecting the levels of the oxidative markers (headspace

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oxygen, headspace hexanal and MDA) during the incubation at 60 °C. The digestion fate of

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Pickering emulsions was investigated in simulated GI tract at 37°C by checking microstructural

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evolution of the Pickering emulsion and the extent of lipid hydrolysis and oxidation. Besides, we

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also discussed the potential linkage between the interfacial characteristics of Pickering emulsions

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stabilized by gliadin/PA hybrid particles (GPHPs, duel-function particles) and their fates in the 5



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simulated GI tract.

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MATERIALS AND METHODS

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Materials. Gluten was purchased from Fengqiu Hua Feng powder Co., Ltd (Fengqiu, China).

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Food-grade corn oil was bought in a local supermarket (Guangzhou, China) and purified by

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molecular sieve to remove initial antioxidants before use.

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JianFeng Natural product R & D Co., Ltd (Tianjin, China), with the degree of polymerization (DP)

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of 2.72 ± 0.07. 29 Bile bovine (B3883), mucin (from porcine stomach, Type II), porcine lipase

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(L3126, 100-500 U/mg, type II), porcine pepsin (P7000, ≥250 U/mg), porcine pancreatin (P7545,

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8×USP specifications) and metmyoglobin (from equine skeletal muscle) were bought from Sigma–

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Aldrich (Shanghai, China). Other chemical reagents were of analytical purity.

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PA (85% OPCs) was offered by

Gliadin Extraction. The extraction was performed following the method described previously.

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The mixture was stirred at room temperature for 2 h. The obtained mixture was centrifuged (8,000g,

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20 min) at 4 °C. The resultant supernatant (rich in gliadin) was dialyzed in dialysis bags with the

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molar weight cut-off of 12000 Da at 4 °C against de-ionized water (day 1), 0.05 M acetic acid (day

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2), and de-ionized water (day 3) again. Gliadin powder was obtained by freeze-drying the dialysate.

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Preparation of Gliadin/PA Hybrid Particles (GPHPs). GPHPs were synthesized following a

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facile anti-solvent precipitation. Precisely, gliadin (0.25 g) was dissolved in ethanol-water mixture

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solvent (40 mL, 70:30, v/v), and PA were added to 1% acetic acid solution (100 mL) at

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PA-to-gliadin mass ratios of 0, 1, 2, 5, 10% (marked as GCPs, GPHPs-1, GPHPs-2, GPHPs-5,

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GPHPs-10, respectively). Then, aliquots of gliadin solution were added dropwise into 1% acetic

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acid solution under shearing (6000 rpm) for 6 min by a T25 homogenizer (IkA, Germany). Next,

In short, gluten powders (100 g) were admixed with 1 L of aqueous ethanol solution (70%, v/v).

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ethanol in the particle dispersion was removed by rotary evaporation (RV 10, IkA, Germany).

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Gliadin content in GPHPs solution was eventually adjusted to 0.5%.

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Pickering Emulsion Preparation. Pickering emulsions (GPHPEs) were produced at pH 4.0

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using GPHPs as particulate emulsifiers. The volume fraction in Pickering emulsions was fixed at

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50%. That is, 4 mL of corn oil and 4 mL of aqueous GPHPs dispersions was mixed in a glass bottle,

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and then the mixture was sheared (15000 rpm, 4 min) to yield Pickering emulsions (GPHPEs) using

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an T10 homogenizer with a 5-G head (IkA, Germany). PA-free gliadin colloid particle (GCPs) was

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used to produce GCPEs as the control via the above-mentioned protocol.

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Particle Size and Zeta Potential (ζ-potential) Measurements. The size and size distributions

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as well as ζ-potential of GPHPs were characterized at 25 °C by a Malvern Zetasizer Nano ZS

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(Worcestershire, UK). GPHPs dispersions were thinned to 1 mg/mL (pH 4.0) prior to the test. Each

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result reported is the mean and standard deviation (SD) of triplicate tests.

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Particle Morphology. The morphology of GPHPs was characterized by a Multimode 8 atomic

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force microscopy (Bruker, German). The samples were diluted to 30 µg/mL, and 5 µL of diluted

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GPHPs was dripped on mica disks, then dried overnight. For each samples, at least two preparations

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were examined.

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Fourier-Transform Infrared Spectroscopy (FTIR). IR of PA, gliadin and GPHPs-2 were

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measured with a Fourier transform infrared instrument (Bruker Co., Germany) at a resolution of 2

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cm−1. For each type of particles, the spectrum was obtained by overlapping 128-scans, from 400 to

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4000 cm−1.

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Determination of Droplet Size and Size Distributions as well as Zeta Potential of

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GPHPEs. Droplet size and size distributions of GPHPEs freshly prepared and sampled during the

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simulated GI digestion were investigated by Malvern Mastersizer 3000 (Malvern Instruments, UK). 7



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Pump speed was 2500 rpm. The refractive index (RI) of water (the dispersant) and corn oil was

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1.330 and 1.467, respectively. The absorption parameter was 0.001. Droplet size was reported as

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surface mean diameter (D3,2) and volume-mean diameter (D4,3). Zeta potentials of freshly prepared

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Pickering emulsions were assayed by the same protocol described previously.

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Optical Microscopy Measurements. The micrographs of Pickering emulsions before and/or

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after every digestion step were obtained using a MX-4000 light microscope (Meiji Techno., Japan).

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The GPHPEs samples were mildly shaken by a vortex for a few seconds prior to the test to

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guarantee the homogeneity. The resultant specimens (GPHPEs) were dripped in the center of slide

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glasses which were then covered by cover slips.

