gum Arabic

Food Hydrocolloids 100 (2020) 105381

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One-step preparation of high internal phase emulsions using natural edible Pickering stabilizers: Gliadin nanoparticles/gum Arabic

T

Li Maa, Liqiang Zoua,∗, David Julian McClementsb,∗∗, Wei Liua,c a

State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang, 330047, Jiangxi, China Biopolymers & Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA, 01003, United States c National R&D Center for Freshwater Fish Processing, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Gliadin nanoparticles Gum Arabic High internal phase emulsions Pickering emulsions β-carotene Bioaccessibility

Pickering high internal phase emulsions (HIPEs) were prepared using a simple one-step process that involved blending an aqueous solution (15 vol%) containing gliadin nanoparticles (GNP, 2 wt%) and gum arabic (GA, 0, 1, 2, and 4 wt%) with corn oil (85 vol%). The impact of GA addition into HIPEs on the microstructure, rheology, stability, photodegradation and bioaccessibility of the encapsulated β-carotene was determined. The mean particle diameter of the HIPEs decreased as the GA concentration was raised. HIPEs stabilized by GNP/GA had a higher apparent viscosity and storage modulus than those stabilized by GNP alone. HIPEs stabilized by GNP/GA were formed a compact three-dimensional network that was relatively stable to pH, ionic strength, and temperature changes. Moreover, the GA improved the stability of β-carotene encapsulated within the oil droplets and did not affect lipid digestion or carotenoid bioaccessibility. In summary, our results suggest that GNP/GA complexes may be used as effective Pickering stabilizers for creating HIPEs that could be used in the food and nutrition industries.

1. Introduction HIPEs are semi-solid viscoelastic materials that can be formulated to have relatively high stabilities to droplet aggregation and creaming (Liu, Gao, et al., 2019a, b). Moreover, they can be used to encapsulate functional food ingredients within the oil droplets or the surrounding aqueous domains (Wei & Huang, 2019a, 2019b; Weigel, Weiss, Decker, & McClements, 2018). These traits make them suitable for various application in the food, cosmetics, personal care, and pharmaceutical industries (Caldero, Llinas, Jose Garcia-Celma, & Solans, 2010; Lee, 2003; Wijaya, Van der Meeren, Dewettinck, & Patel, 2018). If they are going to find application within commercial products, then HIPEs must be carefully formulated to prevent them from breaking down during storage and utilization (Zank et al., 2006). For food applications, they must also be capable of being produced economically from edible ingredients. Pickering stabilizers, which typically consist of solid or semi-solid particles, can be used to form stable HIPEs (Zamani, Malchione, Selig, & Abbaspourrad, 2018). Pickering HIPEs have some advantages over the conventional type, such as a high resistance to Ostwald ripening, coalescence, and phase separation (Xiao, Li, & Huang, 2016). This kind of

∗

HIPE has become a research hotspot because of their applications in a broad range of commercial products (Jiao, Shi, Wang, & Binks, 2018; Menner, Ikem, Salgueiro, Shaffer, & Bismarck, 2007). Traditionally, inorganic solid particles have been used for stabilizing Pickering HIPEs, such as those comprised of silica (Kim et al., 2017) or graphene oxide (Yi, Wu, Wang, & Du, 2016). This type of particle is, however, unsuitable for many food applications because consumers are demanding “clean” label products. For this reason, there has been profound interest in developing Pickering HIPEs prepared using food-grade particles, such as those consisting of proteins and/or polysaccharides (Xiao et al., 2016; Xu, Tang, Liu, & Liu, 2018). Gliadin is a wheat protein containing hydrophilic amino acids (glutamine and proline) in its central region and hydrophobic amino acids in its terminal regions. The terminal regions of gliadin are generally more hydrophobic than the central region, which leads to an amphiphilic character that enables it to become nanoparticles by selfassembly (Banc et al., 2007; Kasarda et al., 1984; Veraverbeke & Delcour, 2002). These protein nanoparticles can be utilized to prepare emulsions by Pickering mechanism (Joye & McClements, 2013). Additionally, gliadin nanoparticles (GNPs) can be used to promote the formation and enhance the stability of foams by absorbing to gas-liquid

Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, Jiangxi, China. Corresponding author. Department of Food Science, University of Massachusetts, Amherst, MA, 01003, USA. E-mail addresses: [email protected] (L. Zou), [email protected] (D.J. McClements).

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https://doi.org/10.1016/j.foodhyd.2019.105381 Received 8 June 2019; Received in revised form 2 August 2019; Accepted 11 September 2019 Available online 12 September 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.

