Fabrication and characterization of novel Pickering emulsions and Pickering high internal emulsions stabilized by gliadin colloidal particles

Food Hydrocolloids 61 (2016) 300e310

Contents lists available at ScienceDirect

Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Fabrication and characterization of novel Pickering emulsions and Pickering high internal emulsions stabilized by gliadin colloidal particles Ya-Qiong Hu a, Shou-Wei Yin a, c, *, Jian-Hua Zhu b, Jun-Ru Qi a, Jian Guo a, Lei-Yan Wu d, Chuan-He Tang a, Xiao-Quan Yang a a

Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou, 51640, PR China Food Science and Engineering Institute, Shaoguan University, Shaoguan, 512005, Guangdong, PR China c State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, 510640, PR China d College of Food Science and Engineering, JiangXi Agricultural University, Nanchang, 330045, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 November 2015 Received in revised form 29 April 2016 Accepted 23 May 2016 Available online 24 May 2016

In this paper, we demonstrate for the first time the use of gliadin colloid particles (GCPs) as an effective particulate stabilizer of oil-in-water emulsions of natural oils and water. For this purpose, we fabricated GCPs through a facile anti-solvent precipitation procedure and demonstrated their uses in the formation of Pickering emulsions as well as Pickering high internal phase emulsions (HIPEs). We found that unmodified GCPs can produce stable, surfactant-free o/w emulsions with microscale droplet sizes under experimental mixing conditions at pH 4 and above. In contrast, the emulsions were not stable against coalescence at ~pH 3.0. The microstructures, e.g., interfacial framework, GCPs partition between the continuous phase and interfacial region, and state of the droplets, of Pickering emulsions as a function of pH were visualized by optical microscopy and confocal laser scanning microscopy (CLSM), confirming that in addition to Pickering stabilization, the GCPs-based network and/or dispersed droplets-based network also contributed to the stabilization of the emulsions, in a pH-dependent manner. Clear correlations exist between colloid properties of the GCPs dispersions and the emulsion characteristics. Interestingly, stable surfactant-free Pickering HIPEs were fabricated by a facile shearing emulsification. This study opens a promising route based on Pickering HIPEs to transform liquid oils into viscoelastic emulsion gels with zero trans-fat and less saturated fat. The Pickering HIPEs possess promising potentials to replace solid fat in food formulations, which outline new directions for future fundamental research. © 2016 Elsevier Ltd. All rights reserved.

Keywords: GCPs Pickering emulsions Pickering HIPEs Microstructure Physical performance

1. Introduction Oil-in-water emulsions have wide applications in pharmaceuticals, foods, and personal care products (Lomova, Sukhorukov, & Antipina, 2010). Surfactants and/or amphiphilic polymers can kinetically stabilize the emulsions, both decreasing interfacial tension and creating steric hindrance or electrostatic repulsions between dispersed droplets (Destribats, Rouvet, Gehin-Delval, Schmitt, & Binks, 2014). Synthetic surfactants get more and more

* Corresponding author. Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou, 51640, PR China. E-mail address: [email protected] (S.-W. Yin). http://dx.doi.org/10.1016/j.foodhyd.2016.05.028 0268-005X/© 2016 Elsevier Ltd. All rights reserved.

limited due to the increasing consumer and legal requirements such as nature, nontoxicity, biocompatibility and high ecological acceptability (Lam, Velikov, & Velev, 2014). Manipulating interfacial structures via solid particles provides a promising and green alternative to manufacture stable emulsions. The “surfactant-free” character makes them more suitable for various applications particularly in food, pharmaceutical and cosmetic formulations (Schrade, Landfester, & Ziener, 2013). Pickering emulsion is an emulsion stabilized by solid particles instead of surfactants or polymers. Surfactants form fluid interfaces with a substantial surface lateral diffusion coefficient, and the adsorption and desorption occur simultaneously (Berton-Carabin & €en, 2015). Unlike surfactants, once solid particles adsorb at an Schro oilewater interface they are irreversibly anchored therein, forming

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

stable Pickering emulsions (Binks, 2002). The high resistance to coalescence is a major benefit of Pickering emulsions (Binks & Horozov, 2006; Dickinson, 2010). Their applications in food formulations were realized only recently, possibly due to the promising potentials for texture modification, calorie reduction, and vehicles of functional ingredients (Rousseau, 2013). The focus is gradually shifting from inorganic particle (e.g., SiO2) to biological particles to fabricate emulsions with varied end-uses, particularly in food, pharmaceutical and cosmetic formulations (Lam et al., 2014; Rayner et al., 2014). However, it is still a key technological challenge to manufacture Pickering emulsions using edible colloid particles (Dickinson, 2010, 2012a; Rousseau, 2013). Nowadays, a few works has been available on Pickering emulsions stabilized by a wide range of biological micro- and nano-particles, such as modified starch (Yusoff & Murray, 2011), cellulose nanocrystal (Kalashnikova, Bizot, Cathala, & Capron, 2011), chitin nanocrystal (Tzoumaki, Moschakis, Kiosseoglou, & Biliaderis, 2011), chitosan particles (Liu, Wang, Zou, Wei, & Tong, 2012) and flavonoids (Luo et al., 2012). Limited information is available on Pickering emulsions stabilized by protein-based particles, such as hydrophobic zein colloid particles (de Folter, van Ruijvena, & Velikov, 2012; Wang et al., 2015; Zou, Guo, Yin, Wang, & Yang, 2015), kafirin nanoparticles (Xiao, Li, & Huang, 2015), as well as hydrophilic whey protein microgel particles (Destribats et al., 2014), soy protein nanoparticle aggregates (Liu & Tang, 2013). Gliadins, one of the most abundant storage proteins in cereals, are prolamine-type proteins in wheat. Today, no studies have described Pickering stabilization by gliadin colloid particles. Gliadin is not soluble in water or oil, but is soluble in an aqueous alcohol solution. It is characterized by high levels of glutamine and proline, but low content in basic amino acid (Bietz, Huebner, Sanderson, & Wall, 1977; Kasarda, Autran, Lew, Nimmo, & Shewry, 1983). The terminals of gliadin molecules are generally more hydrophobic than the repetitive domain, making gliadins amphiphatic (Banc et al., 2007; Kasarda et al., 1984; Okita, Cheesbrough, & Reeves, 1985). Amphiphilicity is one of the main driving forces for self-assembly. Thus, gliadins can self-associate to form a wide range of mesostructures. GCPs are usually produced for drug or bioactive delivery (Ezpeleta et al., 1996; Wang, Hu, Yin, & Yang, 2014), but their usage as Pickering emulsifier to stabilize oil-water interface has not been explored. In theory, Pickering emulsifiers should remain insoluble in both phases, and maintain intact over the longevity of a Pickering system (Dickinson, 2010; Gao et al., 2014). Therefore, GCPs possess promising potentials to fabricate Pickering emulsions with food-grade status. Solid-like hydrophobic matrices have a large range of applications in food and pharmaceutical formulations, cosmetics, and others (Nikiforidis & Scholten, 2015). Organogel formation is a strategy to impart solid-fat functionality to liquid oils in food and pharmaceutical industry (Co & Marangoni, 2012; Sahoo et al., 2011). An alternative approach is the use of emulsions in a form of concentrated internal phase, known as high internal phase emulsions (HIPEs) (Nikiforidis & Scholten, 2015). HIPEs are twophase systems with the internal phase fraction over 74% (v/v) (Lissant, Peace, Wu, & Mayhan, 1974). Conventional HIPEs are usually stabilized by large amounts of surfactant (5e50 vol%) (Barbetta & Cameron, 2004). Pickering HIPEs are an alternative that substitutes for sugar hazardous surfactants and provides additional and/or improved properties to final products (Ikem, Menner, & Bismarck, 2008; Menner, Ikem, Salgueiro, Shaffer, & Bismarck, 2007). However, phase inversion usually occurs in HIPEs stabilized by particles. Binks and co-workers have experimentally demonstrated that Pickering emulsions phase-invert between internal phase volume fractions of 0.65 and 0.70 (Binks & Lumdson, 2000). No information about protein-based Pickering HIPEs was

