Novel bilayer emulsions co-stabilized by zein colloidal particles and

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Food and Beverage Chemistry/Biochemistry

Novel bilayer emulsions co-stabilized by zein colloidal particles and propylene glycol alginate. Part 1:Fabrication and characterization Yang Wei, Cuixia Sun, Lei Dai, Like Mao, Fang Yuan, and Yanxiang Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03240 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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Journal of Agricultural and Food Chemistry

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Novel bilayer emulsions co-stabilized by zein colloidal particles and propylene

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glycol alginate. Part 1:Fabrication and characterization

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Yang Wei, Cuixia Sun, Lei Dai, Like Mao, Fang Yuan, Yanxiang Gao*

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Beijing Advanced Innovation Center for Food Nutrition and Human Health,

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Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of

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Functional Food from Plant Resources, College of Food Science & Nutritional

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Engineering, China Agricultural University, Beijing, 100083, P. R. China

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10 11 12 13

*Corresponding author.

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Tel.: + 86-10-62737034

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Fax: + 86-10-62737986

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Address: Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083,

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China

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E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: In this study, both zein colloidal particles (ZCPs) and propylene glycol

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alginate (PGA) were simultaneously applied to prepare novel bilayer emulsions using

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the method of layer-by-layer (LBL) electrostatic deposition. The effects of different

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concentrations of PGA as well as incorporating sequences of ZCPs and PGA on

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physical stability and microstructure of bilayer emulsions were investigated.

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Furthermore, optical microscopy as well as confocal laser scanning microscopy

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(CLSM) showed that the oil droplets presented uniform spheres and a compact

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network appeared in bilayer emulsion. Compared to the Pickering emulsion stabilized

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by ZCPs alone, novel bilayer emulsions exhibited simultaneous and long-term

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stability against creaming, coalescence and Ostwald ripening due to the unique

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interface framework of a particle-polysaccharide hierarchical structure. Novel bilayer

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emulsions synergistically stabilized by collodial particles and biopolymers were

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designed by using interfacial engineering and a promising pathway was found to

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produce stable bilayer emulsions for the delivery of bioactive compounds.

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KEYWORDS: Zein collodial particles, Proplylene glycol alginate, Bilayer emulsion,

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Physical stability, Microstructure

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INTRODUCTION

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Low-molecular weight surfactants, amphiphilic proteins and hydrocolloids have

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been studied extensively to stabilize the traditional oil-in-water (O/W) emulsions for

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various applications in food industry. However, it is hard for a single emulsifier to


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produce uniform droplets and avoid droplet aggregation under various environmental

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stresses. A common strategy is to apply the LBL technique to fabricate a multilayer

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emulsion whose O/W interface composed of multiple layers of proteins,

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polysaccharides and/or lipids. In addition, the multilayer emulsions can be designed to

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protect and deliver bioactive ingredients from chemical degradation,1 and to release

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them in response to specific triggers.2

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The characteristics of droplets covered by multilayered biopolymers are largely

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dominated by the properties of the outer biopolymer layer (e.g. electrostatic properties

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and spatial structure ).3 Once the stability of outer biopolymer layer is influenced by

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environmental conditions, the emulsion might tend to be unstable (aggregation,

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coalescence & creaming). Compared to β-lactoglobulin stabilized nanoemulsions with

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the isoelectric point of 4.5-5.0, emulsion droplets coated by β-lactoglobulin-chitosan

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complexes have a better stability against aggregation, which was ascribed by the

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elevated electrostatic attraction and steric repulsion between droplets.4 However, the

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positive charge of chitosan was decreased at a relatively high pH value (>6.5) and

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tended to be water-insoluble, and desorbed from interfaces of droplets and led to the

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destabilization of bilayer emulsions at neutral pH.