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hypothesized that the formation and stability of the Pickering HIPEs would be improved by the one step preparation method. The rheology, microstructure, stability, and encapsulation properties of the Pickering HIPEs were measured. The knowledge produced in this study could lead to the development of a new class of edible soft-solid materials that could be used in foods and other products to create desirable functional attributes.

interfaces (Peng et al., 2017). Pickering emulsions prepared from protein nanoparticles (like zein or gliadin) are often highly unstable on certain conditions. For instance, Hu, et al. (2016) reported that Pickering HIPEs prepared from gliadin nanoparticles were unstable under acidic conditions. The performance of protein nanoparticles as Pickering stabilizers can, however, be improved by modifying their surface properties (Li et al., 2019; Sedaghat Doost et al., 2019; Yuan et al., 2017). For example, modifying the surface properties of gliadin nanoparticles by adding cationic chitosan improved their ability to form and stabilize Pickering HIPEs (Yuan et al., 2017). Even so, this general approach of improving the performance of protein nanoparticles in Pickering HIPEs does have some limitations. The incorporation of surface modifiers often promotes aggregation of the protein nanoparticles through strong electrostatic attraction (Zhou et al., 2018). Consequently, the fabrication conditions have to be rigorously controlled to avoid this problem. Moreover, some surface modifiers reduce the ability of the protein nanoparticles to form and stabilize Pickering HIPEs by creating unfavorable surface wettability characteristics (Li et al., 2019). Consequently, it is important to develop alternative methods to prepare stable Pickering HIPEs, which can overcome these potential limitations. Gum arabic (GA) is an amphiphilic polysaccharide that can adsorb on the surfaces of oil droplets and enhance their resistance to pH, ionic strength, and temperature changes (Ozturk, Argin, Ozilgen, & McClements, 2015). However, GA is not a strongly surface-active molecule and high concentrations are typically needed to produce stable emulsions. This problem can be overcome by forming GA-protein complexes. The proteins rapidly adsorb on the surfaces of oil droplets formed during homogenization, thereby leading to the production of small oil droplets at low protein levels. The GA then forms a hydrophilic shell that provides good stability by generating strong steric and electrostatic interactions. This strategy has been used to prepare and stabilize emulsions using combinations of GA and various types of proteins, such as soybean, wheat, rice, milk, and egg proteins (Li et al., 2019; Niu et al., 2017; Wang, Wang, Li, Adhikari, & Shi, 2011; Wei & Huang, 2019a, 2019b; Xu, Luo, Liu, & McClements, 2017). Wu, Kong, Zhang, Hua, and Chen (2018) reported that the stability of gliadin nanoparticles could also be improved by adding GA. We hypothesized that GNP/GA complexes may also be suitable for the fabrication of Pickering HIPEs. And for all we know, there has been no previous research on this subject. One of the problems associated with this approach is that extensive aggregation occurs when GNPs are mixed with GA at the relatively high concentrations required for preparing Pickering HIPEs. For this reason, a one-step preparation method means that add GNPs and GA solution to oil phase at the same time while emulsification, which was developed to fabricate Pickering HIPEs stabilized by GNP/GA complexes to overcome this problem. Compared with conventional methods of modifying nanoparticles, the one-step preparation could avoid extensive aggregation before preparing emulsions. One of the potential applications of HIPEs is to encapsulate and deliver functional ingredients, such as nutraceuticals, vitamins. β-carotene is a natural nutraceutical reported to exhibit biological activities (Paiva & Russell, 1999). The use of this carotenoid as a nutraceutical in many foods, however, is limited due to its poor water solubility and its sensitivity to chemical degradation when exposed to oxygen or light (Boon, McClements, Weiss, & Decker, 2010). Previous studies have indicated that O/W emulsions can be used as effective colloidal delivery systems for β-carotene, which can be designed to enhance its waterdispersibility, bioaccessibility and/or chemical stability (Dai, Sun, Wei, Mao, & Gao, 2018; Mao, Wang, Liu, & Gao, 2018; Tan et al., 2017). In this study, β-carotene was selected as a model hydrophobic nutraceutical to evaluate the encapsulation, protection, and delivery capabilities of the Pickering HIPEs. In the present study, the one-step procedure was used to prepare the Pickering HIPEs involved adding aqueous GNP and GA solution to corn oil and immediately blending with a high-shear mixer. We