301

reported. Therefore, there is a huge challenge to fabricate Pickering HIPEs using GCPs. Meanwhile, fabrication of protein-based Pickering HIPEs is a promising pathway to expand the application of emulsion-based food ingredients. In this work, we reported, for the first time, a facile method to fabricate stable, soap-free Pickering emulsions using fully natural colloidal particles (GCPs) as a particulate emulsifier. The role of pH on emulsion formation and stability was studied and these observations were related to the colloidal properties of GCPs. The microstructure, including interfacial framework, GCPs partition, and aggregated state of the droplets in Pickering emulsions as a function of pH were characterized to relate to their physical performances. In particular, this work succeeded in fabricating stable Pickering HIPEs using food-grade protein-based colloid particles. This study opens a promising pathway for producing edible Pickering emulsions and/or Pickering HIPEs using protein-based colloid particles as potential vehicles of functional ingredients and/or food texture modifiers. 2. Materials and methods 2.1. Materials Nile Red and Nile Blue A were obtained from SigmaeAldrich, Inc. (St. Louis, MO, USA). Gluten was obtained from Fengqiu Hua Feng powder Co., Ltd (Fengqiu, Henan, China). Corn oil was obtained in a local supermarket (Guangzhou, China). All other chemicals used were of analytical grade. 2.2. Gliadin extraction Gliadins were extracted according to the procedure described by Ezpeleta et al. (1996). Gluten powders (100 g) were dispersed gently in 1 L of ethanol-water mixture (70/30 v/v) for 2 h at room temperature. The suspension was centrifuged (8,000g, 20 min). The supernatant fraction, i.e., gliadins, was dialyzed firstly against deionized water (24 h), and then against 0.05 M acetic acid (24 h), finally against de-ionized water (24 h). The dialysate was freezedried to yield gliadin powder in which the amount of proteins was around 85% (w/w) and the proportions of the different gliadin groups were 55% w/w for a/b-gliadins, 15% w/w for g-gliadin, and 15% w/w for u-gliadin (Ezpeleta et al., 1996). 2.3. Particle synthesis Gliadin colloid particles (GCPs) were prepared using a facile anti-solvent procedure. Gliadin powder (2.5 g) was dissolved in 100 mL of aqueous ethanol binary solvent (70% v/v) to form a gliadin stock solution. Gliadin solution was trickled into 1% acetic acid solution within 4 min, under continuous shearing (6000 rpm) using an Ultraturax T25 homogenizer (Janke & Kunkel, Germany). After shearing for another 10 min, the remaining ethanol in GCP dispersions was removed at 40
C in a RV 10 digital rotary evaporator (IkA-Works Inc, Germany). Finally, gliadin concentration in the GCPs dispersion was 2%. Particle size and z-potential of fresh GCPs dispersions were characterized prior to the emulsification. 2.4. Pickering emulsion preparation GCPs were employed as particulate emulsifiers to produce Pickering emulsions at pH between 2.9 and 9.0. The pH of GCPs dispersions were adjusted by drop-wise adding HCl or NaOH solution to yield a series of solutions with pH, 2.9, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, respectively. The emulsions were prepared at equal volume fraction of water and oil phases. In brief, 10 mL of corn oil

302

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

was added to 10 mL of aqueous GCPs suspensions in a glass vial and the oil-water mixture was sheared using an Ultraturax T25 homogenizer (Janke & Kunkel, Germany) at 20,000 rpm for 2 min at room temperature to yield GCPs-stabilized emulsions. The emulsions were kept at room temperature over 3 months to check the stability. Pickering emulsions with varied GCPs concentrations were prepared according to the above-mentioned procedure. In this series, the pH of GCPs solutions was 5.0, and GCPs concentrations were adjusted to 0.2, 1.0 and 2.0%, respectively. Finally, selected GCPs concentration (2.0%) was used to prepare Pickering HIPEs. In brief, 8 mL of corn oil was added to 2 mL of aqueous GCPs suspensions in a 20 mL beaker and the oil-water mixture was sheared using an Ultraturax T25 homogenizer (Janke & Kunkel, Germany) at 20,000 rpm for 2 min at room temperature to yield Pickering HIPEs. The emulsions were kept at room temperature over 3 months to check their stabilities. 2.5. Physical performance of Pickering emulsion 2.5.1. Emulsion type The type of emulsion (either O/W or W/O) was inferred by observing what happened when a drop of an emulsion was added to either oil or water. For O/W emulsions, when a drop was added to water, it dispersed. In contrast, the emulsion droplets remained agglomerated when placed in oil. 2.5.2. Centrifugation stability Emulsion stability was checked by centrifugation for 2 min at 10,000 g, and the thickness of the creaming layer was measured with a digital caliper. 2.5.3. Creaming stability The stability against creaming of the emulsions was analyzed via creaming index (CI). Emulsion samples were poured into glass tubes (2.8 cm in diameter 7.5 cm in height), and the tubes were sealed to prevent moisture evaporation. The samples were monitored for 2 months. The CI values were calculated according to the equation below. CI (%) ¼ Hs/He where Hs is the serum layer height and He is the total emulsion height.