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CLSM. CLSM technique was used to investigate the microstructure of GPHPEs according to

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the protocol described previously,21 using a LSM 710 confocal microscopy (Carl Zeiss, Germany).

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An aliquot (1 mL) of the emulsions were dyed using 80 µL Nile Red solution (in isopropyl alcohol)

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and/or 80 µL Nile Blue A solution (in isopropyl alcohol). One drop of dyed GPHPEs (about 20 µL)

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was added on a concave slide, following by covered it with a coverslip. The fluorescence was

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excited at 488 nm (for Nile Red) or 633 nm (Nile Blue A), as well as simultaneously excited at 488

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nm and 633 nm to obtain overlapped CLSM images.

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Dynamic Oscillatory Measurements. Small amplitude oscillatory measurements were

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performed to evaluate the viscoelastic properties of GPHPEs by a HAAKE RS600 Rheometer

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(Karlsruhe, Germany). In short, amplitude sweep (0.1 to 100 Pa, frequency as 1 Hz) and frequency

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sweep (0.01 to 10 Hz, stress as 1 Pa) were performed out at 25 °C. Frequency scanning was

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achieved within the linear viscoelastic region of GPHPEs.

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Measurements of Lipid Oxidation Stability in Pickering Emulsions. Oxygen Consumption.

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Gas chromatograph (GC) was used to monitor the oxygen consumption of GPHPEs during the 8


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thermally accelerated oxidation process using the protocol reported by Kim et al.,30 with a slight

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modification. Briefly, headspace vial (22.4 mL) containing 3.0 mL GPHPEs samples were

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hermetically sealed with the silicon/Teflon septum and aluminum crimp cap. Next, the sealed

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headspace vial was incubated at 60 °C for 51 h in the dark. The evolution in headspace oxygen was

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monitored periodically (0, 3, 6, 16, 23, 40 and 51 h) by a series 580 gas chromatography

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(GOW-MAC Instrument Co, US) united with a CTR1 column and a thermal conductivity detector.

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The flow rate of nitrogen (99.99%) as the carrier gas was 40 mL min−1. Oven temperature was 60°C

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for 10 min to reach equilibrium state, injector and detector temperature were 120°C. Corresponding

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O2 peak area in 1.0 mL of headspace air was measured as the control. Amount of O2 in vial was

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then calculated based on headspace volume and theoretical O2 concentration in ambient air, O2

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depletion after a period of time was the remaining O2 subtracted from that in vial headspace at

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initial time. The final data were reported in mmol O2 kg−1oil.

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Secondary Lipid Oxidation Products. Hexanal Measurements. Fresh GPHPEs samples (3.0

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mL) was added in headspace vial (22.4 mL), then incubated in the dark at 60°C for 35 d. An Agilent

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7890B GC (Agilent, USA) united with flame ionization detector (FID) was used to monitor

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periodically the headspace hexanal of the vials, using the procedures reported previously,31 with a

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minor revision. Samples were equilibrated at 60 °C for 10 min at 450 rpm in autosampler heating

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block before collecting. An aliquot (1000 µL) of headspace gas was injected into GC system. GC

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analysis was performed by a TR-5MS capillary column (30m×25 mm×0.25µm). The injector

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temperature, oven temperature and flame ionization detector temperature was 250, 60, and 250°C,

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respectively. Duration of each test was 10 min. The tests for each sample were performed in

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triplicate.

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Malondialdehyde (MDA) Measurements. Measurements of MDA levels in the GPHPEs were 9



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followed the procedure reported by Obando et al.32 Aliquots of trichloroacetic acid (TCA) were

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mixed with the emulsions sample to precipitate gliadin. The centrifuge at 8000 rpm for 10 min was

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performed to yield MDA-rich supernatant. The TBA reagent (20 mM) was used to derive the

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extracted MDA, and then the resultant mixtures were cooled in an ice bath. Finally, an aliquot of

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reaction mixture (10 µL) was injected into a C18 HPLC column maintained at 35 °C. The flow rate

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of mobile phase (water and acetonitrile, 82:18, v/v) was l mL/min. Emission and excitation

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wavelengths of the fluorometric detector were 560 and 525 nm, respectively. MDA standard curve

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were prepared by 1, 1, 3, 3-tetraethoxypropane in the range of 0.1-50 µg/mL.

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The In Vitro Digestion. The fate of the GPHPEs under the simulated GI digestion was

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characterized according to the model reported previously.33,

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(GCPEs, GPHPEs-1, GPHPEs-5) were digested in the SGF and SIF in sequence.

Three typical emulsion samples

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Gastric Phase. SGF was made up using the formula described by Shah et al.35 It consisted of

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pepsin (3.2 mg/mL), NaCl (2 mg/mL) and HCl (0.7% v/v) with the pH of 2.0. An aliquot of

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Pickering emulsion samples (2 g) was put into 20 mL SGF. The pH of the mixture was re-adjusted

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to 2.0 if necessary by 1.0 M HCl. The resultant mixture was shaken for 1 h at 37 °C and 95 rev

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min−1 to complete the digestion under SGF. The pH of the mixture was adjusted to 7.0 to stop the

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digestion.

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Small Intestinal Phase. The pH-stat protocol was performed to monitor the release of free

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fatty acids (FFA) in simulate small intestinal phase.33, 34 The bolus from gastric phase was mixed

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with bile extract (20 mg/mL) and salt solutions (10 mM CaCl2, 120 mM NaCl). The pH of the

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digesta was re-adjusted to 7.0 if necessary by 1M NaOH. Then, aliquots of fresh digestive enzyme

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dispersions (5 mL) including pancreatin (2.4 mg/mL) and lipase (3.6 mg/mL) were added into

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digestive juice. The pH of digestive juice was kept at 7.0 by dripping 0.1 M NaOH, the volume of 10


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NaOH (in mL) consumed to titrate FFA liberated was monitored within the 120 min of the digestion.