2. Materials and methods 2.1. Materials Wheat gluten (protein content ≥ 85%) was provided by Xunxian Tianlong Flour Co., Ltd. (Hebi, China). Gum arabic and β-carotene (≥96%) were purchased from Aladdin Industrial Corporation (Shanghai, China). Corn oil was purchased from Yi Haikerry Grain and Oil Food Company (Jiangxi, China). Mucin (M2378), pepsin (P7125; enzymatic activity of ≥400 units/mg protein), lipase (L3126; enzymatic activity of 100–500 units/mg protein using olive oil), pancreatin from porcine pancreas (P1750; 4 × US Pharmacopeia (USP) 98 specifications), Nile red (72,485) and Nile blue A (N0766) were purchased from the Sigma Chemical Company (St. Louis, MO). All other reagents were OF analytical grade. 2.2. Fabrication of GNPs and GA solution Gliadin with a protein purity of 92% was extracted from the wheat gluten using a 70 vol% ethanol solution (the ratio of wheat gluten/ ethanol solution was 1:10). Then, this solution was stirred for 5 h at 35 °C and centrifuged (3355×g, 15 min) to remove the insoluble components. The supernatant was gathered and ethanol was eliminated by vacuum-rotary evaporation at 50 °C. Finally, gliadin protein powders were obtained after freeze-drying for 48 h. Gliadin molecular weight profile was analysed by SDS-PAGE (Fig. S1). The bands around 32 kDa are the deepest, indicating gliadin mainly contains α-gliadin, β-gliadin and γ-gliadin (28–35 kDa). The polypeptide composition of the gliadin is similar to the results of Chen et al. (2019). GNPs were fabricated by an anti-solvent precipitation method based on our previous studies (Chen, McClements, Wang, et al., 2018a). Briefly, gliadin was dissolved in 70 vol% ethanol solution. This solution was then injected into deionized water (pH 4.0) at the volume ratio of 1:4. Then this solution was stirred continuously to form the nanoparticle suspensions (1000 rpm, 10 min). The ethanol in these suspensions was then removed by rotary evaporation (55 °C). Then, the resultant nanoparticle suspensions were adjusted to a concentration of 20 mg/mL using distilled water and the final pH was adjusted to 4.0 by HCl or NaOH solutions. The average size and zeta potential of GNPs were 251.3 ± 5.1 nm and 29.3 ± 1.5 mV. GA solutions (0, 1, 2, and 4 wt%) were prepared by stirring GA into deionized water until it fully dissolved. The resulting GA solutions were then adjusted to pH 4.9 using HCl or NaOH solutions as described previously (Wu et al., 2018). 2.3. Preparation of pickering HIPEs Pickering HIPEs were prepared on the basis of a method in previous study (Zhu, Chen, McClements, Zou, & Liu, 2018). Briefly, aqueous GNP (2 wt%) and GA solution (0, 1, 2, and 4 wt%) at 1:1 ratio (15 vol%) were added to corn oil (85 vol%) and then immediately blended with a high-shear mixer (ULTRA TURRAX® T18 digital, IKA, Staufen, Germany) at 12,000 rpm for 2 min. 2.4. Characterization of pickering HIPEs 2.4.1. Particle size and ζ-potential measurements The particle size of the HIPEs was measured using a static light 2

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using the analytical centrifuge and CLSM methods described earlier (Section 2.5.1). The initial phases were recorded after equilibrate the samples for 6h at different salt concentrations.

scattering instrument (Malvern 3000, Malvern Instruments Ltd, Worcestershire, UK). The particle size values were expressed by surfaceweighted mean diameters (d3,2). A particle electrophoresis instrument (Zetasizer Nano ZSP, Malvern Instruments, Worcestershire, UK) was used to measure the ζ-potential of the HIPEs. All measurements were taken at 25 °C. The results reported represent an average of at least three independent readings per sample.

2.5.3. Thermal-stability The influence of thermal treatment on the properties of the HIPEs was also examined. HIPEs were held at 90 °C for 30 min and then cooled to 25 °C. Any variations in visual appearance of the HIPEs after heating treatment were recorded by camera. The stability of the HIPEs was also characterized using the analytical centrifuge and CLSM methods described above (Sections 2.5.1 and 2.5.2).

2.4.2. Microstructure Microstructures of the HIPEs were observed by a confocal laser scanning microscope (CLSM) (Carl Zeiss LSM710, Jena, Germany) using a 63 × objective at 25 °C, according to a method described previously (Qiu, Zhao, & McClements, 2015). Briefly, oil phase and protein phase were stained Nile Red (1 mg/mL) and Nile Blue A (1 mg/mL), respectively. The samples were mixed with dye at 50:1 ratio. The HIPEs were then prepared and poured into the glass bottom of cell culture dish. The HIPEs were observed by two laser excitation sources (488 nm and 633 nm) and two reception channels.

2.6. Photodegradation of encapsulated β-carotene The degradation of the encapsulated β-carotene when exposed to UV-light was measured. Initially, β-carotene (0.1 mg/mL) was added to the corn oil and then this sample was stirred continuously at 95 °C for 30 min with sonication treatment for 5 min. The β-carotene-loaded HIPEs were then prepared using the same procedure described in Section 2.3. The UV stability of the carotenoids was measured using the method reported by Lu, Mao, Cui, Yuan, & Gao (2019). Briefly, 6 g of β-carotene-loaded HIPEs were spread onto a 60 × 10 mm (diameter × height) dish. The samples were then exposed in a UV chamber equipped with four UV lamps with a power of 15 W and wavelength of 312 nm at room temperature. 1 mL of dimethylsulfoxide (DMSO) and 2 mL of n-hexane were then used to extract the β-carotene from 0.2 g βcarotene-loaded HIPEs after incubation for 0, 24, 42, 50, 66 and 72 h. The extraction of β-carotene was repeated twice using the same amount of n-hexane. The extracts were then combined, and the absorbance was measured at 450 nm using a UV–visible spectrophotometer. The carotenoid concentration was then determined from a calibration curve prepared by dissolving known quantities of β-carotene in n-hexane.