2.8. Confocal laser scanning microscopy CLSM experiments were performed according to the procedure of Ma, Tang, Yin, & Yang (2013) using a Leica TCS SP5 CSLM (Leica Microsystems Inc., Heidelberg, Germany). Various specimens of the Pickering emulsions were dyed with a mixed fluorescent dye solution consisting of 1 mg mL1 Nile Red and 1 mg mL1 Nile Blue A (in isopropyl alcohol). The dyed emulsions were put on concave slides, and then the concave slides were covered with coverslips. Finally, glycerol was coated around the coverslips to seal the samples. The fluorescent dyes were excited by either an argon laser at 488 nm for Nile Red or a helium neon (HeNe) laser at 633 nm for Nile Blue A. 2.9. Optical microscopy measurements Optical microscopic observation of the emulsions was performed using a Meiji Techno MX-4000 light microscope (Japan) to estimate the droplet size and aggregate state. An aliquot of the emulsion was placed in the hole (0.5 mm deep) at the center of a slide glass and covered with a cover glass. 2.10. Dynamic oscillatory measurements The dynamic viscoelastic properties of the Pickering HIPEs were evaluated using a HAAKE RS600 Rheometer (HAAKE Co., Germany), under a small amplitude oscillatory frequency sweep mode. The trials were employed at 25
C. The frequency was oscillated from 0.1 to 10 rad s1 and the measurements were performed within the identified linear viscoelastic region and made at 1 Pa. The elastic modulus (G0 ) and loss modulus (G00 ) were recorded by RheoWin 3 Data Manager. 2.11. Statistics Statistical analyses were performed using an analysis of variance (ANOVA) procedure of the SPSS 13.0 statistic analysis program, and the differences between means of the trials were detected by a least significant difference (LSD) test (P < 0.05). 3. Results and discussion 3.1. Zeta potential and particle size characterization Fig. 1 shows the evolutions in the zeta potential, z -potential, of

2.6. Particle size and zeta potential measurements

2.7. Particle size distribution of Pickering emulsion Droplet size distributions of Pickering emulsion were measured immediately after the emulsification using a Mastersizer 3000 (Malvern Instruments Ltd., UK). The samples were diluted in deionized water at 2000 rpm until an obscuration rate of 10% was obtained. The Mie theory was applied by considering the following optical properties for corn oil droplets: a refractive index of 1.46 and absorption of 0.001, and for the dispersant (deionized water), a refractive index of 1.330. Weight mean diameter (D4,3) was reported.

Zeta potential (mV)

25 The particle size and zeta-potential of the GCPs dispersions was measured using a Zetasizer Nano (Malvern Instruments Ltd., UK) at 25
C. The results reported represent an average of at least three independent readings trials.

20 15 10 5 0 -5 2

3

4

5

6

7

8

9

pH Fig. 1. Zeta potentials of gliadin colloid particles (GCPs) as a function of pH.

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

GCPs at 25
C as a function of pH. The z -potential of GCPs gradually decreased from 21.6 ± 0.8 mV at pH 2.9 to 4.7 ± 0.3 mV at pH 9.0, a zero value of z -potential was attained at ~pH 6.0, which is usually referred to as the isoelectric point (IEP). The abovementioned z -potential amplitude and their evolution upon adjusting pH are consistent with the fact that gliadins are low in the ionic amino acids, and glutamic and aspartic acids exist almost entirely as amides (Gianibelli, Larroque, MacRitchie, & Wrigley, 2001). Therefore, the amplitude of z epotential at ~pH 6.0 and above was very low, in the range of a few mV. We investigated the colloidal stability of 2.0% GCP dispersions at selected pH, as shown in Fig. 2. Three main pH domains can be identified, the particles formed stable transparent dispersions at pH 2.9 and 3.0. The dispersions changed to be translucent and unstable between pH 4.0 and 6.0. They precipitated to a thin deposit at the bottom of the vessel after 1 h of incubation at pH 4.0 and pH 5.0, while it sedimented immediately after the pH adjustment to pH 6.0 due to the aggregation of the colloid particles (Fig. 2). The particles quickly aggregated to form visual agglomerates that adhered on the electrode of pH meter or the inner wall of beakers when the pH was adjusted to 7.0 or above. The size evolution of the aggregates was found to be related to the aqueous pH value as well as with amino acid composition of gliadins. This aggregation behavior of the GCPs upon adjusting its pH toward basic circumstance was further confirmed by dynamic Light Scattering (DLS) analyses, indicating that particle size of the GCPs evolved from nanoscale particles to microscale ones (Table 1). This behavior was directly correlated with the variation of the zeta potential of the particles as a function of pH (Fig. 1). The particles display a polyampholyte character with an IEP at ~pH 6.0, where their overall charge is zero, and two pH domains above and below this critical value where the colloid particles are negatively and positively charged, respectively. This surface charge evolution is related to the balance between the dissociation of the carboxylic and amino groups of the GCPs constitutive gliadins. The colloidal stability of the dispersions is then mainly triggered by their overall charge, the electrostatic repulsion between particles being high enough to ensure dispersion stability for pH < 4.0 and too low for pH 6.0 and above to counterbalance the attractive interactions that lead to GCPs aggregation and ultimately sedimentation. 3.2. Physical performance of Pickering emulsions 3.2.1. Visual appearance and creaming Storage stability is a key parameter of any formulation because it determines to some degree if a product is suitable for its intended

303

Table 1 Particle size and polydispersity index (PDI) of the GCPs as a function of pH. pH

Particle size (nm)