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Percent of free FFA released was calculated according to Equation (1):

 VNaOH (t ) ∗ M NaOH ∗ MWTG   FFA(%) = 100 ∗  mTG ∗ 2  

(1)

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Where VNaOH(t), the volume of NaOH consumed (in mL) at t moment; MNaOH, the molarity of

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NaOH consumed to neutralize FFA released; MWTG, the mean molecular weight of corn oil (872

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g/mol); mTG, the mass of corn oil added under digestion process (g). Control trials were carried out

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on Pickering emulsions with dodecane as the internal phase. In addition, particle size and

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microstructural evolution was monitored using the protocol described previously.

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Lipid Oxidation under simulate GI Digestion Process. An in vitro digestion model was

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constructed to evaluate lipid oxidation under simulated GI digestion according the procedure

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described previously.36 In short, 1 mL of GPHPEs or 0.5 mL of corn oil was mixed with 1.89 mL

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SGF which contained 6.7 g/L pepsin and 2.7 g/L mucin in a headspace vial (20.5 L), and then 1 mL

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of 20 µM metmyoglobin (in SGF) was put into the headspace vial. An aliquot of 1 M HCl (32.2 µL)

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was added to adjust the pH of the mixture to 2.5. Subsequently, headspace vials were sealed and

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covered by aluminium foil paper. Finally, they were shaken at 90 rev min−1 and 37°C in the dark.

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After 60 min, 380 µL of NaOH (1 M) was added to raise the pH of the juice to 6.5 to stop gastric

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digestion, and then 3.5 mL of simulated intestinal fluid containing pancreatic lipase (0.56 g/L) and

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pancreatin (1.12 g/L) as well as 1.26 mL of bile extract (18.63 g/L) were added by syringes into the

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digestive juice. Headspace vials were shaken for additional 120 min at the same condition. MDA

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generation was determined after simulated digestion according to the above-mentioned procedure.

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Statistics. For each sample or determination, triplicate tests were carried out. The results were

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showed as the mean and standard deviation (SD). SPSS 13.0 program was used to detect the

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differences between the mean under ANOVA analysis using the Tukey test (P < 0.05). 11



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RESULTS AND DISCUSSIONS

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Colloid Properties of Gliadin/PA Hybrid Particles (GPHPs). Polyphenols are prized for the

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physiological activities as well as binding affinity to protein.37 PA are well known to bind the

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proteins (rich in proline) through secondary interactions.25 Mazzaracchio et al. verified that

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anthocyanidins could be adsorbed by gliadin.38 In this work, we investigated the evolutions in

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ζ-potential of gliadin particles and PA in the pH range of 2−6 at 25 ℃ (Figure 1C). ζ-potential of

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GCPs gradually decreased from 18.80 ± 1.28 mV (pH 2.6) to −4.2 ± 0.04 mV (pH 6.0), while

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ζ-Potential of PA decreased from 2.15 ± 1.20 mV (pH 2.0) to −9.10 ± 0.98 mV (pH 6.0). Thus it

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can be inferred from Figure 1C that there was electrostatic attraction forces between gliadin

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molecule and PA at the pH value from 3.0 to 5.0. FTIR spectra of gliadin, PA and GPHPs in Figure

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1D clearly showed peak of amide A was shifted from 3303 cm−1 (gliadin) to 3419 cm−1 (hybrid

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particles), verifying the formation of hydrogen bonds between proton donors (PA) and proton

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acceptors (carbonyl groups of gliadin).38 Our results confirmed the fact that proline-rich gliadin

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exhibited binding affinities to polyphenols through electrostatic interactions and hydrogen bonds,

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hydrophobic interactions may also exist. Herein, grape seed PA was utilized to affect gliadin

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self-assembly process preparing hybrid particles (GPHPs) and manipulate colloidal properties of the

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resulting GPHPs.

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The colloidal property of GPHPs dispersions (0.5%, w/v) was examined at pH 4.0, as a

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function of PA loadings. Initially, hybrid particles dispersions was transparent (Figure 1A), then

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changed to translucent and flocculated to form precipitates after incubating for 24 h at room

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temperature (Figure 1S). The low zeta-potential (Table 1) was responsible for the observed

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precipitation of particles dispersions (Figure 1S). In addition, PA loading impacted the

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self-assembly of gliadin during the anti-solvent process authenticated by hydrodynamic diameter 12


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(Table 1) increasing from 120.07 ± 2.73 nm (GCPs) to 364.63 ± 8.93 nm (GPHPs-2), then

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decreasing to 86.60 ± 1.15 nm (GPHPs-10), this result was corroborated by AFM images (Figure

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1B). Zeta potentials of GPHPs were almost the same as gliadin particles (about 10 mV)

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demonstrating the incorporation of PA did not affect the surface charge of gliadin colloid particles

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(Table 1 and Figure 1C).