2.4.3. Rheological properties A Rotary Rheometer (MCR302, Anton Paar, Germany) with a plateand-plate geometry (pp-50, a diameter of 50 mm) was used to analyze the rheological properties of Pickering HIPEs based on a method described previously (Xi, Liu, McClements, & Zou, 2019). HIPEs were uniformly applied to the measurement cell and waited for 5 min to achieve the set temperature (25 °C). The linear viscoelastic region (LVR) was confirmed by a dynamic strain sweep. The strain was increased logarithmically from 0.1% to 100% at a constant frequency of 1 Hz. The frequency sweeps were performed from 0.1 to 100 rad/s at a fixed strain of 1%. The apparent viscosity was recorded as the shear rate was increased from 0.1 to 100 s−1. The elastic modulus (G′) and loss modulus (G″) were measured using oscillatory frequency sweep measurements from 0.1 to 100 rad/s. All HIPEs were measured in triplicate and the average value is reported.

2.7. Stability and bioaccessibility of β-carotene during in vitro digestion 2.7.1. In vitro digestion model The potential gastrointestinal fate of the HIPEs was determined by a simulated GIT model based on the method described previously (Liu, Wang, McClements, & Zou, 2018; Minekus et al., 2014). The preparation of simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) was the same as described in our previous study (Zou et al., 2015). All solutions were heated to 37 °C before the experiment and kept at 37 °C during the experiment. Mouth stage: Mucin (3.0 mg/mL) was dissolved in SSF around 1 h earlier. This solution (7.5 mL) was added to the initial samples (7.5 mL), adjusted pH of this solution to 6.8 and then stirred at 100 rpm for 10 min. Gastric stage: Pepsin (3.2 mg/mL) were also dissolved in SGF in advance. The samples (15 mL) from the mouth stage were mixed with this solution (15 mL). This mixed solution was then adjusted pH to 2.5 and stirred for 2 h (100 rpm) to simulate gastric conditions. Small intestine stage: The pH of digest (30 mL) after the gastric stage was adjusted to 7.0. SIF (1.5 mL), bile salt solution (3.5 mL), lipase solution (2.5 mL) and pancreatin solution (2.5 mL) were then added to the samples. Bile salts and enzymes were dissolved in phosphate buffer solution (5 mM, pH 7.0). Then, the pH of this solution was monitored and kept at pH 7.0 by titrating 0.1 M sodium hydroxide standard solution into the vessel for 2 h.

2.5. Environmental stability of HIPEs 2.5.1. pH-stability The influence of pH (3–7) on the properties of the HIPEs was examined. The pH was adjusted by adding different amounts of either HCl or NaOH solution. The pH stability of the HIPEs was performed after equilibrating the samples with different pH values for 6h. Any variations in visual appearance of the HIPEs were recorded by camera. The physical stability of the HIPEs were determined by an analytical centrifuge (LUMiFuge, LUM GmbH, Berlin, Germany) (Shimoni, Shani Levi, Levi Tal, & Lesmes, 2013). The intensity of transmitted light measured by this instrument as a function of position and time of samples stored in specific test tubes (the optical path was 2 mm). The HIPEs were injected into the test tubes and centrifuged at 2000 rpm for 2 h. The “instability index” was recorded and calculated by the instrument software (SepView 6.0, LUM, Berlin, Germany), which is a dimensionless number between 0 (very stable) and 1 (very unstable). The influence of pH on the structure of the HIPEs was further examined by CLSM as described in Section 2.4.2. The initial phases were recorded after equilibrate the samples for 6h at different pH values. 2.5.2. Salt-stability The influence of salt concentration on the properties of the HIPEs was also examined. HIPEs containing different NaCl concentrations (0–200 mM) were formed by mixing the Pickering HIPEs with different ratios of NaCl stock solution (5 mol/L). The Pickering HIPEs were then gently stirred for 1 min to make them homogeneous. The salt stability of the HIPEs was performed after equilibrating the samples with different NaCl concentrations for 6h. Any variations in visual appearance of the HIPEs before and after storage were recorded by camera. The physical stability of HIPEs with different ionic strengths was also characterized

