Pectin Hybrid Particle-Stabilized Pickering High

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Food and Beverage Chemistry/Biochemistry

Fabrication of Zein/Pectin Hybrid Particles Stabilized Pickering High Internal Phase Emulsions (HIPEs) with Robust and Ordered Interface Architecture Fu-Zhen Zhou, Xiao-Nan Huang, Zi-ling Wu, Shou-Wei Yin, Jian Hua Zhu, Chuan-He Tang, and Xiaoquan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03714 • Publication Date (Web): 01 Oct 2018 Downloaded from http://pubs.acs.org on October 2, 2018

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Journal of Agricultural and Food Chemistry

Fabrication of Zein/Pectin Hybrid Particles Stabilized Pickering High Internal Phase Emulsions (HIPEs) with Robust and Ordered Interface Architecture Fu-Zhen Zhou †, Xiao-Nan Huang†, Zi-ling Wu†, Shou-Wei Yin†,‡*, Jian-hua Zhu§, Chuan-He Tang†,‡, Xiao-Quan Yang†,‡ †

Research and Development Center of Food Proteins, School of Food Science and

Engineering and ‡Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou 510640, PR China. §School of Ying-Dong Food Sciences and Engineering, Shaoguan University, Shaoguan 512005, PR China.

Running title: Pickering HIPEs stabilized by zein/pectin hybrid particles

* Corresponding author Yin, S. W. Phone: +86-2087114262. Fax: (+86)-20-87114263. E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Diets containing partially hydrogenated oils (PHOs) expose human body to trans fatty acids

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thus endangering cardiovascular health. Pickering HIPE is a promising alternative of PHOs.

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This work attempted to construct stable Pickering-HIPEs by engineering interface

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architecture through manipulating the interfacial, self-assembly, and packing behavior of zein

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particles using the interaction between protein and pectin. Partially wettable zein/pectin

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hybrid particles (ZPHPs) with three-phase contact angles ranging from 84° to 87° were

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developed successfully. ZPHPs were irreversibly anchored at the oil−water interface,

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resulting in robust and ordered interfacial structure, evidenced by the combination of

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LB-SEM and CLSM. This situation helped to hold a percolating 3D oil droplet network,

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which facilitated formation of Pickering HIPEs with viscoelasticity, excellent thixotropy (>

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91.0%) and storage stability. Curcumin in HIPEs was well protected from UV-induced

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degradation, and endowed HIPEs with ideal oxidant stability. Pickering HIPEs fabricated

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possess charming application prospect in foods and pharmaceutical industry.

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Key words: Zein/pectin hybrid particles, interface, Pickering high internal phase emulsions,

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viscoelastic, oxidation

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INTRODUCTION

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Nowadays, human beings are facing with many health risks, including cardiovascular

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diseases, diabetes, high blood pressure, cancer etc. Among these, cardiovascular disease with

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the highest morbidity and mortality1 was believed to be related to the intake of trans fatty

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acids2. With the development of the food industry, PHOs have become the major source of

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trans fatty acids3. Now that the U.S. Food and Drug Administration (FDA) has announced the

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ban on the use of PHOs in processed foods since June 18, 2018. It is imminent for food

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manufacturers and researchers to seek for comparable and healthy alternative of PHOs.

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Currently, HIPEs development is one of the promising ways to directly turn liquid oil into

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solid fat without PHOs. Traditional HIPEs are usually stabilized by high concentrations of

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surfactants. Pickering emulsion is a kind of solid particle stabilized emulsions where

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interfacial absorption of particles is nearly irreversible. The high resistance to coalescence

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and surfactant-free character are major benefits of Pickering emulsion4. Certainly, inorganic

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particles5-7 has been used to prepare Pickering-HIPEs, but they are usually not suitable for

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food usages. Besides, earlier studies have shown that when internal volume fraction was

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beyond a threshold (e.g., 70%), Pickering emulsions tended to phase inversion or oiling-off8, 9.

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Up till now, few studies about development of HIPEs by food-grade particles10-13 were

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reported, it is not conducive to the wide application of HIPEs in food industry. Thus, it is

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significant to prepare appropriate and effective food-grade particles to tackle the key

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technological challenge for Pickering-HIPEs development.

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Zein is the major storage protein of corn or maize, and more than 50% of its amino acids

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are nonpolar contributing to the unique solubility behavior and amphipathy of zein14.

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Particles formed by zein self-assembly have been used as an encapsulation and delivery

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system for nutrients or drugs15, as a stabilizer for Pickering emulsion is another major use. De

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Folter et al.16 first reported the utilization of zein as solid particle stabilizer for Pickering O/W

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emulsions, but the stability of emulsions was not satisfactory. Since then, more and more

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studies focused on zein stabilized Pickering emulsion17-23. In our previous studies19, we found

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that solely zein particles were too hydrophobic to effectively stabilize oil-water interface,

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consequently, coating materials (e.g., sodium stearate17, chitosan19, 20) were used to improve

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the wettability of zein particles to fabricate stable emulsions. The oil fraction used was

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mainly limited to 50%20. When the internal phase fractions were increased to 70%, the

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droplets deformed to nearly merge together, suggesting that this oil fraction was approaching

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the threshold value of phase separation and/or inversion for Pickering emulsions stabilized by

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zein-based colloid particles20. To the best of our knowledge, no research about Pickering

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HIPEs stabilized by zein-based particles has been reported until now. This work provided a

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new insight and cut-in point to develop stable zein-based Pickering HIPEs via the delicate

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interaction between protein and polysaccharide.