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Preparation and Physical properties of Pickering Emulsions. The appearance of GPHPEs

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after 10 d of storage at ambient temperature as a function of PA loadings is shown in Figure 2. In

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this series, 0.5% (w/v) GPHPs dispersions were used to produce GPHPEs with a fixed oil volume

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fraction of 50%. Freshly prepared emulsions were homogeneous while the creaming occurred upon

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storage due to density gradient, and the creaming indices21 were decreased from 30% (GCPEs) to

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25% (GPHPEs-10) after 10 d of storage (data not shown). Visually, sublayer water was clear,

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except for GPHPEs-10 where it was reddish due to the presence of free PA. Importantly, GPHPEs

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were stable at room temperature for more than 1 year. Figure 3 shows the droplet size distributions

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of Pickering emulsions. Obviously, mono-modal droplet size distribution profiles were observed for

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GPHPEs, and droplet size were range from 10 up to 100µm. Zeta potentials of GPHPEs (~20 mV)

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were larger than that of GPHPs (~10 mV) at pH 4.0 (Table 2), indicating that the

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micro-environment change of the particles from bulk aqueous phase to interface led to an adaptive

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conformation of adsorbed gliadin-based particles.39 In brief, GPHPs were effectively adsorbed and

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settled at the dispersed droplet surface, accounting for the formation of stable Pickering emulsions.

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Microstructure of Pickering emulsions. Optical Photographs. Figure 4 showed optical

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micrographs of typical GPHPEs. The drop characteristics of the emulsions evolved with PA

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loadings. In GCPEs, most droplets were isolated from each other or presented discrete, slightly

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flocculated state. At low PA concentrations (GPHPEs-1 and GPHPEs-2), emulsions were composed 13



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of smaller, moderately flocculated drops. In contrast, extensively flocculated drops were observed

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with increased PA loadings (GPHPEs-5 and GPHPEs-10). Herein, good correlation between PA

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amounts and the Pickering emulsion microstructure characteristics was established. In GPHPEs, the

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droplets aggregated to form flocs, but they remained individual rather than merged or coalesced,

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confirming that the steric hindrance of particle-based interface architecture prevented the

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destabilization phenomena. This behavior benefits the formation of interconnected tridimensional

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network, leading to gelled products. Interestingly, the emulsions produced were stable against

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coalescence over 1 year, although the extensive flocculation occurred.

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CLSM. Emulsion microstructures e.g., interfacial architecture, particle location (or partition),

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and droplet flocculation significantly play a dominant role in the physical stability at ambient

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temperature and GI digestibility of emulsions. They were characterized by CLSM technique to

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obtain more information. Figure 5 shows typical CLSM micrographs of selected samples where oil

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droplets and gliadin colloid particles were dyed with Nile Red (green) and Nile Blue A (red),

299

respectively. The incorporation of PA facilitated the adsorption and deposition of gliadin-based

300

particles at the interfacial region of emulsions. This deduction was supported by the gradual

301

increase of red fluorescence (protein) at the surface of dispersed droplets upon increasing PA

302

loadings. CLSM trials also revealed that PA promoted the flocculation of particle-coated dispersed

303

droplets which flocculated to form closely packed droplet-based networks (Figure 5). In brief,

304

addition of PA endowed GPHPEs with robust and compact interfacial structure.

305

Rheological Properties. The rheological property impacts the physical property of the

306

Pickering emulsions, for example, appearance, texture and stability. Figure 6 shows storage

307

modulus (G’) and loss modulus (G”) of GPHPEs as a function of stress (A) or frequency (B) at

308

selected PA loadings. The incorporation of PA led to a gradual increase in G′. This situation may be 14


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309

attributed to the effective interfacial adsorption and accumulation of the hybrid particles due to the

310

special affinity between proline-rich gliadins and PA, along with the emergence of a percolated

311

network architecture derived from the delicate interaction between particle-coated droplets (Figure

312

4D/E and 5). For all Pickering emulsions, G′ was higher than G″ at lower stress. In contrast, a

313

recognizable crossover point was detected for each emulsion at higher stress. The corresponding

314

crossover point was increased from 10 Pa for GPHPEs-1 to 63 Pa for GPHPEs-10 (Figure 6A). As

315

expected, frequency sweep of Pickering emulsions also confirmed that the introduction of PA

316

strengthened the emulsions solid-like behaviors (Figure 6B). In brief, G′ was greater than the

317

corresponding G″ in the entire frequency range tested. The G′ increased upon the increase in PA

318

loadings at a given frequency (Figure 6B). Rheological properties supported the finding of visual

319

appearance and microstructure that GPHPEs prepared were viscoelastic and self-supporting. The

320

formation of interconnected 3D network via the particle-coated droplets and free particles as

321

building blocks (Figure 4D/E and 5) contributed to the rigidity or mechanical strength of Pickering

322

emulsions and was crucial to tune lipid oxidation and digestion profiles.

323

Lipid Oxidation of GPHPEs. To illuminate the impact of PA loading on oxidative stability of

324

GPHPEs, three typical samples (GCPEs, GPHPEs-1, and GPHPEs-5) were subjected to thermally

325

accelerated incubation in the dark at 60 °C for 35d, the content of the markers (oxygen uptake,

326

hexanal and MDA) were monitored. Headspace oxygen of GPHPEs was measured to display initial

327

lipid oxidation rate in Pickering emulsions. In GCPEs, oxygen uptake reached maximum value (38

328

mmol/kg oil) after 23 h and then remained unchanged over time, PA loading delayed oxygen uptake

329

in GPHPEs, which reached only 31 and 26 mmol/kg oil for GPHPEs-1 and GPHPEs-5 after 51 h,

330

respectively (Figure 7A). Comparatively, Pickering emulsions developed consumed less oxygen

331

than conventional emulsions, e.g., Berton, et al. reported that oxygen uptake in the emulsions 15



332

stabilized with BSA (or Tween 20) was approximately 110 (or 80) mmol/kg oil after incubating at

333

33 °C for 75 h.40 Oxygen uptake in emulsions stabilized by protein was about 130 mmol/kg oil after

334

incubating in the dark at 33 °C for 78 h.41 Therefore, the combination of interfacial cargo of PA and

335

Pickering principle is a potent route to control initial lipid oxidation rate and level.