PDI

2.9 3.0 4.0 5.0 6.0 7.0 8.0 9.0

120.1 ± 5.1 124.9 ± 9.6 476.4 ± 11.9 3865.5 ± 45.8 4256.5 ± 57.5 e e e

0.755 0.711 0.243 0.203 0.293 e e e

± ± ± ± ±

0.113 0.105 0.003 0.003 0.006

use. Phase separation is catastrophic to any emulsion-based product. Here, storage stability is firstly evaluated visually by creaming, as it is often a precursor to flocculation and coalescence. Fig. 2 shows visual appearance of Pickering emulsions stabilized by GCPs. Visual inspection states that the fresh emulsions remain homogeneous. They do not as they separate upon storage. Lipid droplet-enriched cream layer gradually appeared as a result of gravitational separation of the emulsion samples during the storage, in a pH-dependent manner. After 2 h of storage, the drainage phenomenon occurred for all the formulations, and the creaming indices were ~25% for the emulsions except the ones produced at pH 9.0 where it was 16.7%. Upon increasing storage times, the height of the interface further increased in a pH-dependent manner. The creaming indices gradually increased to ~45% for the emulsions produced at pH 2.9 and 3.0 after 5 days of storage, while those at pH 4e9 remain unaffected upon the storage for 2 months. Stabilities of emulsions against droplet coalescence varied as a function of pH (Fig. 2). For the emulsions produced at highly acidic condition (~pH 3.0), free oil (slightly yellow) was detected at the creaming of the vessel after 10 d of storages, indicating phase separation occurred due to extensive coalescence of dispersed droplets. In contrast, the emulsions produced at pH 4.0e9.0 remained stable after 2 month of storage. In fact, surface charges of GCPs (21.6 ± 0.8 mV) at pH ~3.0 contributed to some degree to the instability phenomenon. Previous studies pointed out that highly charged particles cannot stabilize emulsions because a charged particle sees an “image charge” at the oil-water interface and experiences a repulsive energy barrier when it comes close to the interface (Danov, Kralchevsky, Ananthapadmanabhan, & Lips, 2006; Wang, Singh, & Behrens, 2012). For highly charged GCPs (pH ~3.0), this repulsive force may be higher than the convective forces pushing them toward the interface during emulsification. Therefore, it is difficult for them to adsorb at the oil-water interface, resulting in unstable emulsions. Similarly, the emulsions stabilized

Fig. 2. Appearance photographs of the GCPs dispersions (up) and the corresponding o/w Pickering emulsions (down) 10 d after preparation as a function of pH.

304

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

by zein nanoparticles with the zeta potentials of approximate þ60 mV were not stable (de Folter et al., 2012). On the other hand, the aggregation behavior of the GCPs upon adjusting its pH toward basic circumstance was confirmed by dynamic Light Scattering (DLS) analyses, indicating that particle size of the GCPs evolved from nanoscale particles to microscale ones (Table 1). For spherical particles of radius r, the free energy of spontaneous desorption (DG) is proportional to r2 (Dickinson, 2010). This means that the increased particle size of the GCPs when the pH was adjusted toward basic circumstance also contributed to the enhanced physical stability of the GCPEs.

emulsions were solidified, and composed of big and slightly flocculated drops. At relatively high pH (pH 8.0 or 9.0), extensively flocculated drops were observed. Herein the good correlation between the colloid properties of GCPs aqueous dispersion and the corresponding emulsion characteristics was worth noting and suggests that the particles adopted different adsorption modes depending on their surface charges. Importantly, the emulsions produced at pH 4e9 are stable against coalescence with no sign of phase separation over 1 month, although extensive flocculation occurrence. This is quite remarkable contrast to normal surfactant systems.

3.2.2. Centrifugation stability Centrifugation accelerates the creaming process of emulsions, forcing the droplets to concentrate. The excess water is then excluded from the emulsion, leading to close packing conditions. The creaming index after the centrifugation was also used to reflect the stability. After centrifugation, the CI was about 50% for all emulsions, which was higher than that after 2 month of storage, reflecting that excess water was excluded from the emulsion in the course of centrifugation. The tested emulsions produced at pH 4.0e9.0 were not “broken up”, whereas those at pH ~3.0 were broken up, and thin oil layer was observed at the top of the emulsions. This result was consistent with storage stability results, further indicating that the stability of the gliadin colloid particlesstabilized emulsions (GCPEs) were dependent on the aqueous pH, and those at highly acidic pH did not resist against the stress induced by the centrifugation.

3.3.2. CLSM Emulsion microstructure, e.g., interfacial framework, GCPs partition, and network structure in continuous phase contributed to the formation and stabilization of emulsions, and they were assessed by CLSM technique. Corn oil was stained with Nile Red (green) and gliadin was marked by Nile Blue A (red). Fig. 4 shows CLSM micrographs of selected GCPEs samples. Microscopic images were obtained in overlap fluorescence field. The CLSM observations strongly indicated that the emulsions exhibited marked microstructure differences, in a pH-dependent manner. In brief, strong protein signals (red) were detected in the aqueous continuous phase of the emulsions produced by GCPs at pH 5.0 or lower. In contrast, there was not a distinct perimeter around dispersed droplets, but the formation GCPs-based continuous networks in the continuous phase. The combination of Pickering stabilization and GCPs-based continuous networks contributed to the stabilization phenomenon for the emulsions at pH 5.0. In the case of the emulsion made at pH 8.0 and/or 9.0 where the emulsions presented distinct microstructures. In brief, green fluorescence from Nile Red resided in spherical droplets while the red fluorescence of Nile Blue A was observed mainly at the perimeter of the emulsion (Fig. 4). The colloidal stability of the dispersions is then mainly triggered by their overall charge, and the change of pH toward to 8 or 9.0 endowed the particles with partially wettability, facilitate the interfacial absorption. This kind of interfacial

3.3. Microstructure 3.3.1. Optical photographs Optical microscopy observation of the emulsions revealed that the drop characteristics also evolved with pH. Fig. 3 shows microscopy images of typical emulsions in the three pH domains. At relatively low pH, emulsions were fluid with discrete, small, and slightly poly-dispersed drops. At intermediate pH (pH 5.0), the

Fig. 3. Microscopy images (scale bar: 20 mm) of Pickering emulsion samples. Pictures were taken 1 h after preparation. (A) pH 2.9, (B) pH 5.0, (C) pH 8.0, and (D) pH 9.0.