2.7.2. Lipid digestion For the lipid digestion experiments, the HIPEs were adjusted to a final corn oil content of 0.5 wt% using ultrapure water. A pH-stat automatic titration appliance (Metrohm 907 Titrando, Metrohm, Switzerland) was utilized to monitor the free fatty acid (FFA) released during the intestine stage as described in an earlier study (Chen, McClements, Zhu, et al., 2018b; Ma, Tu, Wang, Zhang, & McClements, 3

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creaming and phase separation. In addition, their mean particle diameter (d3,2) decreased from around 9.4 to 6.0 μm (Fig. 1a). These results show that the presence of the GA led to smaller oil droplets being generated during the production of the HIPEs. Possible reasons for this phenomenon are: (1) during homogenization, GNP/GA flocculation formed by electrostatic attraction and adsorbed to the oil-water interfaces, which promoted droplet disruption and stabilization; (2) after homogenization, GA increased the viscosity of the emulsions, thereby preventing the droplets from coming close together and inhibiting droplet coalescence (Thanasukarn, Pongsawatmanit, & McClements, 2004). The ζ-potential of the HIPEs was moderately cationic (+22 mV) in the absence of GA but strongly anionic (around −40 mV) in its presence, with the magnitude of the negative charge increasing with GA concentration (Fig. 1b). The fact that the charge went from positive to negative suggests that the oil droplets were coated by a layer of anionic GA. Similar results were also reported by other researchers (Dai et al., 2018; Hu & McClements, 2015). Consequently, there is likely to be a strong electrostatic and steric repulsion between the oil droplets, which may be another reason for the decrease in particle size observed in the presence of GA (Li et al., 2019).

2018). The percentage of free fatty acids (FFA %) released from the HIPEs was calculated from the amount of NaOH standard solution used. The percent of FFA released during the digestion was calculated by this equation: FFA (%) = (CNaOH*VNaOH*Moil*100) / (2*moil) Here, CNaOH is the molarity of the NaOH solution (M), VNaOH is the volume of NaOH solution (L) used, Moil is the mean molecular weight of the oil (g/mol) and moil is the total mass of oil (g). 2.7.3. Stability and bioaccessibility of β-carotene For β-carotene bioaccessibility determination, a weighed amount of raw digesta was centrifuged (15,000 g) at 4 °C for 30 min and then the middle micelle phase was collected. The extraction process of β-carotene was based on previous studies (Park, Mun, & Kim, 2018). Then, the extracted β-carotene were combined for UV–visible spectrophotometer analysis (450 nm). The stability and bioaccessibility of βcarotene were calculated using the following expressions described in previous studies (Lin, Liang, Ye, Singh, & Zhong, 2017; Zhang et al., 2016). Stability (%) = 100 × CD/CI

(1)

Bioaccessibility (%) = 100 × CB/CD

(2)

3.1.2. Microstructure The impact of GA addition on the microstructure of the HIPEs was studied using confocal fluorescence microscopy. The oil and protein phases were stained green and red, respectively, using fluorescent dyes (Fig. 2). In the absence of GA, the oil droplets appeared fairly big and there were large spaces between them. There also appeared to be an appreciable fraction of non-adsorbed GNPs dispersed within the aqueous phase (Liu, Gao, et al., 2019a). Conversely, in the presence of GA, the oil droplets were smaller and more closely packed together. Moreover, the GNPs all seemed to be located at the oil droplet surfaces in this case. At 1 and 2 wt% GA, the majority of oil droplets had a spherical appearance, but at 4 wt% GA many of the larger oil droplets were deformed into polygonal shapes. This effect was probably because the GA molecules occupied more space and so the droplets were forced more closely together.

Here, CD, CI, and CB are the β-carotene concentrations in the raw digesta, initial samples, and in the clear middle layer (bioaccessible form), respectively.

2.8. Statistical analysis Each experiment was conducted in at least triplicate and results are expressed as the mean ± standard deviations. All of the statistical analysis was proceed using Origin 2017 64 Bit software. 3. Results and discussion 3.1. Preparation and characterization of HIPEs

3.1.3. Rheological properties Shear rheology measurements were used to provide information about the impact of GA addition on the mechanical properties of the HIPEs. Extensive shear-thinning was observed in all the HIPEs, i.e., the apparent viscosity decreased appreciably with increasing shear rate (Fig. 3a). The apparent viscosity increased appreciably when 1 wt% of GA was added to the HIPEs, but did not change further when higher levels were added. This effect suggests that the impact of the GA on the

3.1.1. Particle size and ζ-potential Images of HIPEs prepared with a fixed concentration of GNPs but varying concentrations of GA (0, 1, 2, and 4 wt%) were recorded (Fig. 1b). The GNPs-stabilized HIPEs were not stable and a thin oil layer appeared on the top of tubes, this phenomenon was also consistent with Fig. 2. However, the appearance of the HIPEs did not change noticeably as the GA concentration increased from 1 to 4 wt% and showed no

Fig. 1. The average particle size (d3,2) (a) and visual appearance and ζ-potential (b) of HIPEs prepared by GNP with GA (0, 1, 2 and 4 wt%). Different superscript letters among bars denote significant difference (P < .05) according to Turkey's test. 4

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Fig. 2. CLSM images of HIPEs prepared by GNP with GA (0, 1, 2 and 4 wt%): (A) oil stain (excitation at 488 nm); (B) protein stain (excitation at 630 nm); (C) combined image of panels A and B.