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Surfactants or amphiphilic biopolymers are applied to stabilize emulsions by

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lowering interfacial tension, while soild particles can stabilize Pickering emulsions by

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providing a steric hinderance against droplet coalescence.5 Due to the high binding

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energy of solid particles at the interface, Pickering emulsions with an oil droplet



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diameter above 100 µm can also exhibited a long-term stability.6-8 The formation of

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Pickering emulsions is usually affected by particle properties like wettability, size,

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shape, surface property and concentration.9 When particles have a proper wettability

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(approximately 90 °) and are bigger than a certain size (around 10 nm), their

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adsorption at oil-water interface is almostly irreversible because the desorption energy

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of particles reaches several thousand kT.10 In contrast, the desorption energy of

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low-molecular weight surfactants can be less than 10kT.11 Meanwhile, Pickering

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emulsions have attracted a widely interest due to potential applications in texture

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modification, calorie reduction, and bioactive compound encapsulation and delivery

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in the future.1 Many studies have studied serveral kinds of bio-particles capable of

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fabricating Pickering emulsions, such as cellulose nanocrystals,2 chitosan,12 zein, 13

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kafirin, 14 gliadin15 and soy protein.16 However, there are also some drawbacks of

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Pickering emulsions during practical applications. On one hand, it’s hard to find

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suitable biocompatible particles with a great wettability to stabilize emulsions. Many

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biopolymer-based nanoparticles are too hyrophiphilic or hydrophobic to fabricate

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stable Pickering emulsions. On the other hand, many researchers have reported a lipid

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droplet-enriched cream layer and a serum layer gradually occurs because of the

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gravitational separation of emulsions.17 As negative characteristics of Pickering

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emulsions, creaming and serum release have serious impact on commercial

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applications of Pickering emulsions.


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Zein，main storage proteins of maize, approved for oral use by Food and Drug

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Administration (FDA)18 can be easily reconstructed into nanoparticles to deliver

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bioactive ingredients due to its inherent hydrophobicity. Propylene glycol alginate

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(PGA) belongs to one distinct group of surface-active food grade polysaccharides

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because of its propylene glycol groups,20 which has many attractive properties in food

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industry including viscosity enhancement, emulsion stabilization and film

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formation.21-22 Typically, food colloids contain mixtures of particles and polymers,

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present together as multicomponent species in aqueous media, or adsorbing at solid or

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fluid interfaces competitively.48 These polymer-particle interactions dominate the

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stability, rheology and interfacial properties of emulsion systems.49-51

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In this study, zein collodial particles (ZCPs) and PGA were combined to prepare

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bilayer emulsions using LBL electrostatic deposition technique. The oil/water

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interface of the bilayer emulsion consisted of ZCPs and PGA. The aim of this study

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was to utilize interfacial engineering to modulate the properties of bilayer emulsions.

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The effects of various PGA concentrations and incorporation sequences on physical

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stability and microstructure of novel bilayer emulsions were investigated

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systematically. Optical microscopy and confocal laser scanning microscopy (CLSM)

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were utilized to observe the morphological characteristics of bilayer emulsions.

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Results from this work would provide a new insight into fabricating the bilayer

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emulsion stabilized by the combination of particles and biopolymers.

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MATERIALS AND METHODS



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Materials. Zein with a protein content of 91.3% (w/w) was purchased from

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Sigma-Aldrich (USA). Propylene glycol alginate (PGA) (esterification content: 87.9%)

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was generously provided by Hanjun Sugar Industry Co. Ltd. (Shanghai, China).

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Medium-chain triglycerides (MCT, Miglyol 812N) were purchased from Musim Mas

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(Medan, Indonesia). Absolute ethanol (99.99%), solid sodium hydroxide and liquid

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hydrochloric acid (36%, w/w) were obtained from Eshowbokoo Biological

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Technology Co.,Ltd. (Beijing, China). All other chemical agents were analytical

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grade.

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Preparation of ZCPs and PGA solution. ZCPs were prepared by solvent

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evaporation following the method described in our previous study23 with some

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modifications. Specifically, 4 g zein was dissolved in 800 mL 70% v/v aqueous

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ethanol solution (5 mg mL−1) and stirred at 600 rpm for 3 h at 25 ℃. The remaining

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ethanol in solutions was removed with a rotary evaporator at 45 ℃ for 30 min and the

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dispersions acquired were diluted with deionized water to 200 mL. The ZCP

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dispersions were centrifuged (Sigma 3k15, Germany) at 1000 rpm for 10 min to

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separate large particles and aggregates if any. Finally, the supernatant obtained were

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adjusted to pH 4.0 using 0.1 M HCl solution and the samples were stored in the

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refrigerator at 5 ℃ for further analysis. PGA solution with different concentrations

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(0.01%, 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1.0%, 1.25% and 1.5%, w/v) were

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prepared by dissolving PGA in deionized water and stirring at 600 rpm overnight to

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warrant complete hydration. Then pH was adjusted to 4.0 by 1 M NaOH.