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

305

Fig. 4. Selected CLSM images (scale bar: 10 mm) of GCPEs as a function of pH. a, 2.9; b, 5.0; c, 8.0; d, 9.0. Corn oil was stained with Nile Red, and GCPs was stained by Nile Blue A. The fluorescent dyes simultaneously excited at 488 nm for Nile Red (green) and at 633 nm for Nile Blue A (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. The formation and performance of Pickering emulsions at pH 5.0

phases and varied concentrations of GCPs (0.2, 1.0 and 2.0%) was prepared. The size distributions of the emulsion drops were determined by a Mastersizer 3000, as shown in Fig. 6. GCPEs showed a minomodal droplet size distribution, and the maxima of the profiles shifted towards low size upon increasing particle concentrations. The mean particle size was used to quantitatively characterize the size of Pickering emulsions, reflecting that all preparations were micrometer-sized. The D4, 3 of the emulsions stabilized by 0.2% GCPs were 92.5 ± 0.5, and it was decreased to ~60 mm, upon increasing GCPs concentrations to 1.0 or 2.0%. In Fig. 7 the optical microscopy images show the droplet size evolution of the emulsion as a function of the initial particle concentration. As expected, the emulsions consisted of slightly flocculated drops, and relatively big drops were observed in the image of the emulsion samples at 0.2% GCPs relative to that at 1.0 or 2.0% GCPs. It could be seen that the emulsion stabilized by GCPs (0.2, 1.0 and 2.0 wt %/v) showed creaming and serum release, and the creaming index was approximate 25%, It was slightly affected by GCPs concentrations, and the height of interface slight decreased upon increasing GCPs concentrations from 0.2 to 2.0%. Interestingly, the Pickering emulsions remained constant in the prolonged 5 months of storage (data not shown). Actually, these emulsions transformed from the liquid state to the solid state and exhibited arrested dynamics since they were not pourable after inverting the cups (data not shown). This finding encourages us to further increase internal phase in Pickering systems, so as to form Pickering HIPEs.

3.4.1. Effect of GCPs concentration A series of emulsions with equal volumes of water and oil

3.4.2. Pickering HIPEs and their microstructure The strategy of structuring liquid oils into solid-like oil gels is

frameworks contributed to the aforementioned storage stabilization of the emulsions. In addition, those emulsions flocculated to form aggregated droplets. This was consistent with the observation by optical microscopy (Fig. 3). That is, the adjacent droplets were closely packed with each other to form network structure. The combination of Pickering mechanism and droplet-based network contributed to the stabilization of the emulsions. This phenomenon is well in agreement with the data of physical performances (Fig. 2), reflecting that the difference in the pH of GCPs dispersions affected the partition of protein-based colloid particles between oilewater interfaces and aqueous continuous phase, the charge of formed interfacial framework as well aggregated state of dispersed droplets. At intermediate pH (pH 5.0), the emulsions did not resist against creaming phenomenon, but the formed creaming layer were solidified and composed of big, and slightly flocculated drops. The creaming didn’t lead to irreversible flocculation for the emulsions, and those emulsions were stable over 5 months of storages, evidenced by similar droplet size and size distribution in CLSM images of the emulsions fresh and after 5 months of storages (Fig. 5). Therefore, the formation and physical performances of GCPsstabilized emulsions at pH 5.0 were further studied, including the role of GCPs concentration and the potential for the formation of Pickering HIPEs.

306

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

Fig. 5. CLSM images (scale bar: 10 mm) of Pickering emulsions produced at pH 5.0 before (up) and after (down) 5 months of storage.

Volume (%)

15

0.2% pH=5 1.0% pH=5 2.0% pH=5

10

5

0

1

10

100

1000

Particle size (μm) Fig. 6. Particle size distribution of GCPEs as a function of GCP concentrations.

very meaningful, because fat-structured foods, such as chocolate, ice cream, spreads and shortening, are usually created by colloidal networks of fat crystals or hydrogenated vegetable oils, which is composed of saturated and/or trans fatty acids (Rogers, Wright, & Marangoni, 2009). It is known to all that excessive consumption of saturated and/or trans fatty acids has deleterious effects to human health, e.g., negative effects on lipoprotein profiles and the increase in incidence of heart disease and metabolic syndrome (Aro, Jaughiainen, Partanen, Salminen, & Mutanen, 1997). Protein colloid particles-based Pickering HIPEs was used as a novel route to transform liquid oils into solid-like emulsion gels with zero transfat and less saturated fat as an alternative to solid fats. Pickering HIPEs were prepared by mechanically shearing a mixture containing corn oil and GCPs dispersion for 2 min with an Ultra-Turrax T25 homogenizer (10 mm head) at 20,000 rpm. The appearance of emulsions fresh preparation and after 2 months of storage is shown in Fig. 8. Pickering HIPEs stabilized by GCPs are o/ w emulsions, in contrast to the usual water-in-oil (w/o) HIPEs (Ikem et al., 2008; Menner et al., 2007), for which large fractions of

expensive surfactants (5e50%) are often required to stabilize HIPEs effectively (Akartuna, Studart, Tervoort, & Gauckler, 2008). For HIPEs with internal phase of 80 vol%, the emulsions not only exhibited remarkable stability against extensive coalescence but also inhibited creaming of the oil droplets and in particular they were self-standing, indicating that the GCPs are suitable for manufacturing Pickering HIPEs (Fig. 8). Confocal microscopy images taken before and after centrifugation (10,000 g, 2 min) remained quite similar, further confirming that the Pickering HIPEs were stable against coalescence (Fig. 9). In addition, the dependence of the HIPEs response to the applied frequency (i.e., the rate of deformation) was characterized by subjecting the samples to a constant stress (1 Pa) which was within the region of linear response (0.1e10 Hz). For the HIPEs, the elastic moduli (G0 ) are higher than the corresponding viscous moduli (G00 ) at all frequencies ranging from 0.5 to 10 rad s1 (Fig. 10) demonstrating the gel-like behavior in the range. GCPs are amphiphilic, which thus leads to the adsorption on the oil droplet surface during the forceful mixing process, which provides a steric hindrance to dropletedroplet coalescence. GCPs also form three-dimensional network in the continuous phase. In that way, the formed oil droplets are trapped inside the network as soon as agitation is stopped. Most importantly, significant structuring phenomenon occurred in concentrated emulsions due to the oil droplets being crowded together at high packing fractions (Dickinson, 2012b; Knudsen, Øgendal, & Skibsted, 2008; Mezzenga, 2007), as well as strong inter-droplet attractions since the droplets carried few net charge. Viscoelastic feature of emulsions contributed to their creaming and coalescence stability, so that the covered droplets did not raise particle size via undergoing the coalescence even after centrifugation. This work established a facile method to produce solid-like emulsion gels with zero trans-fats and less saturated fats via a surfactant-free Pickering HIPEs approach. 3.5. Schematic illustration Interestingly, the creaming of the encapsulated droplets didn’t lead to irreversible flocculation for the emulsions produced at

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

307

Fig. 7. Microscopy images (scale bar: 20 mm) of Pickering emulsion samples as a function of GCPs concentration (A) 0.2%, (B) 1.0%, (C) 2.0%. Pictures were taken 1 h after preparation.