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Pectin extracted from plant cell wall is a natural and safety polysaccharide with high

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molecular weight and multiple hydroxyl groups. Pectin possess a lot of functional attributes,

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e.g., gelling and thickening properties, beneficial effect on health24, immunomodulating

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activities25, digestive resistance26. Therefore, it is a long time for the utilization of pectin in

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foods, pharmaceutical and cosmetic industries, generally used as a texturizer, stabilizer,

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gelling agent or drug carrier. In addition, pectin is able to interact with proteins influencing

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the colloidal binding and coagulation of proteins.27, 28 Actually, the combination of zein and

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pectin has already been reported, in previous studies, pectin/zein complex hydrogel beads29

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and particles30 had been developed as drug delivery system, emulsions stabilized by

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pectin-zein complexes also has been prepared. However, most of these emulsions were

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mainly stabilized by pectin (conventional emulsions) not by zein particles (Pickering

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mechanism), and the volume fractions of oil phase were no more than 30%31, 32. In a very real

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sense, oil in water Pickering HIPEs stabilized by zein/ pectin complex particles has not been

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developed yet.

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In this work, our overall objective was to construct stable Pickering-HIPEs with ideal

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properties by engineering interface architecture through manipulating the interfacial behavior,

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self-assembly, and packing of zein particles using the interaction between protein and pectin.

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Firstly, we investigated the influence of pectin/zein ratios and pH conditions on colloidal

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properties of ZPHPs, including size and zeta potential. Wettability, one of the crucial factors

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affecting the interfacial adsorption of ZPHPs was evaluated by optical contact angle meter.

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Next, the combination of Langmuir-Blodgett and scanning electron microscope (LB-SEM)

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was used to characterize interfacial adsorption and assembly behavior of ZPHPs at different

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pectin/zein ratios and pH conditions. Lastly, the physical performances, rheological

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properties, microstructure, oxidant stability, and protective effect of Pickering-HIPEs for

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curcumin were investigated. The relationship between interfacial adsorption/packing

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behavior of ZPHPs and physicochemical properties of HIPEs was also been explored. Our

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work will provide a new visual angle and cut-in point to the studies on interaction of protein

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and polysaccharide, also has guiding significance for the construction of complex particle

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stabilized Pickering emulsion.

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MATERIALS AND METHODS Materials. Zein (product Z 3625) and Fluorescent dyes (Nile Red, Nile Blue A, Calcofluor

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white) were purchased from Sigma–Aldrich (Shanghai, China). High ester pectin (YM-115-H)

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extracted from citrus peel was offered by CP Kelco (Limeira, Brazil). Commercial corn oil

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was produced by Yihai Kerry group (Guangzhou, China), and further purified according to

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our previous method33. All other chemicals used were of analytical grade.

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Preparation of Zein/Pectin Hybrid Particles (ZPHPs). ZPHPs or solely zein particles

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(ZPs) were fabricated via an anti-solvent precipitation operation. Accurately, zein (2 g) was

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previously mixed with ethanol (56 mL)-water (24 mL) binary solvent, pectin was dissolved in

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deionized water (0.2 L) by stirring, the mass ratios of pectin-to-zein were 0:10, 1:10, 2:10,

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and 5:10 (marked as ZPs/ZPHPs-0, ZPHPs-1, ZPHPs-2, ZPHPs-5, respectively). Next, zein

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was quickly poured into the water followed by uniformly shearing at 7000 rpm within 5 min

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using an Ultra-Turrax T25 homogenizer (IkA, Germany). Then, ethanol together with the

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excess water was dislodged via evaporating, until the volume of the solution was 100 mL

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with zein concentration of 2%. The pH of particle dispersions was adjusted by HCl or NaOH

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solution when necessary.

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Particle Size and ζ-potential Measurements. Particle size and ζ-potential of ZPHPs were

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determined at a 20 times diluted concentration (0.1%, w/v) by Zetasizer Nano (Malvern

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Instruments Ltd., UK) at 25 °C. Each result showed is the average and standard deviation of

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three tests.

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Particle Morphology. The microstructure of ZPHPs was surveyed by Multimode 8 atomic

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force microscopy (Bruker, German). ZPHPs were diluted by 1000-fold (20 µg/mL), and 10

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µL of diluted liquid was dripped on mica disks, then dried overnight and captured by AFM.

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Fourier Transform Infrared Spectroscopy (FTIR). Pectin, ZPs and ZPHPs-2

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dispersions were lyophilized, then characterized by FTIR (Bruker Co., Germany). For each

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sample, 128-scans were accumulated from 400 to 4000 cm−1 at a resolution of 2 cm−1.

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Three-phase Contact Angle. The freeze-dried ZPHPs (0.1g) were pressed into cylindrical

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tablets (10 mm×2 mm), and three-phase contact angle (θ) was measured by optical contact

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angle meter (OCA 20, DataPhysics Instruments GmbH, Germany). Briefly, the tablet was

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immersed in purified corn oil, then 10µL of ZPHPs dispersions was slowly excluded from the

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syringe and quickly attached to the surface of tablet. The shape of ZPHPs drop was recorded

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by a camera, θ was calculated from Laplace−Young equation.

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Dynamic Surface Tension. The adsorption behavior of ZPHPs at water-oil interface was

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characterized by OCA 20 (DataPhysics Instruments GmbH, Germany). First, ZPHPs

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dispersion (0.5% zein, w/w) was loaded in a syringe, then 15µL of ZPHPs was injected into

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purified corn oil. The adsorption process was captured by a CCD camera, one point was

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recorded within every 2 s and lasting for 3 h.

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Langmuir-Blodgett and Scanning Electron Microscope (LB-SEM). A Langmuir trough

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was used to obtain monolayer film of ZPHPs. Concisely, 30µL of ZPHPs dispersions was

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gently dropped onto sub-phase (300 mL pH 3.8 deionized water) surface, after waiting for 20

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minutes to reach an equilibrium state, the compression was performed at a constant surface

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pressure of 25 mN/m and rate of 8 mm/min. Then, ZPHPs film was transferred to mica sheet

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at 2 mm/min by vertical pulling, and air dried under room temperature. To accurately capture

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the microstructure of ZPHPs at water-air interface, the mica sheet was sprayed a gold layer

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and observed by Field Emission Scanning Microscope (FE-SEM, Zeiss, Oberkochen,

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Germany).