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Preparation of the novel bilayer emulsions co-stabilized by ZCPs and PGA.

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The minimum ZCPs content required to fabricate stable emulsions was determined in

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preliminary experiments. Bilayer emulsions co-stabilized by ZCPs and PGA were

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prepared by different adding sequences (Fig. 9). The total volume of emulsions was

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set to 30 mL and the final emulsions were prepared with 50% (v/v) MCT oil as the

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dispersed phase. Two different adding sequences were described as follows. Method

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I: Primary emulsion (the ZCPs stabilized Pickering emulsion) was prepared by

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mixing 7.5 mL ZCP (2.0%, w/v) dispersion with 15 mL oil phase (MCT) at a speed of

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12,000 rpm using a blender (Ultra Turrax, model T25, IKA Labortechnic, Staufen,

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Germany). After the complete addition of MCT, the mixtures were further

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homogenized for another 5 min to acquire primary emulsions. Secondary emulsions

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(the ZCP/PGA bilayer emulsions) were fabricated by mixing primary emulsions with

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7.5 mL PGA solution (0.01% ~ 1.5%, w/v). Method II: Primary emulsions (the PGA

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stabilized emulsions) were prepared by mixing 7.5 mL PGA solution (0.01% ~ 1.5%,

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w/v) with 15 mL oil phase (MCT) at a speed of 12,000 rpm using a

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blenddddddddddddddder (Ultra Turrax, model T25, IKA Labortechnic, Staufen,

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Germany). After the complete addition of MCT, the mixtures were further

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homogenized for another 5 min to acquire primary emulsions. Secondary emulsions

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(PGA/ZCPs bilayer emulsions) were prepared by mixing primary emulsions with 7.5

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mL ZCP dispersions (2.0%, w/v). These emulsion systems were stirred for another 5

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min after the addition of ZCP dispersions. Such emulsions fabricated by Method II



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were named as PGA/ZCPs bilayer emulsion and termed as 0.01p-z, 0.05p-z, 0.1p-z,

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0.25p-z, 0.5p-z, 0.75p-z, 1.0p-z, 1.25p-z and 1.5p-z.

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ZCPs stabilized (2.0%, w/v) Pickering emulsion of 50% (v/v) oil phase was

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prepared by mixing 15 mL ZCP (2.0%, w/v) dispersion with 15 mL oil phase (MCT)

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at a speed of 12,000 rpm using a blender and the mixture was further homogenized for

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another 5 min. The Pickering emulsion stabilized by ZCPs was termed as “zein” as

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the control in comparison with the bilayer emulsions. All emulsions were then stored

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at ambient temperature (25 ℃) for 12 h before analysis.

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Particle size, zeta (ζ)- potential and polydispersity index (PDI) of ZCPs.

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Particle size, ζ-potential and PDI of ZCPs were determined by Dynamic light

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scattering (DLS) and particle electrophoresis instrument (ZetasizerNano-ZS90,

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Malvern Instruments Ltd., Worcestershire, UK) at 25 °C. Samples were diluted to an

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appropriate concentration before detecting.

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Wettability measurement of ZCPs. The oil-in-water contact angle (θo/w) of

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ZCPs was determined with an OCA 20 AMP (Dataphysics Instruments GmbH,

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Germany) by the method25 with some modifications. The powder obtained from

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freeze-dried ZCPs was compressed to a thin tablet with thickness of 2 mm and

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diameter of 13 mm. Then the tablets were immersed in the MCT in an optical glass

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cuvette. Deionized water (2 µL) was gently placed on the surface of the tablets by the

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tip of a bended needle. After reaching the equilibrium, the images of droplets was


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recorded and the contour of imaged drop was simulated with LaPlace−Young

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equation to acquire contact angle θo/w. Measurements were averaged in triplicate.

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Field emission scanning electron microscope (FE-SEM). Zein colloidal

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particles (ZCPs) were imaged for their morphology and size by a field emission

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scanning electron microscope (FE-SEM) system (SU8010, Hitachi) at an accelerating

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voltage of 5.0 kV. Powdered sample was coated with gold prior to examination.