336

Headspace hexanal was monitored by GC analyses as an important marker of volatile

337

secondary lipid oxidative products of GPHPEs. As shown in Figure 7B, largest relative abundance

338

of hexanal with an identified peak at 3.6 min was recorded in GC profile of GCPEs after 35 d of

339

storage at 60 °C, and peak area of hexanal was decreased gradually with increasing PA loadings.

340

Table 3 shows more detailed evolution of hexanal content in GPHPEs during thermally accelerated

341

storage. As expected, peak of hexanal with an area of 12.57 firstly appeared in GCPEs on the 11th

342

day, while a weak signal (peak area was 1.76) measured in GPHPEs-1 on the 15th day. In the case

343

of GPHPEs-5, there was no detectable hexanal signal after 15 d, and a hexanal peak with an area of

344

6.73 was detected until 20 d of incubation. Evolution trend of headspace hexanal was consistent

345

with the oxygen uptake upon thermally accelerated oxidation at 60 °C (Figure 7A).

346

The combination of TBARS assay and HPLC was performed to determine MDA

347

concentrations in GPHPEs, Table 4 shows that MDA content was decreased from 25.40 ±

348

0.44 µmol/ kg of oil in GCPEs to 20.86 ± 1.60 and 19.07 ± 0.73 µmol/kg in GPHPEs-1 and

349

GPHPEs-5, after 35 d of storage at 60 °C. Comparatively, MDA content detected was lower than

350

the equivalent of biopolymer stabilized conventional emulsions. Huang et al. used soy protein

351

isolate and pectic enzyme treated pectin to stabilize soybean oil forming emulsions. The MDA

352

content in the emulsions was about 5 mmol/kg oil after the incubation at 50 °C for 10 d.42 Lomova

353

et al. investigated the oxidant stability of the linseed oil-based emulsions, which stabilized by

354

bovine serum albumin and biocompatible polyelectrolytes complexes, MDA content were 40−100 16


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355

mmol/kg oil during the incubation at 37 °C for 0−15 d. 1 In short, the MDA content in this work

356

was lower than that of the fore-mentioned emulsions by roughly 2−3 orders of magnitude.

357

Therefore, Pickering emulsions we fabricated were stable against lipid autoxidation in our

358

experiments. Besides, the results of MDA combined with oxygen consumption and hexanal

359

generation proved that Pickering emulsion alone, and the combination with PA effectively

360

improved the oxidative stability of emulsified systems. Consequently, development of antioxidant

361

interface through co-assembled GPHPs provides a powerful strategy to control lipid oxidation in

362

emulsified oil systems.

363

Lipid peroxidation usually speeds up in o/w binary systems when compared with bulk oil. In

364

an emulsion, lipid peroxidation initiates and prevails at the interfacial region.5-7 Herein, Pickering

365

emulsions with tunable interfacial structure (Figure 5) were constructed. Interestingly, the results of

366

oxygen uptake and the secondary oxidative products (hexanal and MDA) generation in Pickering

367

emulsions corroborated with each other and effectively identified that engineering interfacial

368

structure via PA-modified gliadin colloid particles is a powerful strategy to restrict lipid oxidation

369

of emulsified oils. Several factors contributed this finding. Effective adsorption of hybrid particles

370

resulted in robust antioxidant shell around droplets to scavenge free radicals and prevent the close

371

contact between pro-oxidants and LH.43, 44 The particle-coated droplets as building units resulted in

372

the occurrence of structuring phenomenon Pickering emulsions (Figure 4 and 5), and this situation

373

restrained the diffusion of pro-oxidant with toward the interfacial region where amphiphilic primary

374

oxidation products existed.

375

The in vitro Digestive fates of Pickering emulsions stabilized by GPHPs. Lipids are usually

376

untaken in various forms, and their digestion, adsorption and metabolism are really important in

377

directing food formula.1 17



378

Evolution in Droplet Size Distribution and Microstructure. The digestion fate of GPHPEs

379

under simulated GI tract was investigated by checking structural integrity as well as flocculation

380

and/or coalescence behavior of the droplets, by measuring the extent of lipid hydrolysis and

381

oxidation. Particle size and size distribution along with microstructure of the digesta were

382

periodically evaluated. Originally, mean particle diameters of GPHPEs were below 25 µm (D3, 2)

383

with mono-modal particle size distribution (Figure 8), and slightly flocculate (Figure 9) at the

384

beginning of digestion, possibly due to the pH shift to highly acidic condition.21 Shortly, GCPEs

385

underwent extensive droplet coalescence. The mean particle size of Pickering emulsions increased

386

from 22.00 to 94.75 µm (Table 5) after 10 min of incubation in SGF, which was well captured by

387

the light microscope, as shown in Figure 9 where larger droplets were clearly observed.