Fig. 3. Rheological properties of HIPEs prepared by GNP with GA (0, 1, 2 and 4 wt%). Apparent viscosity of the HIPEs with shear rate from 0.1 to 100 1/s (a); Frequency sweeps curves at fixed stain (1%) with frequency ranging from 0.1 to 100 rad/s (b). 5

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were highly susceptible to droplet creaming, coalescence, and oiling off from pH 3 to 5, but were stable at pH 6 and 7 (Fig. 4a). Conversely, HIPEs formed using GNP/GA showed good physical stability at all pH values evaluated. After 7 days storage, there were no obvious changes in their appearance, with no visible evidence of creaming or oiling off. Moreover, the samples did not flow to the bottom of the test tubes when they were inverted, indicating that they maintained their mechanical integrity. The instability index of the HIPEs was measured using a centrifugation method to provide further insights into the impact of pH on emulsion properties. In the absence of GA, the instability index of the HIPEs was much higher from pH 3 to 5 than from pH 6 to 7 (Fig. 4b). Moreover, the microstructure images showed that the oil droplets were relatively far apart from pH 3 to 5, but closely packed together at pH 6 and 7 (Fig. 4c). These effects can be attributed to a strong electrostatic repulsion between the highly cationic gliadin-coated oil droplets at low pH values (which prevents them from coming close together), but a weak electrostatic repulsion at high pH values due to their low surface potential near the isoelectric point (which promotes their flocculation) (Zhu et al., 2018). In the presence of GA, the instability index was relatively low at all pH values, indicating that the GA enhanced the pH-stability of the HIPEs (Fig. 4b). Moreover, the microstructure images indicated that the oil droplets were relatively small and closely crowded together at all pH values (Fig. 4c). A previous study also reported that GA improved the pH-stability of Pickering emulsions (Dai et al., 2018). The good pHstability of the HIPEs in the presence of GA may have been a result of various phenomena. First, the GA may have formed a protective coating around the individual oil droplets, thereby protecting them from coalescence, leading to small oil droplets that were closer together. Second,

rheology of the HIPEs was mainly due to its ability to form a coating around the surfaces of the oil droplets, rather than its ability to thicken the aqueous phase. Indeed, it is well known that GA is a relatively small compact molecule that is not particularly effective at thickening aqueous solutions (Bai et al., 2017). The influence of GA addition on the storage modulus (G′) and loss modulus (G″) of the HIPEs was also measured as a function of frequency (Fig. 3b). The G′ and G″ values increased substantially when 1 wt% GA was added, but did not change appreciably when further amounts were added. For all samples, G′ was much larger than G″ over the frequency range studied, indicating that the HIPEs were predominantly elastic in both the absence and presence of GA. The higher G′ and G″ of the HIPEs containing GA may have been due to their smaller droplet size, stronger droplet-droplet interactions, and/or stronger interfacial coatings (Jain, Winuprasith, & Suphantharika, 2019). This interfacial coating would thicken as the increase of GA concentration as shown in the CLSM images. However, it is the formation of the coating, not the thickness, that plays a significant role in the G′ and G″ of HIPEs. In the following experiments, HIPEs were formed using a fixed GA content (4 wt%) because of the their smaller particle size and polygonal shapes.

3.2. Environmental stability of HIPEs 3.2.1. pH-stability Different foods have different pH values, and foods experience alterations in pH when they pass through different regions of the human gut. It is, therefore, useful to evaluate the impact of pH on the physical stability of the HIPEs prepared in this study. The visual appearance, stability, and microstructure of HIPEs with different pH values were therefore measured. After 7 days storage, HIPEs formed using only GNP

Fig. 4. The visual appearance (a), instability index (b) and microscopy images (c) of GNP HIPEs and GNP/GA HIPEs at different pH values. Different superscript letters among bars denote significant difference (P < .05) according to Turkey's test. 6

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Fig. 5. The visual appearance (a), instability index (b) and microscopy images (c) of GNP HIPEs and GNP/GA HIPEs at different salt concentrations. Different superscript letters among bars denote significant difference (P < .05) according to Turkey's test.

there may have been some bridging flocculation between the GA and GNP-coated oil droplets, leading to the formation of a stronger gel network that inhibited droplet movement. To sum up, the relative stable GNP Pickering HIPEs at pH 7 would be applied to the following experiments.

