Fabrication and Characterization of Antioxidant Pickering Emulsions

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Fabrication and Characterization of Antioxidant Pickering Emulsions Stabilized by Zein/Chitosan Complex Particles (ZCP) Lijuan Wang, Ya-Qiong Hu, Shou-Wei Yin, xiaoquan Yang, Furao Lai, and Si-Qi Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505227a • Publication Date (Web): 30 Jan 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

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Fabrication and Characterization of Antioxidant Pickering Emulsions Stabilized

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by Zein/Chitosan Complex Particles (ZCP)

4 Li-Juan Wang†‡,Ya-Qiong Hu†, Shou-Wei Yin†*, Xiao-Quan Yang†*, Fu-Rao Lai†, Si-Qi Wang†

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†

Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, PR China

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‡

College of Grain Engineering and Technology, Shenyang Normal University, Shenyang 110034, PR China

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Running title: Fabrication and characterization of antioxidant Pickering emulsions

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

*Co-corresponding author Yang, X. Q. Phone: +86-2087114262. Fax: (+86)-20-87114263. E-mail: [email protected]

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ABSTRACT

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Lipid peroxidation in oil-in-water emulsions leads to the rancidity and carcinogen formation.

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This work attempted to protect lipid droplets of emulsions from the peroxidation via manipulating

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the emulsions’ interface framework using dual-function zein/CH complex particles (ZCP). ZCP

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with intermediate wettability was fabricated via a simple antisolvent approach. Pickering emulsions

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were produced via a simple and inexpensive shear-induced emulsification technic. ZCP was

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irreversibly anchored at the oil–water interface to form particle-based network architecture therein,

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producing ultrastable o/w Pickering emulsions (ZCPE). ZCPE was not labile to lipid oxidation,

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evidenced by low lipid hydroperoxides and malondialdehyde levels in the emulsions after thermally

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accelerated storage. The targeted accumulation of curcumin, a modal antioxidant at the interface

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was achieved using the ZCP as interfacial vehicles, forming antioxidant shells around dispersed

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droplets. The oxidative stability of ZCPE was further improved. Interestingly, no detectable hexanal

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peak appeared in headspace gas chromatography of the Pickering emulsions. The novel interfacial

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architecture via the combination of steric hindrance from ZCP-based membrane and interfacial

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cargo of curcumin endowed the emulsions with the favorable oxidative stability. This study opens a

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promising pathway for producing antioxidant emulsions via the combination of Pickering

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stabilization mechanism and interfacial delivery of antioxidant.

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KEYWORDS: Pickering emulsion; dual-functional ZCP; interfacial delivery; oxidant stability;

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microstructure, physical performance.

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INTRODUCTION

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Oil-in-water emulsions are important ingredients of a wide range of functional foodstuffs,

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consumer care products, as well as drugs.1 Lipid oxidation of the emulsions is a major concern for

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food manufactures and consumers since it leads to food deterioration and carcinogen formation.2

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Lipid oxidation of emulsified oil in a diphasic system (i.e. oil-in-water emulsion) proceeds faster

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when compared with bulk oil due to the high oil-water interface area. 1 The emulsion droplet surface,

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coming into close proximity with pro-oxidants of the aqueous continuous phase, is the place where

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the chain reaction of lipid peroxidation is usually initiated and prevalent. Lipid hydroperoxides, the

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primary oxidant products are surface-active compounds and tend to accumulate at the oil−water

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interface of emulsion droplets.3 Transition metals, a kind of the prevailing pro-antioxidants initiate

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the lipid peroxidation via the breakdown of lipid hydroperoxide into free highly reactive radicals

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such as alkoxyl and peroxyl radicals, further promoting lipid oxidation in oil-in-water emulsions.3

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Therefore, engineering the interface makeup of emulsions to fabricate physical barrier that

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segregates hydroperoxides from pro-oxidants, i.e. iron, one of the major pro-oxidants, is a

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promising strategy to enhance the oxidation stability of the emulsions.

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The droplet interfacial characteristics (charge, composition and thickness) are the key factors that

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can impact iron’s ability and oxidant stability of emulsions.4 The utilization of electrostatic

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screening effect between positively charged droplet surfaces and positive iron in the continuous

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phase became an important strategy to retard oxidative degradation of lipid in emulsions.5

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Layer-by-layer (LBL) electrostatic deposition technique was utilized to encapsulate emulsion

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droplets to improve their physical and oxidation stability.1,6.7 However, LBL technique has some

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limitations such as high tendency for the droplets to flocculate.6 The size of surfactant polar head

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groups determined to some extent the thickness and strength of interfacial layer of oil-in-water 3



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emulsions, therefore influenced the oxidative stability of the emulsions.8,9 Manipulating interfacial

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structures is a promising strategy to enhance oxidant stability of emulsified oil.