388

Interestingly, the introduction of PA prevented droplet coalescence of GPHPEs during the gastric

389

digestion. Visually, droplet coalescence occurred after 1 h of gastric digestion with droplet size

390

increasing from 19.73 to 102.88 µm (GPHPEs-1). Furthermore, the mean particle diameter

391

remained constant when GPHPEs-5 passed through the SGF, evidenced by optical microscopy

392

pictures (Figure 9). After the gastric digestion, some of oil droplets in GCPEs and GPHPEs-1

393

coalesced with each other losing their integrity and resulting in serious oiling-off, which was well

394

captured by camera (Figure 2S). Destabilization phenomena were also reported for Pickering

395

emulsions stabilized by Kafirin particles.45 Finally, only a few scattered small oil droplets

396

(0.13−0.19 µm) were observed due to the lipid hydrolysis after simulated GI digestion. In summary,

397

GPHPEs with robust interface architecture (Figure 5) were less labile to coalescence when exposed

398

to SGF.46

399

Figure 3S shows the changes of ζ-potential for Pickering emulsions in different digestion

400

stages. Zeta potential of all samples were decreased when exposing in SGF from approximately 20 18


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401

mV (pH 4.0) (Table 2) to 8 mV resulting from pH change and electrostatic screening effect of salt

402

ion in SGF (pH 2.0). Zeta potentials of all samples were shifted to about -25 mV after the intestinal

403

digestion. The pH shift (from 2.0 to 7.0), electrostatic screening effect and interfacial substitution

404

by bile salts or lipid hydrolysate (e.g. monoglyceride, diglycerides) might responsible for this shift.

405

FFA Release. The bolus of gastric digestion was subjected to SIF digestion in turn to clarify the

406

potential influence of the interfacial architecture made up by gliadin/PA hybrid particles on the lipid

407

digestion. This model imitates the route in which excipient emulsions are orally untaken.45 Lipid

408

hydrolysis was determined using pH-stat protocol. Figure 10 shows the FFA release profiles of

409

GPHPEs with bulk corn oil as the control. A gentle release of FFA was detected within the initial

410

digestion (approximately 10 min), followed by a more rapid lipolysis, and then a slower lipolysis

411

was observed, whether corn oils were emulsified or not. The profiles of FFA release were fairly

412

similar for GCPEs and GPHPEs, but the extent of lipolysis was inversely proportional to PA

413

content (Figure 10). The lipolysis degree of bulk oil reached 87.9% after 2 h of digestion at 37 °C.

414

In contrast, it was largely limited when the surface of droplets was covered by gliadin/PA hybrid

415

particles, as a function of PA loadings. The FFA release was decreased by approximately 43% from

416

87.9% (bulk oil) to 50.2% (GCPEs). Lipid lipolysis was further depressed in GPHPEs where PA

417

were included, and FFA release were 45.5% and 39.5% for GPHPEs-1 and GPHPEs-5, respectively.

418

Surface area and the interfacial physicochemical properties are vital to lipid digestion. In the case of

419

bulk oil, the bare water-oil interface could be readily exposed to bio-surfactants (bile salts), lipase

420

and co-lipase during the digestion, the lipolysis was limited by relative small interface area

421

available for lipase.47 In the case of GCPEs, lipase had to diffuse across the interfacial architecture

422

constituted by the hybrid particles to react with lipid substrate. Particularly, in the case of GPHPEs

423

with robust interface structure derived from co-assembled PA-gliadin hybrid particles, the 19



424

accessibility of lipase to oil was further depressed. Overall, the extent of lipid hydrolysis in

425

GPHPEs (39.5−50.0%) was much lower than that in bulk oil (87.9%) or conventional emulsions

426

(≥90%).35,

427

activate the so-called ileal brake,9 thus restrict the intake of oil as well as other high energy-dense

428

foods and potentially benefit to fight obesity. In summary, our research suggested that manipulating

429

interfacial architecture may aid to limit lipolysis of emulsion system, as a result potentially benefit

430

to fight obesity.

48

Most importantly, Pickering emulsions slow down digestion rate of lipids might

431

Lipid Oxidation of Emulsions during the in Vitro Digestion. Lipid oxidation occurs not only

432

in food production, transportation and storage, but also under the GI digestion. However, the works

433

on the oxidative fate of lipid under the digestion were still very scarce. So, we exposed GPHPEs to

434

the simulated GI fluids to assess lipid oxidation profile by monitoring MDA content with bulk oil as

435

the control. As shown in Figure 11, MDA concentration for bulk oil was the largest after in vitro

436

simulated GI digestion for 3 h, and it decreased by 49% from 232.21 ± 0.00 (bulk oil) to 117.95 ±

437

12.18 (GCPEs) µmol/kg oil. PA loadings further inhibited lipid oxidation in GPHPEs with MDA

438

content of 67.17 ± 14.34 µmol/kg oil (GPHPEs-5), approximately 28.9% of that in bulk oil. When

439

subjected to SIF, bulk oil was emulsified by bile salts, and the increased interfacial area promoted

440

the lipid oxidation, in view of the increased interaction incidence between LH and pro-oxidants.

441

Interfacial membrane constructed by antioxidant particles served as a valid barrier against lipid

442

oxidation by preventing the interaction between LH and pro-oxidants, as well as blocking the chain

443

reaction of lipid peroxidation through the free radical scavenge route. In particular, the structuring

444

behaviour in the robust GPHPEs prevented the transfer of pro-oxidants from the aqueous

445

continuous phase to the interface region.

446

MDA originated from bulk corn oil in SIF (14.69 µM) within the range of concentrations 20


Page 20 of 47

Page 21 of 47


447

(10-100 µM) for increasing mutation risk of human fibroblasts.3 In contrast, emulsified oil in

448

Pickering emulsions experienced less oxidation with lower MDA quantities (9.74µM in GCPEs,

449

6.69 µM in GPHPEs-5). Robust antioxidant interface shell derived from antioxidant hybrid particles

450

benefited to the strong oxidative stability of emulsions under storage and simulated digestion. Our

451

work provides a workable strategy for protecting PUFAs from oxidation, under the storage or in the

452

GI tract. In short, the association of Pickering principle and shell PA is a facile and potent solution

453

to limit lipid oxidation of emulsified system during the storage and digestion.