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Pickering HIPEs Preparation. ZPHPs were used as particulate stabilizer to prepare

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Pickering HIPEs (ZPHPEs). Oil phase volume fraction for Pickering HIPEs development was

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adopted as 0.8. In other words, corn oil (4 mL) and ZPHPs dispersions (1 mL) were added

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into the same glass bottle, then the immiscible liquids were sheared at 10000 rpm for 2 min to

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form HIPEs by a T10 homogenizer (IkA, Germany). Pristine zein colloid particle (ZPs) and

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pectin solution were also used to fabricate HIPEs using the same protocol.

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Particle Size Distribution of Pickering HIPEs. Drop size of ZPHPEs was measured by

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static light scattering technique (Malvern Mastersizer 3000, Malvern Instruments, UK).

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ZPHPEs were dispersed in deionized water, refractive index (RI) of corn oil and water were

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set as 1.467 and 1.330, absorption parameter was 0.001. Results reported were the average of

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six times repeated measurements.

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Centrifugation Stability. HIPEs were centrifuged at 10,000 g for 2 min to investigate the centrifugation stability, the samples were photoed by a camera.

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Optical Microscopy Measurements. An aliquot of ZPHPEs (50 µL) was placed at a slide

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glass and covered with a cover glass. Then, the microstructure of ZPHPEs was captured by

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optical microscope (OG100-3B41L-IPL, Jiangxi Phoenix Optical Co., China).

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CLSM. Microstructure of ZPHPEs was observed by a TCS SP5 confocal microscopy

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(Leica Microsystems Inc., Germany) based on previous method33. Corn oil, zein and pectin in

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ZPHPEs were dyed with 0.1 % of Nile Red, Nile Blue and Calcofluor white, respectively,

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and 20 µL of dyed ZPHPEs were taken out to be analyzed. Emitted light activated at 405 nm

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(Calcofluor white), 488 nm (Nile Red), 633 nm (Nile Blue) were observed in the field of

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vision.

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Dynamic Oscillatory Measurements. Rheology properties of ZPHPEs were studied with

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a HAAKE RS600 Rheometer (HAAKE Co., Germany), a series of experiments including

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small amplitude or stress (0.1-100 Pa, frequency = 1 Hz), frequency (0.1-10 Hz, stress = 5 Pa,

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within linear viscoelastic region) sweeps, flow (shear rate = 1-100 s-1) and three interval

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thixotropy analysis were carried out at 25 °C. The elastic modulus (G’) and loss modulus (G”)

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were obtained by RheoWin 3 Data Manager.

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Protective Effect of Pickering HIPEs on Curcumin Against UV Radiation. 15 mL of

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ZPHPEs stabilized by ZPHPs-2 at pH 3.8 containing curcumin were exposed to UV radiation

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(15 W, 340 nm) at 25 °C, samples were collected periodically to extract curcumin with

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methanol. The centrifugal supernatant was detected at 425 nm, and the residual curcumin

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were quantified by a standard curve. Bulk corn oil and Tween 20-stabilized emulsions

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containing curcumin were used as control.

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Lipid Oxidation Measurements. Samples were placed at 40 °C for 21 d to evaluate lipid

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oxidation, 150 mg of samples were taken out to determine the amount of lipid hydroperoxide

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(LH) and malondialdehyde (MDA) according to the method described previously10. Simply,

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LH was extracted by 2-propanol/isooctane (1:3, v/v) mixed solution, then mixed in

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proportion with methanol/1-butanol mixture (1:14), NH4SCN and Fe2+ were added to render

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color, LH were detected at 510 nm and quantified by an external standard (H2O2). TBA

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solution consisting of thiobarbituric acid (0.375%, w/v) and trichloracetic acid (15%, w/v)

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together with the samples were boiled to detect MDA which was quantified by external

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standard (1, 1, 3, 3-tetraethoxypropane).

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Statistics. Each result (mean ± standard deviation) reported were of at least 3 times of

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repeated tests. Statistical analyses were finished by SPSS 13.0 statistic analysis program, and

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the differences between trials were detected by Tukey test (P < 0.05).

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RESULTS AND DISCUSSION

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Colloidal Performances of ZPHPs. As expected, ZPHPs possessed spherical shape and

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smooth surface (Figure 1B), more information can be seen in Figure 1S. Besides, the fresh

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ZPHPs dispersions were homogeneous, while a thin layer of particles deposited at the bottom

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after 24 h (Figure 1A), indicating indirectly the interaction between ZPHPs, which may

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facilitate the preparation of stable Pickering emulsions. Figure 1C shows that the ζ-potential

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of ZPs decreased from 48.8 mV at pH 3.0 to -11.3 mV at pH 6.0 with pI at about 5.6.

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Therefore, there was an electrostatic attraction between zein and pectin (anionic

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polysaccharide) at pH 3.0 to 5.6, but weak electrostatic repulsion happened at pH 6.0. Figure

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1D shows FT-IR spectra. Pectin exhibited a typical hydrophilic carbohydrate broad peak at

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3349 cm-1 assigned to O-H stretching vibrations34, a carbonyl stretching peak (C=O) at 1741

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cm-1 and carboxylate ion band (COO‒) at 1656 cm-1. In spectrum of zein, characteristic peaks

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of C=O stretching vibration at 1655 m-1 (Amide I) and N-H bending vibration at 1541 cm-1

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were observed. A broad peak at 3389 cm-1 attributed to hydroxy stretching vibration was

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shifted to 3367 cm-1 after the complex with pectin, indicating hydrogen bonds was formed

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between amide groups of glutamine in zein and carboxyl or hydroxyl groups in pectin. So far,

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we proved the hydrogen bonding and electrostatic interaction between zein and pectin.

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Certainly, hydrophobic interactions also existed between methoxy groups of pectin and

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hydrophobic groups of zein. These interactions may be used as key factors for regulating the

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colloid properties and wettability of ZPHPs.