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Determination of emulsion types. Emulsion type was determined following the

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method with some modifications.26 A drop of an emulsion were added to either oil or

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water. If the droplets were dispersed rapidly in water and remained agglomeration in

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oil phase, it was regarded as an O/W emulsion. Otherwise, the emulsion was regarded

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as a W/O emulsion.

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Size and surface charge of oil droplets. The average droplet size and size

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distribution were measured after preparation of emulsions for 12 h with a laser

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scattering size analyzer (LS230®, Beckman Coulter, USA). The samples were diluted

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with deionized water at 3000 rpm until an obscuration rate between 9% to 12% was

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obtained. The optical properties were applied as followed: a refractive indice of 1.52

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for MCT and absorption of 0.001, and a refractive indice of 1.33 for the disperant

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(deionized water). The volume-area (D3,2) average diameters were calculated using

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the following equation:



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D 3, 2

∑nd = ∑nd

i i

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3

i i 2

The ni is the number of particles with a diameter of di.

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The surface charge of emulsion droplets was determined by measuring the

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direction and velocity of droplet movement in a well-defined electric field using a

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Zeta sizer NanoZS90 (Malvern Instruments, Worcestershire, UK). Emulsions were

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diluted to a final oil droplet concentration of 0.005 wt% with pH-adjusted deionized

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water (pH 4.0) to minimize multiple scattering effects.39 The data were collected from

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at least 10 sequential reading per sample after 120 s of equilibration and calculated by

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the instrument using the Smoluchowski model. All measurements were performed in

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triplicate.

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Optical microscopy. Microstructure of the emulsions was observed at 25 °C

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with a optical microscope (Leica DMD 108, Leica Microsystems Inc., Heidelberg,

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Germany) equipped a camera. The emulsions were diluted 10 times with the

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deionized water (pH 4.0). An aliquot of the emulsion was placed at the center of a

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slide glass and covered with a cover glass.

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Confocal laser scanning microscope (CLSM). CLSM (Leica TCS SP5, Leica

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Microsystems Inc., Heidelberg, Germany) was used to indentify the interfacial

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structure of the emulsion droplets. A mixed fluorescent dye solution consisting of

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Nile blue (ZCP) and Nile red (oil phase) was used to pre-stain the emulsions. The


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dyed emulsions were then deposited on concave confocal microscope slides and

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covered with a cover slip. CLSM was carried out using two laser excitation sources:

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an argon/krypton laser at 488nm (Nile red) and a Helium Neon laser (He-Ne) at 633

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nm (Nile blue).

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Physical stability. The physical stability of emulsions was measured by the

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LUMiSizer (L.U.M. 290 GmbH, Germany) on the base of the principle that employed

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the centrifugal sedimentation to accelerate the occurrence of instability phenomena

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such as sedimentation, flocculation, and creaming.27, 28 The samples went through the

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centrifugal force, while near-infrared light illuminated the whole sample cell to assess

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the intensity of transmitted light as a function of time and position over the entire

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sample length simultaneously. The parameters used for the measurement were set as

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follows: 1.2 mL of emulsion; rotational speed, 3000 rpm; performed time, 3600 s;

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time interval, 20 s; temperature, 25 °C.

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Storage stability. After the preparation of emulsions, the droplet size and

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zeta-potential of the emulsion phase after regular storage periods (1, 7, 14, 21 and 28

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days) were recorded. Visual characteristics of the prepared emulsions at different

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storage periods were also summarized.

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Statistical analysis. All the data obtained were average values of triplicate

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experiments and subjected to statistical analysis of variance using SPSS18.0 for

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Windows (SPSS Inc., Chicago, USA). Statistical differences were determined by



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one-way analysis of variance (ANOVA) with Duncan’s post hoc test, and differences

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were considered to be significant with p < 0.05.