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Fabrication of Pickering emulsions is a promising strategy to enhance the oxidative stability of

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emulsified lipid via the formation of particle-based interfacial architecture. Solid particles tend to

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irreversibly adsorb at the droplet surface and build a physical barrier against pro-oxidants. The high

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resistance to coalescence is a major benefit of Pickering emulsion.10,11 Recently, Pickering

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emulsions have arisen extensively interest due to their promising potential for texture modification,

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calorie reduction and bioactive compound encapsulation and delivery.12 The focus of Pickering

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emulsions is gradually shifting from using inorganic particles to adopting particles of biological

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origin for producing the emulsions with food-grade status.13 However, it is still a key technological

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challenge to manufacture Pickering emulsions using edible colloid particles, especially protein

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particles.14-16,21 Limited information is available on Pickering emulsions stabilized by the particles

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of biological origin, such as cellulose nanocrystals17, chitin nanocrystals18, phytosterol19, modified

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starch20, zein colloid particles.16,21 Those works focused mainly on fabrication and physical

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performances of the emulsions, little is known about their oxidant stability.

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Zein, a prolamin, is the major storage protein in corn or maize. It contains sharply defined

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hydrophobic and hydrophilic domains at its surface, and is capable of self-assembly to form a wide

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variety of mesostructures.22 Zein particles have been studied for delivery and encapsulation23, but

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their usages as Pickering emulsifier are still limited. Theoretically, the Pickering emulsifiers should

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remain insoluble in both phases, and keep intact over the lifetime of the emulsions.12,16 Therefore,

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zein particles with partial wettability possesses promising potential in the manufacture of

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food-grade Pickering emulsions. de Folter et al., reported that zein particles (approximate 80 nm)

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were able to stabilize oil-in-water emulsions, but emulsion stability should be improved since free 4


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oil appeared after 3 days storages.21 Zein has a high proportion of apolar amino acid (> 50%), as a

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result the surface of zein particles seem quite hydrophobic. They tend to aggregate in the aqueous

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circumstance and are hard to arrange at the oil–water interface, leading to unstable Pickering

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emulsions. Some pertinent strategy should be utilized to tune the surface wettability of zein

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particles so as to form stable Pickering emulsions, e.g., the interaction between proteins and

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polysaccharides.

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Curcumin, a hydrophobic polyphenol with yellow color, is a major constituent extracted from the

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dried root of turmeric, the rhizome of the herb Curcuma longa.24 It is an effective and potent

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antioxidant.25 In addition, curcumin is a fluorophore, and its fluorescence can be monitored by

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exciting by an argon laser at 488 nm.26 Curcumin was encapsulated in silica nanoparticle stabilized

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Pickering emulsion to study its fat during the storage and simulated digestion. 27 In this paper, we

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demonstrate the fabrication of stable, soap-free and edible Pickering emulsions based on fully

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natural renewable resources using protein/polysaccharide complex particles. This work attempted to

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protect lipid droplets of emulsions from the peroxidation via manipulating the emulsions’ interfacial

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framework using Pickering mechanism and interface cargo of antioxidant. For this purpose,

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dual-function particles acting as Pickering emulsifier and interfacial transport vehicles of curcumin

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(a modal antioxidant) were fabricated via a simple co-assembly approach. We propose a novel

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strategy to targeted accumulation of antioxidants at the interface via dual-function particles, and

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compared the oxidative stability of encapsulated corn oil via either shell or core antioxidant.

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Pickering emulsions were characterized by confocal laser scanning microscopy (CLSM), particle

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size distribution, Zeta-potential and physical stability measurements. The oxidative status of the

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samples was assessed by monitoring the formation of primary and secondary products during

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thermally accelerated oxidation storage. In addition, a schematic illustration of the formation 5



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pathway of ZCP-stabilized antioxidant emulsions was proposed to correlate the oxidative stability

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of Pickering emulsions with their interfacial architecture.

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MATERIALS AND METHODS

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Materials. Zein (products no. Z 3625) and chitosan were purchased from Sigma Chemical Co.