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To verify our hypothesis, we studied the particle size, surface charge and wettability of

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ZPHPs. As shown in Table 1, particle size increased from 116 nm (ZPs) to 619.9 nm

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(ZPHPs-1), but decreased to 583nm (ZPHPs-2), then increased to 659.1 nm (ZPHPs-5),

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indicating pectin influenced the self-assembly process of zein, not simply formed a coating at

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surface. We also investigated the effect of pH on particles size which increased from 536 nm

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at pH 3.0 to 691nm at pH 5.0, but then decreased to 585 nm at pH 6.0, the opposite

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electrostatic interactions between zein and pectin at pH 6.0 and 3.0 to 5.0 may be responsible

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for this varied trend. Even more interestingly, ζ-potential of ZPHPs were similar to that of

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pectin within pH 3.0-6.0 (Figure 1C), which meant ζ-potential of ZPHPs were dominated by

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pectin indicating once again that the sugar chains of pectin extended to the surface of zein

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particles rather than embedded in hybrid particles or simply wrapped zein particles up. The

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above results stated clearly the interactions between zein and pectin (hydrogen bond,

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electrostatic and hydrophobic interaction) affected the self-assembly nucleation process of

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ZPHPs.

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Wettability. There's no doubt, wettability of solid particles has a profound effect on

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fabrication and properties of Pickering emulsions. The partial wettability facilitates efficient

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adsorption, assembly and packing of colloidal particles at the interface to form steric

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hindrance against the coalescence19. Contact angle measurement is a straightforward

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approach to evaluate the wettability of colloid particles. The wettability of ZPHPs as a

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function of pectin-to-zein ratios and pH were investigated by measuring the θ of ZPHP films

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that were immersed in purified corn oil. For pristine zein particles, three-phase contact angles

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(θ) at corn oil-water interface was 110.64° (Table 1) due to its hydrophobic nature. Similar

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result was observed in our previous work19. The decrease in the θ of ZPHPs proved

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successful surface modification of overly hydrophobic ZPs with hydrophilic pectin (56.68°).

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The θ of ZPHPs-1, ZPHPs-2 at pH 3.0-5.0, ZPHPs-5 were 84.43° to 87.04°. In contrast,

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ZPHPs-2 at pH 6.0 was a little more hydrophilic with θ of 76.32°, which may be associated

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with the electrostatic repulsive between zein and pectin at pH 6.0. The energy (E) needed to

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remove a particle from the interface is given by Eq. (1).

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E = πr2γow(1 - |cosθ|)2

(1)

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Where r is the radius of solid particle, γow is the surface tension of the oil-water interface,

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and θ is the contact angle. Based on the formula35, 36, the particles with too high or low θ are

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not suitable for used as stabilizer to form Pickering emulsions. The contact angle of particles

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around 90° is the ideal choice.

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Therefore, hybrid particles prepared from zein and pectin via hydrogen bond, electrostatic

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and hydrophobic interaction with ideal contact angles close to 90° exhibited promising

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potentials as a stabilizer for Pickering emulsions development. In this section, the hydrogen

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bond, electrostatic and hydrophobic interaction between zein and pectin was used to

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manipulate colloid properties and wettability of ZPHPs, some of which may affect the

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following interfacial self-assembly and structure formation, thus contributing to the

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development and performance of HIPEs.

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Adsorption of ZPHPs to the Interface. Particle properties, e.g., structural flexibility,

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surface charges and wettability significantly influence the adsorption and/or packing

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behaviors of particles at oil-water or air-water interface. The LB films stabilized by ZPHPs at

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air-water interface were characterized by FE-SEM. Typical microstructures are shown in

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Figure 2. For ZPs, its positively charged and overly hydrophobic nature facilitated interfacial

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adsorption and assembly procedure forming large aggregates, a film not spherical particles

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was observed in higher magnification, thus it can also be said that zein particles fused into

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films at interface (Figure 2a), which was also reported by Zou et al.37 This kind of interfacial

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structure stabilized by large zein aggregates and low coverage might be the direct cause of

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the destabilization of Pickering HIPEs. When pectin was added, more orderly network

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consisting of continuously linked particles was captured (Figure 2b and c). In addition, the

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network architecture based on scattered spherical particles was more clearly observed with

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increasing pectin/zein ratios. On the one hand, pectin adjusted the wettability of ZPHPs

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(Table 1), on the other hand, extended sugar chains of pectin formed a protective layer

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around the surface of zein particles. More pectin offered stronger electrostatic and steric

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hindrance, restricting agglomerate behavior of hydrophobic zein particles. Lastly, the

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interaction between zein and pectin weakened hydrophobic interaction between zein

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molecules affecting the adsorption of ZPHPs at the interface. These factors together

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promoted diffusion of ZPHPs at interface forming a more compact and interconnected

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interfacial structure. Surprisingly, ZPHPs-2 at pH 6.0 formed similar interfacial structure with

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ZPs, irregularly distributed aggregates of particles on mica sheets were caught by SEM

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(Figure 2d). Pectin desorbed from the surface of zein particles due to electrostatic repulsive

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interaction between zein and pectin molecules at pH 6.0 (Figure 1C), thus the appearance of

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large aggregates of ZPs via hydrophobic interactions may be the reasons for this

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phenomenon.