454

In Conclusion. The binding affinity between proline-rich gliadin and PA was used to

455

manipulate the self-assembly nucleation process of gliadin, to construct dual-function hybrid

456

particles. This work engineered emulsions’ interface using the hybrid particles, and fabricated

457

Pickering emulsions with robust interfacial architecture. This strategy facilitated interfacial

458

adsorption and deposition of the gliadin-based particles through the special affinity interaction

459

between gliadin and PA. Pickering droplets as building units assembled into interconnected

460

three-dimensional network structure, giving the emulsions viscoelasticity and ultra-stability.

461

Interfacial membrane constructed by antioxidant particles served as a valid barrier against lipid

462

oxidation under storage and simulated GI tract, in a PA dose-dependent manner. FFA release

463

decreased by 55% from 87.9% (bulk oil) to 39.5% (GPHPEs-5), implying engineering interfacial

464

architecture potentially benefited to fight obesity.

465

ACKNOWLEDGEMENTS

466

This work was supported by the National Key Research and Development Program of China

467

(Project No. 2017YFC1600405), the National Natural Science Foundation of China (21406077;

468

31471628), the Pearl River S & T Nova Program of Guangzhou (201506010063), and the

469

Fundamental Research Funds for the Central Universities (SCUT, 2017ZD080). 21



470

ABBREVIATIONS

471

PA: proanthocyanidins; GPHPs: gliadin/proanthocyanidins hybrid particles; GPHPEs,

472

gliadin/proanthocyanidins hybrid particles emulsions; GCPs, GPHPs-1, GPHPs-2, GPHPs-5 and

473

GPHPs-10: gliadin colloid particles with PA-to-gliadin mass ratios of 0, 1%, 2%, 5%, and 10%,

474

respectively; MDA: malondialdehyde; CLSM: confocal laser scanning microscope; TBA:

475

thiobarbituric acid; GI: gastro intestinal; SIF: simulated intestinal fluid; SGF: simulated gastric fluid;

476

FFA: free fatty acids; AFM: atomic force microscopy; LH: lipid hydroperoxides.

477

SUPPORTING INFORMATION

478

Fig.1S. Appearance photographs of the GPHPs dispersions after 24 h incubation. Fig.2S.

479

Photographs of emulsions during simulated gastric digestion. Fig.3S. ζ-Potentials of Pickering

480

emulsions exposed to simulated GI environments.

481

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occurrence, dietary intake and effects on nutrition and health. J. Sci. Food Agric. 2000, 80,

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1094−1117.

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(45) Xiao, J.; Li, C.; Huang, Q. R. Kafirin nanoparticle-stabilized Pickering emulsions as oral

599

delivery vehicles: Physicochemical stability and in vitro digestion profile. J. Agric. Food Chem.

600

2015, 63, 0263−10270.

601

(46) Moreau, L.; Kim, H. J.; Decker, E. A.; McClements D. J. Production and characterization of

602

oil-in-water emulsions containing droplets stabilized by β-lactoglobulin−pectin membranes. J.

603

Agric. Food Chem. 2003, 51, 6612−6617

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(47) Maldonadovalderrama, J.; Wilde, P.; Macierzanka, A.; Mackie, A. The role of bile salts in digestion. Adv. Colloid Interface Sci. 2011, 165, 36−46.

606

(48) Bellesi, F. A.; Martinez, M. J.; Pilosof, A. M. R. Comparative behavior of protein or

607

polysaccharide stabilized emulsion under in vitro gastrointestinal conditions. Food 27



608

Hydrocolloids 2016, 52, 47−56.

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609

Figure captions

610

Figure 1. Appearance photographs of freshly prepared GCPEs (a), GPHPEs-1 (b), GPHPEs-2 (c),

611

GPHPEs-5 (d), GPHPEs-10 (e) dispersions(A), AFM images of GPHPs-2 (B1), GPHPs-10 (B2),

612

ζ-potential of pristine gliadin colloid particles (GCPs) and PA as a function of pH (C), FT-IR

613

spectra of gliadin, PA and GPHPs-2 (D).

614

Figure 2. Appearance photographs of the GCPEs (a), GPHPEs-1 (b), GPHPEs-2 (c), GPHPEs-5 (d),

615

GPHPEs-10 (e) after 10 days of storage at room temperature.

616

Figure 3. Particle size distribution of GPHPEs as a function of PA concentration at pH 4.0.

617

Figure 4. Microscopy images (scale bar: 20 µm) of GCPEs (A), GPHPEs-1 (B), GPHPEs-2 (C),

618

GPHPEs-5 (D), GPHPEs-10 (E). Pictures were taken 10 d after preparation.

619

Figure 5. Selected CLSM images (scale bar: 10 µm) of GCPEs (a), GPHPEs-2 (b), GPHPEs-10 (c)

620

in green fluorescence field (left) and overlap fluorescence field (right). Corn oil was stained with

621

Nile Red, and GPHPs was stained by Nile Blue A. The fluorescent dyes simultaneously excited at

622

488 nm for Nile Red (green) and at 633 nm for Nile Blue A (red). Oil location is shows at left side,

623

while right side shows the colocation of oil and proteins.

624

Figure 6. A: storage modulus (G’) and loss modulus (G”) of GPHPEs as a function of stress; B:

625

storage modulus (G’) and loss modulus (G”) of GPHPEs as a function of frequency.