results suggest that the presence of GA greatly improved the salt-stability of the HIPEs, which was probably due to a similar mechanism to how they improve the pH-stability. 3.2.3. Thermal-stability Foods are often subjected to thermal processes during their manufacture or utilization. For this reason, the influence of a heat treatment (90 °C for 30 min) on the stability of the samples was determined. In the absence of GA, the HIPEs exhibited appreciable creaming and some oiling off after heating (Fig. 6a). Similarly, there was a large increase in their instability determined by centrifugation (Fig. 6b) and large gaps were seen between the oil droplets in the microscopy images (Fig. 6c). Thus, the GNP-HIPEs did not have good resistance to thermal processing, which may have been due to unfolding and aggregation of the gliadin molecules after heating. Conversely, in the presence of GA, the HIPEs were much more stable to the thermal treatment. There was no large change in their appearance (Fig. 6a), instability index (Fig. 6b), or microstructure (Fig. 6c). These results show that GA addition was highly effective at increasing the thermal-stability of the HIPEs, which may have been due to their ability to form a protective coating around the GNP-coated oil droplets, as well as forming a three-dimensional network around the oil droplets.

3.2.2. Salt-stability HIPEs may also be used in commercial products with different salinity, so it is significant to evaluate the impact of salt concentration on their properties. Consequently, the impact of salt addition on the appearance, microstructure, and stability of the HIPEs were determined. In the absence of GA, the HIPEs were highly sensitive to salt, undergoing extensive flocculation even in the presence of relatively low levels, i.e., 50 mM NaCl (Fig. 5a). After 7 days storage at room temperature, extensive oiling off was observed in all the HIPEs containing salt. Centrifugation analysis also indicated that the HIPEs were unstable in the presence of salt (Fig. 5b). Finally, the microstructure images indicated that many of the gliadin nanoparticles aggregated in the presence of salt (Fig. 5c). In summary, these results show that the GNPHIPEs became highly unstable to creaming and oiling off when salt was added. Presumably, the salt weakened the electrostatic repulsion between the oil droplets, allowing them to come into closer contact, thereby promoting coalescence (Dickinson, 2019). Conversely, in the presence of GA, the HIPEs had good stability at all salinity levels (0–200 mM). After 7 days storage at room temperture, there was no change in their visual appearance and they did not flow to the bottom of the test tubes when they were inverted (Fig. 5a). Centrifugation analysis also indicated that the GNP/GA-HIPEs were stable to separation at all salt levels (Fig. 5b). Finally, the microstructure images indicated that the oil droplets in these samples remained relatively small and closely packed together (Fig. 5c). Taken together, these

3.3. Photodegradation of encapsulated β-carotene The ability of the HIPEs to protect carotenoids (β-carotene) from chemical degradation when exposed to UV-light was also examined. The β-carotene content of the HIPEs decreased with increasing exposure time for both systems, but the level of carotenoids remaining was always higher in the HIPEs containing GA (Fig. 7). For instance, the amount of β-carotene remaining in the HIPEs after 72 h exposure to UV radiation was only around 23% in the absence of GA, but about 45% in 7

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Fig. 6. The visual appearance (a), instability index (b) and microscopy images (c) of GNP HIPEs and GNP/GA HIPEs before and after thermal treatment. Different superscript letters among bars denote significant difference (P < .05) according to Turkey's test.

Fig. 8. Visual appearance of HIPEs digesta from each GIT stage.

its presence. The slower rate of β-carotene degradation in the GNP/GAHIPEs might because the encapsulated carotenoids were better protected from pro-oxidants located in the aqueous phase surrounding the oil droplets. Fu et al., (2019) also reported that the good protective effect of WGN-XG Pickering emulsions on β-carotene was due to the

Fig. 7. Residual β-carotene levels in the GNP and GNP/GA HIPEs during UV radiation.

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Fig. 9. (a) FFA release profile for the GNP and GNP/GA HIPEs. (b) Stability and bioaccessibility of β-carotene in the GNP and GNP/GA HIPEs. Different superscript letters among bars denote significant difference (P < .05) according to Turkey's test.

digestion or carotenoid bioaccessibility. These edible soft solids may be useful as functional materials in foods, cosmetics, or other commercial products.

presence of a thick biopolymer layer around the oil droplets. 3.4. Stability and bioaccessibility of β-carotene during in vitro digestion