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Figure 3 compares the difference of surface tension (γ) between ZPHPs with various

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zein/pectin ratios (Figure 3A) and different pH (Figure 3B). The value of interfacial tension

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decreased with time indicating ZPHPs continually adsorbed at the oil-water interface. In

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addition, positively charged and overly hydrophobic ZPs easily adsorbed at interface, that

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arises from the negatively charged nature of air-water or oil-water interface38, high protein

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concentrations (0.5%) we used in this experiment may also contribute to this result. It is

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unexpected that the addition of pectin hardly affected the interfacial tension, this phenomenon

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was consistent with the research of Sahar & Ashkan21, which meant adsorption process of

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ZPHPs at oil-water interface was dominated by zein particles. We also investigated the

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potential influence of pH on adsorption of ZPHPs at the oil-water interface. Unexpectedly, at

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pH 3.0, ZPHPs-2 with least negative charge reduced γ to a lowest value (about 10 mN/m)

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within shortest time (about 2000s), while ZPHPs-2 possessed highest γ at pH 5.0 and 6.0, and

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γ of pH3.8 and 4 were in between. Obviously, the ability of ZPHPs to adsorb at interface was

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impacted by pH conditions. Therefore, the interaction between zein and pectin affected the

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packing behaviors of ZPHPs at interface and the formation of interfacial structure, both of

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which are crucial to the physiochemical properties of HIPEs.

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Storage and Centrifugation Stability of Pickering HIPEs. For the first time, we

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successfully prepared o/w Pickering HIPEs stabilized by zein/pectin hybrid particles, which

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was confirmed by the fact that HIPEs were evenly dispersed in the water but they remained

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intact in the oil (Figure 2S). More surprisingly, HIPEs remained stable at room temperature

280

for at least 1 month. To illuminate the effect of pectin and pH on formation and stability of

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Pickering HIPEs, experiments of emulsion’s storage and centrifugation stability were carried

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out. As shown in Figure 4, ZPs were unable to stabilize emulsions with high internal fraction,

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which may be related to large agglomerates formed by overly hydrophobic ZPs in bulk phase

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and at the interface. Thus, dispersed oil droplets with low surface coverage were prone to

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coalesce, eventually caused phase separation. HIPEs stabilized by pristine pectin were also

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unstable, indicating that pectin alone was not a good choice for HIPEs development.

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Encouragingly, Pickering HIPEs stabilized by ZPHPs-1, ZPHPs-2, ZPHPs-5 remained stable

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and self-supported after 30 days of storage. Maybe, pectin manipulated assembly behavior of

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zein particles at the interface, facilitating formation of more ordered interfacial structure and

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improving coverage rate (Figure 2). Moreover, the interactions between zein and pectin

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molecules, between oil droplets and between droplets and particles in bulk phase also played

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an important part in the stabilization of oil droplets. Furthermore, pectin remained in bulk

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phase as thickening agent, and it may increase viscosity and help to capture oil droplets in the

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continuous phase mesh, these factors worked together, eventually preventing coalescence of

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oil droplets and forming stable emulsions with high internal phase. ZPHPEs-2 fabricated at

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different pH is shown in Figure 5. Figure 5C clearly shows that, ZPHPEs-2 at pH 3.0 - 4.0

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on the bottom of inverted petri dish were stable and kept self-supporting ability after 30 days

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of storage, but slight separation of oil and water phase happened at pH 5.0, and the HIPEs

299

completely destabilized at pH 6.0. When the pH of ZPHPs-2 was adjusted from original value

300

(3.8) to 6.0, electrostatic repulsion between zein and pectin molecules at pH 6.0 (Figure 1C)

301

resulted in desorbed behavior of pectin from the surface of zein. Neither free pectin

302

molecules nor zein particles can stabilize HIPEs (Figure 4). Additionally, different

15



303

electrostatic repulsion between charged particles and like-charged ionic surfactants promoted

304

interfacial adsorption39, on the contrary, similarly charged pectin assisted flocculation of zein

305

particles at pH 6.0 forming large aggregates in bulk phase and at the interface (Figure 2) thus

306

destabilize emulsion.

307

Centrifugation process is another way to characterize the stability of HIPEs. As shown in

308

Figure 6, results of centrifugation were almost the same with that of storage experiments.

309

Pickering HIPEs stabilized by pectin were “broken up”, top oil layer was observed.

310

ZPHPEs-1 at pH 3.8 and ZPHPEs-2 at pH 5.0 were also unstable under centrifugation at

311

10000 rpm × 2 min, while ZPHPEs-5 at pH 3.8 and ZPHPEs-2 at pH 3.0, 3.8, 4.0 were still

312

stable. These results indicated once again that the stability of Pickering HIPEs stabilized by

313

ZPHPs were dependent on the pectin/zein ratio and pH, which play crucial roles on the

314

interfacial structure formed by ZPHPs. Comparatively, HIPEs prepared at high pectin/zein

315

ratio and acidic pH condition possessed more complete and ordered interfacial structure

316

(Figure 2), thus they are stable against centrifugation stress.

317

Microstructure and Droplet Size of ZPHPEs. Microstructural characteristics including

318

interfacial architecture, particle location, and droplet flocculation determined the performance

319

of Pickering HIPEs. We investigated the microstructure of ZPHPEs by CLSM and optical

320

microscopy. A series of typical images are shown in Figure 7 and Figure 3S. ZPHPEs

321

captured by optical microscopy showed representative shape of deformed droplets resulting

322

from compressed packing of drops40 at high internal phase fraction (80%). Purple signal of

323

ZPHPs dispersed around green corn oil droplet reflected again that HIPEs prepared were o/w

324

emulsions and no phase inversion occurred. Intense and brilliant purple signal representing

16


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325

robust and ordered interfacial ZPHPs layer reflected high coverage rate confirming effective

326

adsorption of ZPHP at interface, hindering the coalescence of oil droplets and facilitating the

327

physical stability of ZPHPEs. Meanwhile, protective ZPHPs interfacial layer functioned as

328

structural bones, supported the stacking of oil droplets to form a 3D network contributing to

329

self-supporting properties of HIPEs (Figure 7F). However, oil droplets of ZPHPEs-2 at pH

330

6.0 coalesced into large droplets arrested by CLSM (Figure 7E) intuitively indicating that

331

HIPEs developed at pH 6.0 were unstable from microcosmic perspective, which was in

332

accordance with the storage experimental results. Combined microstructures, stabilities of

333

HIPEs with interfacial adsorption and packing of ZPHPs, we found that Pickering HIPEs

334

stabilized by ZPHPs at high pectin/zein ratio and acid condition with robust and ordered

335

interfacial structure leading to high interfacial coverage rate and sturdy net backbone, thus

336

possessed better stability than those at lower pectin/zein proportion or higher pH value. In

337

particular, the rope shaped ZPHPs in bulk phase connects adjacent oil droplets to develop

338

percolating 3D network architecture which contributed to the stability and self-supporting

339

attribute of ZPHPEs, and endowed the HIPEs with viscoelasticity.