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RESULTS AND DISCUSSION

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Characteristics of ZCPs. Zein colloidal particles (ZCPs) were prepared by the

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solvent evaporation method reported in our previous work with some modifications.23

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The mean hydrodynamic size and poly dispersity index (PDI) of ZCPs were 1356 ±

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26.88 nm and 0.05. This result was different from some previous reports.3, 13 This

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difference was attributed to the concentration of zein in aqueous ethanol solution and

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the final concentration of ZCPs after evaporation of ethanol, and the solvent

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evaporation method was used to fabricate ZCPs instead of classical antisolvent

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precipitation. Wang and Padua29 reported that microspheres self-assembled

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layer-by-layer by adsorption to a central core or nucleus. Radial growth occurred due

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to hydrophobic associations as ethanol was evaporated, elevating the water content

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and hydrophilicity of the solvent. This relatively large size of colloidal particles

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provided sufficient energy for attachment at the oil–water interface (i.e. the energy of

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attachment should be much higher than the thermal energy kBT).3 The isoelectric

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point (pI) of zein is around pH 6.2.30 Therefore, ZCPs has a positive charge (50.13 ±

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0.65 mV) at pH 4.0. The particles can’t readily absorb onto the surface of the droplet

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and easily trigger sedimentation because of large sizes, but they would exhibit a

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long-term stability against creaming, coalescence and Ostwald ripening once the

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absorption completed.


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The micromorphology of ZCPs fabricated by solvent evaporation method was

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observed by FE-SEM and showed in Fig. 1A. ZCPs exhibited a spherical shape with

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the diameter closing to 1 µm. Similar result that zein particles with a large particle

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diameter (1.4 µm) was also reported in our previous study.19 It was easy to distinguish

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individual particles clearly, but the surface of ZCPs was roughness.

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The interfacial wettability of the particles plays a crucial role for the stabilization

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of Pickering emulsions.31 A proper wettability is an important prerequisite for the

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absorption of particles at the oil-in-water interface and provide a physical barrier to

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prevent the droplet coalescence. Nevertheless, it’s hard to fabricate suitable and

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biocompatible particles with a great wettability (about 90°) to stabilize the Pickering

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emulsion.32 The wetting property was determined by the three-phase contact angle

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θo/w of ZCPs, which was immersed in MCT. As shown in Fig. 1B, the θo/w of ZCPs

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was around 68.10°, which might be attributed to more hydrophilic amino residues

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exposed to the surface of ZCPs during the process of solvent evaporation. ZCPs alone

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was unsuitable for preparing a stable emulsion, hence PGA was involved to fabricate

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novel bilayer emulsions co-stabilized by ZCPs and PGA.

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Visual appearance of bilayer emulsions. The ZCPs stabilized Pickering

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emulsion and the ZCPs and PGA co-stabilized bilayer emulsions in this work were

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dropped in water and could be dispersed rapidly in the aqueous phase, indicating that

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they were O/W emulsions. The visual observations of Pickering emulsion stabilized

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by ZCPs and bilayer emulsions co-stabilized by ZCPs and PGA were performed after



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storage at ambient temperature (25 ℃) for 12 h to guarantee either the nanoparticles or

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the emulsifiers absorbing onto the droplet surfaces completely (Fig. 2). The effects of

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different PGA levels and adding sequences of two kinds of stabilizers on visual

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appearance and physical stability of bilayer emulsions could be distinguished clearly.

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The Pickering emulsion stabilized by ZCPs alone was unstable, which showed droplet

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aggregation and the release of oil to the top phase of emulsion shortly. The instability

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of the Pickering emulsion might be ascribed to an inappropriate wettability of ZCPs.

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The result was consistent with previous findings.3,19,31 Interestingly, a lipid

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droplet-enriched cream layer appeared immediately with the low level of PGA in

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ZCPs stabilized Pickering emulsion. It was supposed that the presence of low

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concentrations of PGA was hardly to cover the droplets of ZCPs stabilized Pickering

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emulsion and therefore resulted in briging flocculation among the droplets.

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Meanwhile, the addition of a low PGA concentration reduced zeta-potential value (-

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9.53 mV) at first and was unable to provide sufficient electrostatic repulsion to keep

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the emulsion stable. With the level of PGA elevating from 0.01% to 0.50% (w/v),

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emulsified phase volume of ZCPs/PGA bilayer emulsion was increased continuously.

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Visual observation demonstrated the fresh emulsions remained homogeneous without

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obvious creaming or phase separation when the concentration of PGA reached above

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0.50% (w/v). Compared to the ZCPs/PGA bilayer emulsion, the bilayer emulsion

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using PGA as an inner layer exhibited a more uniform and smooth appearance of

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milky white. The color of PGA/ZCPs bilayer emulsion was gradually lightened from


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faint yellow to milky white. We proposed that the phenomenon was attributed to two

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reasons: i. PGA as a distinct amphiphilic polysaccharide has a higher velocity of

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absorption to the interface and a stronger emulsibility for droplets than ZCPs.23

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Consequently, the emulsion could form more homogeneous and small-sized droplets

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when PGA was added at first. ii. The sufficient coverage of PGA on the droplets

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stabilized by ZCPs improved the stability of droplets and subsequently altered the

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visual appearance of the emulsion.