Fig. 8. Visual appearance of Pickering HPIEs before (A, B1, C2) and after (B1, C2) 2 months of storage at room temperature. Pickering HPIEs with 80% oil phase were stabilized by 2% GCPs with or without 50 mM NaCl.

pH 5.0, those emulsion were stable over 5 months of storages, evidenced by similar particle size in CLSM images for the emulsions fresh and after 5 months of storages (Fig. 5). Stable and selfsupported emulsions were formed by 0.2% GCPs at pH 5.0, and the emulsions transformed from the liquid state to the solid state and exhibited arrested dynamics. Most important, stable Pickering HIPEs were prepared by a facile mechanically shearing route (Figs. 8 and 9). Herein, a schematic diagram for the formation pathway of GCPEs is proposed to correlate the physical performances of the emulsions with their microstructures, including interface frameworks, GCPs partition, and aggregated state of dispersed droplets (Fig. 11). Gliadin molecule consists of three domains, and the nonrepetitive domain is more hydrophobic than others, suggesting an amphiphilic feature of gliadins (Banc et al., 2007; Kasarda et al., 1984). GCPs are amphiphilic as well, which thus leads to the adsorption behavior on the oil droplet surface. The particles (~120 nm) formed stable transparent dispersions at pH ~3.0, and the cream of the emulsions concomitantly resulted in translucent aqueous lower phase (Fig. 2). This situation may be associated with relatively hydrophilic properties of the GCPs at highly acidic pH where the protonation of amino groups in gliadin happen, thus they can be preferentially wetted by water (Fig. 11A). As a consequence, the interfacial absorption of GCPs was restrained to some degree. In theory, particle charges play an important role in the formation and physical performance of Pickering emulsions. Previous studies pointed out that highly charged particles cannot stabilize emulsions because a charged particle sees an “image charge” at the oil-water interface and experiences a repulsive energy barrier when it comes close to the interface (Wang et al., 2012). Therefore, it is difficult for highly charged particles to adsorb at the oil-water interface (Wang et al., 2012), resulting in a sparse coverage at the oil-water interface. Therefore, hydrophilic GCPs may form weak steric barrier in view that they are preferentially wetted by water and most of the particles’ surfaces protrude into the continuous phase of the emulsions (Fig. 11C). Those

308

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

Fig. 9. CLSM images of Pickering HIPEs stabilized by GCPs before (up) and after (down) centrifugation (10,000g 2 min).

G' G''

G'/G''(Pa)

1000

100

10

1

10

Frequency (Hz) Fig. 10. Elastic moduli G0 and viscous moduli G00 of the GCPEs as a function of oscillatory shear frequency.

observations accounted for the instability of the emulsions during the storage and/or centrifugation. The pH value of the aqueous phase triggered the change in the overall surface charge of the GCPs, thus impacted their surface wettability (Fig. 11B). In fact, partial wettability of the surface by water and oil is the origin of the strong anchoring of solid particles at the oilewater interface. When the pH of GCPs dispersion was adjusted to 5.0, the dispersions changed to be translucent, and they settled to produce thin deposit at bottom of the vessel after 1 h of incubation. In fact, the correlation between effective Pickering stabilization and particle flocculating conditions was well established experimentally (Binks & Rodrigues, 2007), and it was also confirmed theoretically (Salari, Leermakers, & Klumperman, 2011). The tendency of GCPs to aggregate facilitates the formation of stable Pickering emulsions. For spherical particles of radius r, the free energy of spontaneous desorption (DG) is proportional to r2 (Dickinson, 2010). This means that the increase in the particle size contributed to the enhanced physical stability of the GCPEs. On the other hand, zeta potential of the GCPs decreased from

21.6 ± 0.8 mV at pH 2.9 to 3.5 ± 0.1 mV at pH 5.0 (Fig. 1). Concomitantly, the above-mentioned “image charge” effect may disappear when less charged GCPs come close to the interface, facilitating the interfacial absorption of the particles. Furthermore, the high detachment energy of adsorbed particle contributed to the stability of the emulsion made at pH 5.0 owing to the partial wettability of the particles at this pH (Fig. 11B and D). Additionally, the emulsions transformed from the liquid state to the solid state and exhibited arrested dynamics. In principle, the formation of an emulsionegel from particle-covered droplets and free particles in aqueous continuous phase could provide additional stabilization against coalescence (Fig. 11D). Therefore, GCPs with a few surface charges and intermediate wettability at pH 5.0 were proved to an effective Particulate emulsifier to stabilize oil-water interface, and a facile approach to produce stable food-grade Pickering emulsions, especially Pickering HIPEs was established in this work. When the pH of GCPs dispersions was further increased, the emulsion made at pH 8.0 and/or 9.0 presented in aggregated state (Fig. 4). The fluorescence trials visualized directly Pickering stabilization phenomenon in the emulsion. In addition, the adjacent oil droplets were closely packed with each other to form network structure. The combination of Pickering mechanism and dropletbased network contributed to the stabilization of the emulsions (Fig. 11E). This result is well in agreement with the evolution of physical performances. When the pH of GCPs dispersions were adjusted 6.0 and above, the amplitude of z epotential decreased to a few mV, leading to hydrophobic GCPs aggregates. This situation facilitated the absorption of GCPs at the oil-water interface and increased their interfacial coverage, producing stable Pickering emulsions with thick interfacial membrane formed by GCPs and/or aggregates (Fig. 11E). However, the particles quickly aggregated to form visual agglomerates during the pH adjustment, and the random aggregation behavior of the GCPs at this pH range affect the homogeneity of the products since free protein-based aggregates were detected in CSLM images of the emulsions produced at pH 9.0. In addition, it is difficult to scale this process up since rapidly extensive sedimentation of particles is difficult to overcome in a large e scale colloid particle production.