340

Mono-modal distribution of drop diameter were observed for ZPHPEs (Figure 7B and C),

341

and the center position of peak moved to the left obviously as pH decreased or the pectin/zein

342

ratio increased. In other words, average diameter (D3,2) of ZPHPE (Table 2) decreased with

343

increased pectin, which may be related to stronger electrostatic interaction and steric

344

hindrance

345

because the change of pH directly affected the ability of ZPHPs to stabilize the interface and

346

the intermolecular interactions between like or unlike molecules. In addition, pectin/zein ratio

41

. In addition, D3,2 of ZPHPEs-2 increased with increasing pH value, possibly

17



347

as well as pH were closely interrelated to interfacial structure, further affected the droplet

348

size.

349

Rheology of Pickering HIPEs. Figure 8 shows storage modulus (G’) and loss modulus

350

(G”) of ZPHPEs as a function of stress at different pectin/zein ratio (A) and pH (B). G’/ G”

351

increased with pectin content resulting from the more efficient rearrangement of ZPHPs at

352

interface into more ordered network (Figure 2), and stronger steric force provided by pectin

353

endowed ZPHPEs with stronger viscoelasticity. Similarly, pH condition affected interfacial

354

packing behaviors of ZPHPs as well as interactions between zein and pectin molecules in

355

bulk phase and at interface, thus leading to stronger network architecture (Figure 7) further

356

endowing ZPHPEs with better viscoelasticity at lower pH. G’ was larger than corresponding

357

G” at test conditions revealed elasticity-dominated attributes of ZPHPEs. A crossover point at

358

higher stress was observed in amplitude sweeps indicating stronger structure formed at higher

359

pectin content or lower pH condition. Frequency sweep (Figure 4S) also verified that

360

ZPHPEs prepared at these conditions possessed sturdier network, which was well caught by

361

CLSM (Figure 7). In addition, ZPHPEs at different pH showed shear thinning response

362

demonstrating breakdown of original structure under tested conditions. To further investigate

363

structure-recovery properties of ZPHPEs, a three-interval time test in rotation were performed.

364

Thixotropic behavior (Figure 8D) showed that percent recovery42 (viscosity at interval 3

365

divide by that of interval 1) of ZPHPEs-2 at pH 3.0, 3.8, 4.0, 5.0 were 94.1%, 93.5%, 91.8%,

366

91.0%, respectively. According to previous studies43, 44 percent recovery larger than 70% was

367

regarded as good thixotropic recovery. Therefore, ZPHPs-stabilized Pickering HIPEs

368

possessed excellent thixotropic recovery, even virtually as good as mayonnaise (99.28%). To

18


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369

sum it up, rheological analysis further confirmed the results of stability experiments and

370

microstructure that HIPEs prepared were viscoelastic and self-supporting. Thus, Pickering

371

HIPEs developed with excellent thixotropic property owned promising application prospect

372

in food industry.

373

Protective Effect of ZPHPEs on Curcumin under Ultraviolet Radiation. Curcumin

374

with beneficially biological activities has promising application in pharmaceuticals and foods.

375

However, curcumin is sensitive to ultraviolet radiation and is prone to degradation, which

376

limits its wide application. In this work, we used curcumin as a model active compound to

377

check out the protective effects of ZPHPEs under ultraviolet (UV) radiation. Bulk oil and

378

emulsions stabilized by Tween 20 were tested as the control. Curcumin was degraded rapidly

379

in bulk oil or Tween 20-stabilized emulsion when compared with that in ZPHPEs (Figure 9).

380

In the end, the residual percentage of curcumin in oil, Tween 20-stabilized emulsion and

381

ZPHPEs were 1.74%, 0.06%, and 15.11%, respectively, after 61 h of UV radiation at 340 nm.

382

In oil, curcumin was readily exposed to the UV light absorbing highest radiation energy

383

accelerating degradation, and Tween 20 dynamically adsorbed on the oil droplet surface, but

384

it did not offer effective protection for curcumin. In contrast, ZPHPs packed into an ordered

385

interfacial layer which shielded curcumin from radiation. Moreover, close packed 3D

386

network of oil drops in Pickering HIPEs (Figure 7) delayed energy transmission, and aroma

387

amino acid residues of zein helped to absorb energy. These factors worked together provided

388

protective effect for curcumin.

389

Lipid Oxidation of ZPHPEs. To figure out exactly the oxidative stability of ZPHPEs, we

390

subjected these samples to thermally accelerated storage at 40 °C for 21 d, the extent of lipid

19



391

oxidation was measured by quantifying the content of lipid hydroperoxide (LH) and

392

malondialdehyde (MDA). LH in oil reached 22.24 mmol/kg oil after a 21-d period, curcumin

393

in bulk oil dramatically delayed LH production and it finally reached 13.98 mmol/kg oil,

394

which confirmed the validity of curcumin to depress oil oxidation. It's astonishing, Pickering

395

HIPEs stabilized by ZPHPEs-2 at pH 3.8 (12.16 mmol/kg oil) without curcumin displayed a

396

slower oxidation rate than that of curcumin-loaded bulk oil (Figure 10A). That is to say, the

397

protect effect of interfacial ZPHPs layer for oil was comparable to curcumin under our

398

experimental conditions, it was without doubt addition of curcumin in ZPHPEs further

399

limited lipid oxidation. Compared to pH 3.0 (7.19 mmol/kg oil) and 3.8 (7.67 mmol/kg oil),

400

ZPHPEs at pH 5.0 (10.43 mmol/kg oil) was found to be less effective in depressing oxidation.