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Size distribution and zeta-potential. The average droplet sizes and

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zeta-potential values of bilayer emulsions with two different adding sequences were

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presented in Fig. 3A and C. In order to reflect the characteristics of emulsion droplets

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more comprehensively, droplet size distribution was also shown in Fig. 3B and D,

295

respectively. It could be found that most of the droplet sizes of bilayer emulsions

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(except for 0.01p-z) were smaller than 80 µm. The emulsion droplets were relatively

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small compared to those of the zein particles stabilized Pickering emulsion enriched

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with pectin,3 which demonstrated that PGA has a better emulsibility than pectin. In

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terms of the ZCPs/PGA bilayer emulsion, the droplet sizes was increased as PGA at

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low concentrations (0.01% and 0.05%, w/v) was added into the Pickering emulsion

301

stabilized by ZCPs. Meanwhile, the zeta-potential value of ZCPs/PGA bilayer

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emulsion was decreased from the positive charge (+ 41.3 ± 3.9 mV) to the negative

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charge ( - 9.53 ± 2.66 mV and - 10.25 ± 2.98 mV), which meant charge reversal

304

occurred when PGA neutralized the opposite charge on the surface of droplets. In



305

terms of electrostatic stabilization, zeta-potential value over |30| mV was required and

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progressive flocculation occurred between |5| and |15| mV.33 Therefore, the droplet

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aggregation was induced due to the insufficient electrostatic repulsion among oil

308

droplets at low concentrations of PGA. The repulsion forces (electrostatic repulsion

309

and steric hinderance) among the droplets provided by bilayered interfaces could not

310

provide sufficient stability against coalescence and creaming. The results were in

311

accordance with the visual appearance of ZCPs/PGA bilayer emulsion which showed

312

that a cream layer appeared with low PGA level. Similarly, Hou reported that the

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droplet aggregation occurred in soybean soluble polysaccharides stabilized emulsions

314

when a low concentration of chitosan was added.34

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However, as the concentration of PGA was elevated from 0.10% to 1.00% (w/v),

316

the average size of droplets was progressively reduced from 36.58 ± 0.64 µm to the

317

minimum value of 12.46 ± 0.03 µm. Meanwhile, the zeta-potential value of droplets

318

was increased from -14.1 ±2.69 mV to a maximum value of -29.63 ± 1.19 mV. When

319

the level of PGA addition was above 1.00% (w/v), neither the droplet size nor zeta-

320

potential exhibited an obvious fluctuation. The results indicated that PGA provided a

321

continuous growth of electrostatic repulsion and steric hindrance among the droplets

322

to stabilize the emulsions against droplet aggregation. Besides, PGA molecules were

323

absorbed on the droplets and greatly increased the efficiency of emulsification under

324

high shear force, which resulted in a progressive reduction of droplet size. It was

325

supposed that some PGA molecules might compete for absorbing on the interface


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with ZCPs and subsequently reduced the droplet size by lowering the surface tension

327

of interface.46,47 When PGA concentration was above 1.00% (w/v), a slight increase in

328

droplet size occurred due to the presence of free PGA in the continuous phase due to a

329

promoting droplet aggregation. This result showed that 1.00% (w/v) might be the

330

minimum PGA level required to fully cover the oil-water interface and stabilize the

331

ZCPs/PGA bilayer emulsion as an outer layer. Similar phenomenon was also found in

332

the formation of SDS-chitosan-pectin stabilized multilayer emulsion.35 At a high

333

concentration, PGA molecules might protrude into the aqueous phase and cause the

334

depeletion flocculation of droplets in the emulsion.36

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Similar but not the same rule was found in the PGA/ZCPs bilayer emulsion. At

336

the low concentration (0.01%, w/v) of PGA, the droplet size of the bilayer emulsion

337

(155.2 ± 2.5 µm) was significantly (p

Novel bilayer emulsions co-stabilized by zein colloidal particles and

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