626

Figure 7. Headspace-oxygen consumption (A) and headspace hexanal (B) of the GPHPEs under

627

accelerated storages at 60 °C.

628

Figure 8. Particle size distribution of GCPEs (a), GPHPEs-1 (b) and GPHPEs-5 (c) exposed to

629

simulated GI fluids. G0, G10, G60 represent 0, 10, 60 min after subjected to SGF, respectively. I120

630

represents 60 min in SGF and followed by 120 min in SIF.

631

Figure 9. Microstructure evolution of GCPEs (a), GPHPEs-1 (b) and GPHPEs-5 (c) during the 29



632

simulated GI digestion at different time. G0, G10, G60 represent 0, 10, 60 min after exposing to

633

SGF, respectively. I120 represents 60 min in SGF and followed by 120 min in SIF.

634

Figure 10. Percentage of free fatty acids (FFAs) released in Pickering emulsions under the in vitro

635

digestion.

636

Figure 11. MDA content of Pickering emulsions after simulated GI digestion. Different letters on

637

the top of columns differ significantly (p < 0.05) according to the Tukey test.

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Table Table 1 Particle size, polydispersity index and zeta potential of the GPHPs as a function of PA loadings at pH 4.0 Sample

PA/gliadin ratio

Particle size (nm) polydispersity index (PDI) Zeta potential/mV

GCPs

0

120.07 ± 2.73d

0.21 ± 0.04

10.06 ± 0.12a

GPHPs-1

1:100

289.70 ± 2.23b

0.21 ± 0.02

10.02 ± 0.69a

GPHPs-2

1:50

364.63 ± 8.93a

0.30 ± 0.04

10.15 ± 0.66a

GPHPs-5

1:20

153.47 ± 1.12c

0.15 ± 0.02

10.63 ± 0.15a

GPHPs-10

1:10

86.60 ± 1.15e

0.35 ± 0.01

11.45 ± 1.48a

Different superscript letters (a-e) within the same column indicated significant difference (p < 0.05)

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Table 2 Particle size and Zeta potential (ζ-potential) of the GPHPEs with different PA loadings produced at pH 4.0 samples

D 4, 3/ µm

D 3, 2/ µm

GCPEs

71.37 ± 0.93c

22.00 ± 0.10bc

19.00 ± 0.66a

GPHPEs-1

52.5 ± 0.26d

19.73 ± 0.06cd

20.23 ± 1.90a

GPHPEs-2

54.97 ± 1.18d

17.53 ± 0.55d

19.17 ± 0.15a

GPHPEs-5

80.40 ± 4.89b

24.10 ± 0.36b

20.73 ± 0.15a

GPHPEs-10

93.97 ± 3.80a

61.50 ± 2.31a

18.23 ± 1.29a

Zeta potential/ mV

Different superscript letters (a-d) within the same column indicated significant difference (p < 0.05)

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Table 3 Peak Area of headspace hexanal generated from the GPHPEs under accelerated storages at 60 °C Peak Area of Hexanal/g lipid Time/d

GCPEs

GPHPEs-1

GPHPEs-5

0

0.00 ± 0.00a

0.00 ± 0.00a

0.00 ± 0.00a

8

0.00 ± 0.00a

0.00 ± 0.00a

0.00 ± 0.00a

11

12.57 ± 2.11a

0.09 ± 0.01b

0.00 ± 0.00b

15

14.40 ± 1.31a

1.76 ± 0.52b

0.00 ± 0.00b

20

18.40 ± 3.21a

13.11 ± 0.00b

6.73 ± 1.04c

35

29.52 ± 0.00a

23.09 ± 2.13b

13.97 ± 5.72c

Different superscript letters (a-c) within the same column indicated significant difference (p < 0.05)

33



Table 4 MDA content of GPHPEs under accelerated storages at 60 °C after 35days Time

35d

samples

MDA/(µmol/kg oil)

GCPEs

25.40 ± 0.44a

GPHPEs-1

20.86 ± 1.60b

GPHPEs-5

19.07 ± 0.73b

Different superscript letters (a-b) within the same column indicated significant difference (p < 0.05)

34


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Table 5 Mean droplet size (D32) and specific surface area (SSA) obtained for initial moment (tG0), at 10 min (tG10), 60min (tG60) of gastric digestion and after intestinal digestion (tI120). GCPEs

GPHPEs-1

GPHPEs-5

Time

D32/µm

SSA(m2/g)

D32/µm

SSA(m2/g)

D32/µm

SSA(m2/g)

tG0

22.00 ± 0.10

0.27 ± 0.00

19.73 ± 0.06

0.30 ± 0.00

24.10 ± 0.36

0.25 ± 0.00

tG10

94.75 ± 9.56

0.07 ± 0.01

16.27 ± 0.58

0.37 ± 0.01

20.50 ± 4.29

0.30 ± 0.07

tG60

25.78 ± 2.09

0.25 ± 0.02

102.88 ± 4.55

0.06 ± 0.00

24.95 ± 3.26

0.24 ± 0.03

tI120

0.14 ± 0.01

43.77 ± 6.13

0.13 ± 0.01

46.42 ± 4.23

0.19 ± 0.04

32.26 ± 5.78

35



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Figure 1

B A

B1

B2 C

D

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Figure 2

a

b

c

d

37


e


Figure 3

38


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Figure 4

A

B

C

D

E

39



Figure 5

a

b

c

40


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Figure 6

41



Figure 7

42


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Figure 8

43



Figure 9

44


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Figure 10

45



Figure 11

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TOC Graphic

47


Development of Pickering Emulsions Stabilized by Gliadin

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