401

Perhaps, ZPHPEs at lower pH accompanied by more favorable interfacial structure and

402

intermolecular interaction in bulk phase thus possessing better stability and viscoelasticity,

403

which retarded lipid oxidation. Iron is well-known to accelerate lipid oxidation through

404

Fenton reaction as well as promote the formation of peroxyl and alkoxyl radicals45, 46. The

405

concentration of iron in blood serum of human adults under normal physiological conditions

406

ranges from 10 to 35 µM. In this work, 0.03 mM and 0.3 mM Fe2+ were tested representing

407

the physiological and the high iron condition, respectively. LH concentration in ZPHPEs-2

408

containing curcumin at pH 3.8 with 0.03 mM Fe2+ was only 1.23 times of that without Fe2+

409

after storage for 7 days, but lipid oxidation was accelerated under 0.3 mM Fe2+ condition.

410

Simultaneously, a 3-day lag phase of oil oxidation was observed (Figure 10A), which was

411

totally different with previous research reported by Kargar et al.47, who observed higher

412

oxidation rate throughout the process at 0.14 mM of ferrous ion in water phase, our results

20


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413

indicating the satisfactory oxidation resistance of ZPHPEs. The MDA trend of all samples

414

were similar to that of LH, most MDA (17.87 µmol/kg oil) was detected in ZPHPEs-cur at

415

pH 3.8 with 0.3 mM Fe2+, MDA in oil was 14.89 µmol/kg oil after 21 d of incubation at

416

40 °C, curcumin-loaded in oil also decreased MDA to 10.62 µmol/kg oil. MDA in ZPHPEs

417

with curcumin at pH 3.8 under 0.03 mM Fe2+ (12.84 µmol/kg oil) lay between

418

curcumin-loaded bulk oil and bulk oil. Compared to biopolymer stabilized conventional

419

emulsions33, MDA content in our work was much lower indicating excellent oxidation

420

resistance of ZPHPEs. There were three main factors leading to these results. Firstly, ordered

421

interfacial ZPHPs layer partly hindered the contact between LH in oil droplets and

422

pro-oxidant in water phase. Secondly, 3D oil drop network in structured HIPEs was not

423

conducive to molecular motion and energy transfer. Finally, the balance between chelation of

424

negatively charged ZPHPs at bulk phase and attraction of ZPHPs at interface to pro-oxidant

425

was also important to the antioxidation of the HIPEs.

426

Schematic Illustration. We investigated the interaction between polysaccharide and

427

protein from an interface manipulation point of view, for the first time, intuitively revealed

428

the regulation of interfacial packing behavior of the zein particles by pectin through LB-SEM

429

technique, and linked it with the physiochemical performance of the HIPEs. Herein, a

430

schematic illustration (Figure 11) for the formation pathway of ZPHPs-stabilized Pickering

431

HIPEs is proposed to correlate the physiochemical properties of the ZPHPEs with their

432

interface frameworks, so as to elucidate the possible stable mechanism for HIPEs. For

433

positively charged and overly hydrophobic ZPs (Figure 11a), its strong intermolecular

434

hydrophobic interaction led to the formation of large aggregates both at the interface and in

21



435

bulk phase (Figure 2). Under this circumstance, oil droplet in HIPEs coalesced incessantly

436

into larger drops, eventually lost the stability and phase separation occurred (Figure 4).

437

When pectin was added to prepare hybrid particles (Figure 11b), it regulated the

438

self-assembly process of zein particles through hydrogen bonds, electrostatic and

439

hydrophobic interactions, thus manipulated the performance of the particles, including

440

potential, particle size and wettability. ZPHPs were hardly distributed in the aqueous phase

441

indicating less hydrophobic ZPHPs facilitating the adsorption and assembly at the interface to

442

produce robust and ordered interfacial architecture (Figure 2 and 7), oil droplets with high

443

interfacial coverage rate relied on structural bones of ZPHPs stacking into a 3D network,

444

which endowed Pickering HIPEs with a series of ideal physicochemical properties, such as

445

viscoelasticity, admirable thixotropy, and oxidation stability. When pH of ZPHPs continued

446

to increase and exceeded the isoelectric point of zein (Figure 11c), it is electrostatic repulsive

447

force with pectin leading to pectin desorb from the surface of ZPs, the disadvantageous

448

membranous interface structure was formed again under this condition (Figure 2). Therefore,

449

Pickering HIPEs developed at pH 6.0 was unstable.

450

In conclusions. The interaction between zein and pectin was used to manipulate the

451

self-assembly nucleation process of zein constructing effective food-grade hybrid particulate

452

stabilizer for Pickering-HIPEs. Our results showed that the physicochemical properties of

453

ZPHPEs were related to the interfacial adsorption/packing behavior of ZPHPs, which was

454

affected by zein-pectin interaction and pH condition. In brief, ZPHPEs prepared at high

455

pectin/zein ratios or low pH value with robust and ordered interface architecture facilitating

456

the stack of oil droplets into a 3D network, which endowed HIPEs with satisfactory

22


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Page 23 of 45


457

viscoelasticity, thixotropy, oxidation/storage stability. This work provides a feasible way to

458

successfully prepare Pickering HIPEs, which will be of great guiding significance to the

459

development of Pickering HIPEs stabilized by food grade particles, also benefit the wide

460

application of HIPEs in food, cosmetics, chemical and pharmaceutical industry.

461

ASSOCIATED CONTENT

462

Supporting Information. The Supporting Information is available free of charge.

463

Representative SEM images of the ZPHPs, Photos of HIPEs, typical microscopy images of

464

ZPHPEs, Frequency sweep of ZPHPEs.