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

309

Fig. 11. Schematic diagram for the formation pathway of GCPEs as a function of pH proposed to relate the physical performance of the emulsions with their microstructure.

4. Conclusion In this paper, we demonstrate firstly the use of gliadin colloid particles (GCPs) as effective particle-stabilizers of oil-in-water emulsions of natural oils and water. We fabricated GCPs through a facile anti-solvent precipitation procedure and demonstrated their usage as a Particular emulsifier in the formation of oil-inwater Pickering emulsions as well as Pickering high internal phase emulsions (HIPEs). The o/w emulsions formed are biocompatible, edible and based on fully natural renewable resources. We found that unmodified GCPs can produce stable, surfactant-free emulsions when the amplitude of z-potential was a few mV where the particles were partially wetted by both phases. The microstructures of GCPEs as a function of pH were visualized by optical microscopy and CLSM, confirming that in addition to Pickering stabilization, GCPs-based network framework and/or dispersed droplets-based network also contributed to the stabilization of the emulsions. Interestingly, stable surfactant-free Pickering HIPEs were fabricated by a facile shearing emulsification. This study opens a promising route based on Pickering HIPEs to transform liquid oils into solid-like viscoelastic emulsion gels as a suitable alternative to solid fats, without introducing trans-fat and saturated fat. This work facilitates the development of formulation over a broader range of practical use that includes food, consumer care, and pharmaceutical related products. Acknowledgments This work was partially supported by The Project supported by the National Natural Science Foundation of China (21406077, 31471628, 31471694, 33004501), and the Pearl River S & T Nova Program of Guangzhou (201506010063). We also appreciate the financial support by Key Projects in the National Science & Technology Program during the Twelfth Five-year Plan Period (2013BAD18B10-4) and State Key Laboratory of Pulp and Paper Engineering (201536). References Akartuna, I., Studart, A. R., Tervoort, E., & Gauckler, L. J. (2008). Macroporous ceramics from particle-stabilized emulsions. Advanced Materials, 20, 4714e4718.

Aro, A., Jaughiainen, M., Partanen, R., Salminen, I., & Mutanen, M. (1997). Stearic acid, trans fatty acids, and dairy fat: effects on serum and lipoprotein lipids, apolipoproteins, lipoprotein (a), and lipid transfer proteins in healthy subjects. American Journal of Clinical Nutrition, 65, 1419e1426. Banc, A., Desbat, B., Renard, D., Popineau, Y., Mangavel, U., & Navailles, L. (2007). Structure and orientation changes of u- and g-gliadins at the air-water interface: a PM-IRRAS spectroscopy and Brewster angle microscopy study. Langmuir, 23(26), 13066e13075. Barbetta, A., & Cameron, N. R. (2004). Morphology and surface area of emulsionderived (PolyHIPE) solid foams prepared with oil-phase soluble porogenic solvents: Span 80 as surfactant. Macromolecules, 37, 3188e3201. €en, K. (2015). Pickering emulsions for food applicaBerton-Carabin, C. C., & Schro tions: background, trends, and challenges. Annual Review in Food Science and Technology, 6, 12.112.35. Bietz, J. A., Huebner, F. R., Sanderson, J. E., & Wall, J. S. (1977). Wheat gliadin homology revealed through N-terminal amino acid sequence analysis. Cereal Chemistry, 54, 1070e1083. Binks, B. P. (2002). Particles as surfactants  Similarities and differences. Current Opinion in Colloid & Interface Science, 7, 21e41. Binks, B. P., & Horozov, T. S. (Eds.). (2006). Colloidal particles at liquid interfaces. Cambridge (: Cambridge University Press. Binks, B. P., & Lumdson, S. O. (2000). Catastrophic phase inversion of water-in-oil emulsions stabilized by hydrophobic silica. Langmuir, 16, 2539e2547. Binks, B. P., & Rodrigues, J. A. (2007). Enhanced stabilization of emulsions due to surfactant-induced nanoparticle flocculation. Langmuir, 23, 7436e7439. Co, E. D., & Marangoni, A. G. (2012). Organogels: an alternative edible oil structuring method. Journal of the American Oil Chemists Society, 89(5), 749e780. Danov, K., Kralchevsky, P., Ananthapadmanabhan, K., & Lips, A. (2006). Particleinterface interaction across a nonpolar medium in relation to the production of particle-stabilized emulsions. Langmuir, 22, 106e115. Destribats, M., Rouvet, M., Gehin-Delval, C., Schmitt, C., & Binks, B. P. (2014). Emulsions stabilised by whey protein microgel particles: towards food-grade Pickering emulsions. Soft Matter, 10, 6941e6954. Dickinson, E. (2010). Food emulsions and foams: stabilization by particles. Current Opinion in Colloid & Interface Science, 15, 40e49. Dickinson, E. (2012a). Use of nanoparticles and microparticles in the formation and stabilization of food emulsions. Trends in Food Science and Technology, 24, 4e12. Dickinson, E. (2012b). Emulsion gels: the structuring of soft solids with proteinstabilized oil droplets. Food Hydrocolloids, 28, 224e241. Ezpeleta, I., Irache, J. M., Stainmesse, S., Chabenat, C., Gueguen, J., Popineau, Y., et al. (1996). Gliadin nanoparticles for the controlled release of all-transretinoic acid. International Journal of Pharmaceutics, 131, 191e200. de Folter, J. W. J., van Ruijvena, M. W. M., & Velikov, K. P. (2012). Oil-in-water Pickering emulsions stabilized by colloidal particles from the water-insoluble protein zein. Soft Matter, 8, 6807e6815. Gao, Z. M., Yang, X. Q., Wu, N. N., Wang, L. J., Wang, J. M., Guo, J., et al. (2014). Protein based Pickering emulsion and oil gel prepared by complexes of zein colloidal particles and stearate. Journal of Agricultural and Food Chemistry, 62, 2672e2678. Gianibelli, M. C., Larroque, O. R., MacRitchie, F., & Wrigley, C. W. (2001). Biochemical, genetic, and molecular characterization of wheat glutenin and its component subunits. Cereal Chemistry, 78(6), 635e646. Ikem, V. O., Menner, A., & Bismarck, A. (2008). High internal phase emulsions