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(St. Louis, MO, USA). According to the manufacturer, the molecular weight of the chitosan was

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approximately 500 kDa and the degree of deacetylation was 90%. Fluorescent dyes including Nile

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Red, Nile Blue A and fluoresceine 5(6)-isothiocyanate, mixed isomers (FITC) were obtained from

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Sigma–Aldrich, Inc. (St. Louis, MO, USA). Chitosan was labeled by FITC to check its distribution

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in emulsions. In short, the FITC solution (in methanol) was added to chitosan solution, and

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FITC-labeled chitosan was precipitated using 0.2 mol L−1 NaOH and obtained via the centrifugation

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(4000×g 15 min) after 1 h incubation at 40°C. The unreacted FITC was removed before

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freeze-drying by dialysis against deionized water under darkness until no free FITC were detected.

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1,1,3,3–Tetramethoxypropane, thiobarbituric acid and trichloroacetic acid were purchased from

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Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Corn oil was purchased from a local

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supermarket (Guangzhou, China). All other chemicals used were of analytical grade.

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Solution Preparation. Zein stock solutions was produced by dissolving 10.0 g of zein powder to

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100 mL of aqueous ethanol binary solvent (70 v/v%). Cucrmin-loaded zein solutions were prepared

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by adding curcumin to the preceding zein solutions at the curcumin-to-zein ratios of 1:100, 1:50 and

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1:20, respectively. Chitosan (CH) solutions were produced by dispersing 0.5 g of chitosan powder

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into 100 mL of 1% acetic acid under magnetic stirring overnight to facilitate chitosan hydration.

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Particle Synthesis. Dual-function ZCP (or ZCP-cur) was fabricated via a so-called antisolvent

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procedure. All operations were at room temperature. Curcumin-loaded zein solutions were trickled

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into CH solutions within 2 min under the shearing at 5000 rpm using an Ultraturax T25 6


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homogenizer. After shearing for another 10 min, the remaining ethanol in the particle dispersions

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was removed by an RV 10 digital rotary evaporator (IkA-Works Inc, Staufen, Germany). Finally,

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the zein and CH concentrations in the dispersion were 7% and 0.35%, respectively. The plain ZCP

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was produced by the same procedure without curcumin addition. Particle size, shape and ζ-potential

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of fresh ZCP and ZCP-cur dispersions were characterized prior to the emulsion preparation.

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Preparation of Pickering Emulsion. The preceding ZCP and ZCP-cur were utilized to prepare

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Pickering emulsions, ZCP stabilized emulsions (ZCPE) and ZCP-cur stabilized emulsions

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(ZCP-curE). All emulsions were prepared using an oil−water ratio of 50 : 50. In brief, 5 mL of corn

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oil was added to 5 mL of the particles suspensions in a glass vial and the resultant mixtures were

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sheared using an Ultraturax T25 homogenizer (IkA-Works Inc, Staufen, Germany) at 6000 rpm for

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5 min at room temperature to yield the Pickering emulsions. For ZCPE-cur, curcumin was

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incorporated in corn oil at curcumin-to-zein ratios of 1:100, 1:50 and 1:20, respectively, prior to the

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emulsion preparation.

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Zeta-potential Measurements. Zeta-potentials of the complex particle dispersions and Pickering

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emulsions were determined by a Zetasizer Nano (Malvern Instruments Ltd., Worcestershire, UK) at

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room temperature. Each value represents the average of at least three independent trials.

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Contact Angle Measurements. Three-phase contact angle (θ) measurements were measured

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using an OCA 20 AMP (Dataphiscis Instruments GmbH, Germany) equipped with a high-speed

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video camera. Colloid particles dispersions were freeze-dried to yield particle powders that were

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compressed to produce tablets with 13 mm in diameter and 2 mm in thickness. The tablets were

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then immersed in corn oil bath, and 2 µL of Milli-Q water was deposited gently on the tablets using

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a high-precision injector. The drop image was photographed using the high-speed video camera at a

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rate of 10 pictures per second, and the LaPlace−Young equation was utilized to fit the profile data 7



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of the droplet to calculate the θ. Measurements were averaged over at least ten droplets.

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Morphology observation. Topographic structures of ZCP were analyzed by a ZEISS Merlin

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Field emission scanning electron microscope (ZEISS, Oberkochen, German). The freeze-dried ZCP

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powers were utilized to investigate their morphological attributes, and they were loosely glued onto

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a conductive adhesive mounted on a stainless steel stage. Subsequently, they were coated with a

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thin (

Fabrication and Characterization of Antioxidant Pickering Emulsions

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