465

ACKNOWLEDGEMENTS

466

This work was supported by The Project granted by the National Natural Science

467

Foundation of China (31471628; 31471694). We also appreciate the financial support by the

468

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

469

ABBREVIATIONS

470

ZPHPs, zein/pectin hybrid particles; ZPHPEs, zein/pectin hybrid particles-stabilized

471

emulsions; ZPs/ZPHPs-0, ZPHPs-1, ZPHPs-2, and ZPHPs-5: zein colloid particles with

472

pectin-to-zein mass ratios of 0:10, 1:10, 2:10, and 5:10, respectively; LB-SEM,

473

Langmuir-Blodgett and scanning electron microscope; LH, lipid hydroperoxide; MDA,

474

malondialdehyde; CLSM, confocal laser scanning microscope; TBA, thiobarbituric acid;

475

AFM, atomic force microscopy.

476

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29


approach.


603

Figure captions

604

Figure 1. Visual appearance (A) of ZPs (a), ZPHPs-1 (b), ZPHPs-2 (c), ZPHPs-5 (d)

605

dispersions at pH 3.8; e, f, g and h were a, b, c and d after 24 h of storage, respectively. AFM

606

images (B) of ZPHPs-2 at pH 3.8. zeta potentials (C) and FT-IR spectra (D) of ZPHPs and

607

pectin.

608

Figure 2. LB-SEM images of air-water surfaces stabilized by ZPHPs with zein/pectin ratios

609

of 1:0.0 (a, a1, a2), 1:0.1 (b, b1, b2), 1:0.2 (c, c1, c2) at pH 3.8 and 1:0.2 at pH 6.0 (d, d1, d2)

610

under a fixed surface pressure of 25 mN/m. a1/a2, b1,/b2, c1/c2 and d1/d2 are the digital

611

magnification images of a, b, c and d.

612

Figure 3. Surface tension (γ) of oil-water interface stabilized by ZPHPs as a function of time

613

with various zein/pectin ratios (A) and different pH (B).

614

Figure 4. Photographs of Pickering HIPEs stabilized by ZPs (a), ZPHPs-1 (b), ZPHPs-2 (c),

615

ZPHPs-5 (d), and pectin (e: 0.1%, f: 0.2%, g: 0.5%, w/v). Panel A: fresh emulsions, Panel B:

616

after 1 months of storage, oil phase fraction of a was 74%, the others were 80%, pH were the

617

original value (pH 3.8) without regulation.

618

Figure 5. Visual appearance of Pickering HIPEs stabilized by ZPHPs-2 at different pH

619

values. Panel A: fresh emulsions, Panel B: after 1 months of storage. Panel C: emulsions

620

(after 1 months of storage) on the bottom of inverted petri dish.

621

Figure 6. Visual appearance of Pickering HIPEs after centrifugation, Panel A: Pickering

622

HIPEs stabilized by ZPHPs-1 (a), ZPHPs-2 (b), ZPHPs-5 (c) at pH 3.8, pectin (d: 0.2%, e:

623

0.5%, w/v), Panel B: HIPEs stabilized by ZPHPs-2 at pH 3.0 (f), 4.0 (g), 5.0 (h).

624

Figure 7. Visual appearance (A) and particle size distribution (B and C) of ZPHPEs at

30


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625

different pH or pectin/zein ratio. Typical CLSM images of fresh ZPHPEs-2 at pH 3.0 (D and

626

F) and pH 6.0 (E), D and E are 2D images, F is 3D images. Pectin, zein and oil was stained

627

with Calcofluor white (color blue), Nile blue (color red) and Nile red (color green),

628

respectively, pectin and zein location is shown on the left side and in the middle, while the

629

right side shows the colocation of corn oil and ZPHPs.

630

Figure 8. Storage modulus (G’) and loss modulus (G”) of ZPHPEs as a function of stress at

631

different pectin/zein ratio (A) and pH (B), viscosity versus shear rate curves (C) and

632

thixotropic recovery (D) for ZPHPEs-2 at different pH.

633

Figure 9. Residual curcumin levels in bulk oil, Tween 20-stabilized emulsion and ZPHPEs-2

634

under UV radiation treatment.

635

Figure 10. Evolution of lipid hydroperoxides (A) and MDA (B) under accelerated storages at

636

40 °C for 21 days.

637

Figure 11. Schematic illustration for the formation of ZPHPs-stabilized Pickering HIPEs

638

proposed to relate physicochemical properties of the HIPEs with their interface frameworks.

639

The letters abc represent interfacial structure formed by ZPs at pH 3.8 (a), ZPHPs-2 at pH 3.8

640

(b), and ZPHPs-2 at pH 6.0 (c).

31



Page 32 of 45

Table 1 Particle size, zeta potential and contact angle of ZPHPs at different pH and pectin/zein ratios Samples

Pectin/Zein ratio

pH

Particle size / nm

Zeta potential / mV)

Contact angle / deg

ZPs

0:1

3.8

116.11 ± 0.50f

51.20 ± 4.24a

110.64 ± 1.39a

ZPHPs-1

1:10

3.8

619.92 ± 4.74c

-14.00 ± 0.30c

84.43 ± 1.36b

ZPHPs-5

5:10

3.8

659.13 ± 9.30b

-23.50 ± 0.87de

85.50 ± 0.48b

3.8

583.74 ± 10.11d

-22.20 ± 0.70d

86.44 ± 2.76b

3.0

536.02 ± 10.32e

-6.61 ± 0.55b

86.48 ± 1.20b

4.0

609.82 ± 7.50c

-25.47 ± 0.40e

87.04 ± 1.43b

5.0

691.03 ± 12.16a

-33.67 ± 0.32f

85.67 ± 1.70b

6.0

585.42 ± 12.44d

-38.33 ± 0.58g

76.32 ± 1.91c

-

-

-

56.68 ± 0.32d

ZPHPs-2

pectin

2:10

1:0

Different superscript letters in the same column indicate the significant difference (p

Pectin Hybrid Particle-Stabilized Pickering High

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