310

Y.-Q. Hu et al. / Food Hydrocolloids 61 (2016) 300e310

stabilized solely by functionalized silica particles. Angewandte Chemie International Edition, 47, 8277e8279. Kalashnikova, I., Bizot, H., Cathala, B., & Capron, I. (2011). New Pickering emulsions stabilized by bacterial cellulose nanocrystals. Langmuir, 27, 7471e7479. Kasarda, D. D., Autran, J. C., Lew, E. J. L., Nimmo, C. C., & Shewry, R. P. (1983). Nterminal amino acid sequences of u-gliadins and u-secalins; Implications for the evolution of prolamin genes. Biochimica et Biophysica Acta, 747, 138e150. Kasarda, D. D., Okita, T. W., Bernardin, J. E., Baecker, P. A., Nimmo, C. C., Lew, E. J. L., et al. (1984). Nucleic (cDNA) and amino acid sequences of a-type gliadins from wheat (Triticum aestivum). Proceedings of the National Academy of Sciences, 81, 4712e4716. Knudsen, J. C., Øgendal, L. H., & Skibsted, L. H. (2008). Droplet surface properties and rheology of concentrated oil-in-water emulsions stabilized by heat-modified blactoglobulin B. Langmuir, 24, 2603e2610. Lam, S., Velikov, K. P., & Velev, O. D. (2014). Pickering stabilization of foams and emulsions with particles of biological origin. Current Opinion in Colloid & Interface Science, 19, 490e500. Lissant, K. J., Peace, B. W., Wu, S. H., & Mayhan, K. G. (1974). Structure of highinternal-phase-ratio emulsions. Journal of Colloid and Interface Science, 47, 416e423. Liu, F., & Tang, C. H. (2013). Soy protein nanoparticle aggregates as Pickering stabilizers for oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 61, 8888e8898. Liu, H., Wang, C., Zou, S., Wei, Z., & Tong, Z. (2012). Simple, reversible emulsion system switched by pH on the basis of chitosan without any hydrophobic modification. Langmuir, 28, 11017e11024. Lomova, M. V., Sukhorukov, G. B., & Antipina, M. N. (2010). Antioxidant coating of micronsize droplets for prevention of lipid peroxidation in oil-in-water emulsion. ACS Applied Materials and Interfaces, 2, 3669e3676. Luo, Z. J., Murray, B. S., Ross, A. L., Povey, M. J. W., Morgan, M. R. A., & Day, A. J. (2012). Effects of pH on the ability of flavonoids to act as Pickering emulsion stabilizers. Colloids and Surfaces B-Biointerfaces, 92, 84e90. Ma, W., Tang, C. H., Yin, S. W., & Yang, X. Q. (2013). Fabrication and characterization of kidney bean (Phaseolus vulgaris L.) protein isolate chitosan composite films at acidic pH. Food Hydrocolloids, 31, 237e247. Menner, A., Ikem, V., Salgueiro, M., Shaffer, M. S. P., & Bismarck, A. (2007). High internal phase emulsion templates solely stabilised by functionalised titania nanoparticles. Chemical Communication, 41, 4274e4276. Mezzenga, R. (2007). Equilibrium and non-equilibrium structures in complex food systems. Food Hydrocolloids, 21, 674e682. Nikiforidis, C. V., & Scholten, E. (2015). High internal phase emulsion gels (HIPEgels) created through assembly of natural oil bodies. Food Hydrocolloids, 43,

283e289. Okita, T., Cheesbrough, V., & Reeves, C. D. (1985). Evolution and heterogeneity of the alpha-/beta-type and gamma-type gliadin DNA sequences. Journal of Biological Chemistry, 260(13), 8203e8213. €o € , M., Dejmek, P., & Wahlgren, M. (2014). Rayner, M., Marku, D., Eriksson, M., Sjo Biomass-based particles for the formulation of Pickering type emulsions in food and topical applications. Colloids and Surfaces A: Physicochemical Engineering Aspects, 458, 48e62. Rogers, M. A., Wright, A. J., & Marangoni, A. G. (2009). Oil organogels: the fat of the future? Soft Matter, 5, 1594e1596. Rousseau, D. (2013). Trends in structuring edible emulsions with Pickering fat crystals. Current Opinion in Colloid & Interface Science, 18, 283e291. Sahoo, S., Kumar, N., Bhattacharya, C., Sagiri, S. S., Jain, K., Pal, K., et al. (2011). Organogels: properties and applications in drug delivery. Designed Monomers and Polymers, 14(2), 95e108. Salari, J. W. O., Leermakers, F. A. M., & Klumperman, B. (2011). Pickering emulsions: wetting and colloidal stability of hairy particles-A self-consistent field theory. Langmuir, 27, 6574e6583. Schrade, A., Landfester, K., & Ziener, U. (2013). Pickering-type stabilized nanoparticles by heterophase polymerization. Chemical Society Reviews, 42, 6823e6839. Tzoumaki, V. M., Moschakis, T., Kiosseoglou, V., & Biliaderis, G. C. (2011). Oil-inwater emulsions stabilized by chitin nanocrystal particles. Food Hydrocolloids, 25, 1521e1529. Wang, L. J., Hu, E. K., Yin, S. W., & Yang, X. Q. (2014). Fabrication and characterization of water-soluble antimicrobial gliadin nanoparticles. Modern Food Science and Technology, 30(5), 1e5. Wang, L. J., Hu, Y. Q., Yin, S. W., Yang, X. Q., Lai, F. R., & Wang, S. Q. (2015). Fabrication and characterization of antioxidant Pickering emulsions stabilized by zein/ chitosan complex particles (ZCPs). Journal of Agricultral and Food Chemistry, 63, 2514e2524. Wang, H., Singh, V., & Behrens, S. H. (2012). Image charge effects on the formation of Pickering emulsions. The Journal of Physical Chemistry Letters, 3, 2986e2990. Xiao, J., Li, C., & Huang, Q. R. (2015). Kafirin nanoparticles-stabilized Pickering emulsions as oral delivery vehicles: physicochemical stability and in vitro digestion profile. Journal of Agricultral and Food Chemistry, 63(47), 10263e10270. Yusoff, A., & Murray, B. S. (2011). Modified starch granules as particle-stabilizers of oil-in-water emulsions. Food Hydrocolloids, 25, 42e55. Zou, Y., Guo, J., Yin, S. W., Wang, J. M., & Yang, X. Q. (2015). Pickering Emulsion gels prepared by hydrogen-bonded zein/tannic acid complex colloidal particles. Journal of Agricultral and Food Chemistry, 63, 7405e7414.