Conflicts of interest

The impact of GA on the stability and bioaccessibility of β-carotene during passage of the HIPEs through a simulated gastrointestinal tract (GIT) were also determined. This was achieved by measuring changes in the appearance, lipid digestion profiles, and β-carotene bioaccessibility of the HIPEs. There were some differences in the visual appearances of the samples after exposure to each GIT stage (Fig. 8). In particular, there appeared to be less particle aggregation and oiling-off in the samples containing the GA in the earlier stages of the GIT model. This phenomenon is probably due to the ability of the GA to improve the pH and salt stability of the oil droplets. The FFA release profiles of the HIPEs were fairly similar in the absence and presence of GA and the final FFA release amount was around 100% (Fig. 9a). This phenomenon might be due to pepsin hydrolysis of gliadin protein in both GNP and GNP/GA flocculation (Cornacchia & Roos, 2011). Therefore, oil droplets floated at the top of the digesta after gastric digestion stage were observed in both HIPEs. Most oil droplets were digested during the small intestine stage, and there was no obvious differences in FFA release profiles of the HIPEs. In addition, the stability and bioaccessibility of the carotenoids were fairly similar in both HIPEs (Fig. 9b). For instance, in the presence and absence of GA, the bioaccessibility was 37.7 ± 0.6% and 33.9 ± 1.4%, whereas the stability was 74.9 ± 0.7% and 69.4 ± 1.1%, respectively. The main reason for these results was that the three-dimensional network of GNP/GA HIPEs was completely damaged with pepsin hydrolysis during stomach stage. Overall, these results suggest that the presence of GA does not adversely affect lipid digestion or carotenoid bioaccessibility.

None. Acknowledgments The authors are grateful for the financial support of this study by the National Natural Science Foundation of China (31860452, 31972071, 31601468) and the Key Project of Natural Science Foundation of Jiangxi Province, China (20171ACB20005). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodhyd.2019.105381. References Bai, L., Liu, F. G., Xu, X. F., Huan, S. Q., Gu, J. Y., & McClements, D. J. (2017). Impact of polysaccharide molecular characteristics on viscosity enhancement and depletion flocculation. Journal of Food Engineering, 207, 35–45. Banc, A., Desbat, B., Renard, D., Popineau, Y., Mangavel, U., & Navailles, L. (2007). Structure and orientation changes of ω- and γ-gliadins at the air-water interface: A PM-IRRAS spectroscopy and brewster angle microscopy study. Langmuir, 23(26), 13066–13075. Boon, C. S., McClements, D. J., Weiss, J., & Decker, E. A. (2010). Factors influencing the chemical stability of carotenoids in foods. Critical Reviews in Food Science and Nutrition, 50(6), 515–532. Caldero, G., Llinas, M., Jose Garcia-Celma, M., & Solans, C. (2010). Studies on controlled release of hydrophilic drugs from W/O high internal phase ratio emulsions. Journal of Pharmaceutical Sciences, 99(2), 701–711. Chen, X., Chen, Y., Zou, L., Zhang, X., Dong, Y., Tang, J., et al. (2019). Plant-based nanoparticles prepared from proteins and phospholipids consisting of a core–multilayer-shell structure: Fabrication, stability, and foamability. Journal of Agricultural and Food Chemistry, 67(23), 6574–6584. Chen, X., McClements, D. J., Wang, J., Zou, L. Q., Deng, S. M., Liu, W., et al. (2018a). Coencapsulation of (-)-Epigallocatechin-3-gallate and quercetin in particle-stabilized W/O/W emulsion gels: Controlled release and bioaccessibility. Journal of Agricultural and Food Chemistry, 66(14), 3691–3699. Chen, X., McClements, D. J., Zhu, Y. Q., Zou, L. Q., Li, Z. L., Liu, W., et al. (2018b). Gastrointestinal fate of fluid and gelled nutraceutical emulsions: Impact on proteolysis, lipolysis, and quercetin bioaccessibility. Journal of Agricultural and Food Chemistry, 66(34), 9087–9096. Cornacchia, L., & Roos, Y. H. (2011). Stability of beta-carotene in protein-stabilized oil-inwater delivery systems. Journal of Agricultural and Food Chemistry, 59(13), 7013–7020. Dai, L., Sun, C., Wei, Y., Mao, L., & Gao, Y. (2018). Characterization of Pickering emulsion gels stabilized by zein/gum Arabic complex colloidal nanoparticles. Food Hydrocolloids, 74, 239–248. Dickinson, E. (2019). Strategies to control and inhibit the flocculation of protein-

4. Conclusions In the present study, Pickering HIPEs were formed by simply blending an aqueous phase and oil phase together in the presence of GNPs and GA. The emulsions formed contained a high concentration of relatively small oil droplets that were packed tightly together, which led to a semi-solid texture and good stability to gravitational separation. The presence of the GA was shown to increase the apparent viscosity, shear modulus, and creaming stability of the HIPEs, as well as enhance their resistance to changes in pH, ionic strength, and temperature. It is proposed that the GA formed an anionic coating around the GNP-stabilized oil droplets, as well as forming a 3-D network of aggregated droplets throughout the emulsions due to polymer bridging effects. Moreover, the GA improved the stability of β-carotene encapsulated within the oil droplets and did not adversely affect lipid 9

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