Edible pickering high internal phase emulsions stabilized by soy glycinin: Improvement of emulsification performance and pickering stabilization by glycation with soy polysaccharide

Journal Pre-proof Edible Pickering High Internal Phase Emulsions Stabilized by Soy Glycinin: Improvement of Emulsification Performance and Pickering Stabilization by Glycation with Soy Polysaccharide

Ze-Zhou Hao, Xiu-Qing Peng, Chuan-He Tang PII:

S0268-005X(19)32560-3

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105672

Reference:

FOOHYD 105672

To appear in:

Food Hydrocolloids

Received Date:

31 October 2019

Accepted Date:

14 January 2020

Please cite this article as: Ze-Zhou Hao, Xiu-Qing Peng, Chuan-He Tang, Edible Pickering High Internal Phase Emulsions Stabilized by Soy Glycinin: Improvement of Emulsification Performance and Pickering Stabilization by Glycation with Soy Polysaccharide, Food Hydrocolloids (2020), https://doi.org/10.1016/j.foodhyd.2020.105672

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Journal Pre-proof TOC Graphic Edible Pickering High Internal Phase Emulsions Stabilized by Soy Glycinin: Improvement of Emulsification Performance and Pickering Stabilization by Glycation with Soy Polysaccharide Yan-Teng Xu, Chuan-He Tang

Journal Pre-proof

Edible Pickering High Internal Phase Emulsions Stabilized by Soy Glycinin: Improvement of Emulsification Performance and Pickering Stabilization by Glycation with Soy Polysaccharide

Ze-Zhou Hao+1, Xiu-Qing Peng+1, Chuan-He Tang1, 2* 1

Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, P. R. China 2

Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), Guangzhou 510640, P.R. China

* To whom correspondence should be addressed. E-mail: [email protected] (C.H. Tang) + Contribute

equally.

1

Journal Pre-proof Abstract: There is fast increasing interest in the development of food grade high internal phase emulsions (HIPEs; with oil fractions > 0.74) stabilized by protein-based particles, due to their potential applications in food, cosmetics and biomedical fields. This work reported that soy glycinin (SG), an important soy storage globulin, exhibited a potential to act as Pickering stabilizers for HIPEs; and the glycation with soy soluble polysaccharide (SSPS) greatly improved the emulsification performance of SG, the gel network formation and stability (against heating or freeze-thawing) of the resultant HIPEs. In the work, two selected glycation incubation periods (24 and 72 h) were applied to obtain the glycated SG. The results indicated that the glycation progressively decreased the minimal concentration required for the formation of gel-like HIPEs, and droplet size, but increased the gel network strength, in an incubation period dependent manner. The improvement of emulsification performance by the glycation was mainly associated with the enhanced adsorption, as well as facilitated subunit dissociation at the interface, during the HIPE formation. All the HIPEs stabilized by untreated or glycated SG exhibited an excellent coalescence stability against a long-term storage. The structural analyses of non-adsorbed and adsorbed SG indicated that although untreated SG showed a high whole structural integrity with no distinct changes in structures after adsorption at interface, it tended to aggregate (due to its high surface hydrophobicity); in contrast, glycated SG easily dissociated into subunits at the interface, but the dissociated subunits exhibited a high structural stability at the tertiary and secondary levels. The findings would be of importance not only for the development of soy protein-based Pickering stabilizers for emulsions or HIPEs, but also for extending the knowledge about the modification of emulsifying properties of proteins by glycation with carbohydrates. 2

Journal Pre-proof Keywords: Soy glycinin; High internal phase emulsions (HIPEs); Glycation; Soy soluble polysaccharide; Pickering stabilizers

3

Journal Pre-proof 1. Introduction The development of biocompatible high internal phase emulsions (HIPEs; usually with a minimal internal phase volume ratio of 0.74), especially those stabilized by protein-based colloidal particles, has attracted fast increasing interest in a variety of potential applications, e.g., as delivery containers for nutraceuticals and templates for scaffolds (Huang, Zhu, Xi, Yin, Ngai & Yang, 2019 a; Li, Xiao, Wang & Ngai, 2013; Patel, 2018; Tan, Sun, Lin, Mu & Ngai, 2014; Xu, Tang, Liu & Liu, 2018), or templetes for biopolymeric oleogels (Wijaya, Sun, Vermeir, Dewettinck, Patel & van der Meeren, 2019). It has been well recognized that the HIPEs stabilized by colloidal particles, or Pickering HIPEs, are much advantageous over those stabilized by conventional small molecular weight surfactants, in terms of better performance, higher stability and safety (Capron & Cathala, 2013; Chen, Zheng, Xu, Yin, Liu & Tang, 2018; Li, Ming, Wang & Ngai, 2009). To date, a lot of solid polymeric particles have been confirmed to act as effective Pickering stabilizers for HIPEs, which can be classified into three catogories: (i) polysaccharide-based (nano)particles, e.g., cellulose or chitin nanocrystals, starch nanocrystals (Capron & Cathala, 2013; Perrin, Bizot, Cathala & Capron, 2014); (ii) protein-based (nano)particles, e.g., gliadin colloidal particles (Hu, Yin, Zhu, Qi, Guo, Wu et al., 2016), peanut protein microgels (Jiao, Shi, Wang & Binks, 2018), crosslinked bovine serum albumin (Li et al., 2013), whey protein (Patel et al., 2014) or its heat-induced microgels (Zamani, Malchione, Selig & Abbaspourrad, 2018); (iii) polysaccharide-protein hybrid complexes, e.g., protein-polysaccharide (nano)complexes (Huang, Zhou, Yang, Yin, Tang & Yang, 2019 b; Wijaya, Van der Meeren, Wijaya & Patel, 2017; Zeng, Wu, Zhou, Yin, Tang, Wu & Yang, 2017), bovine serum albumin-coated cellulose nanocrystals (Liu, Zheng, Huang, Tang & Ou, 2018), or 4

Journal Pre-proof polysaccharide-protein hybrid nanoparticles from Okara (Yang, Li & Tang, 2020). Due to the amphiphilic nature of their surface properties, protein-based (nano)particles or protein-polysaccharide complexes as stabilizers for HIPEs are considered to be more effective in emulsification performance than those consisting of polysaccharides alone.

One prerequisite for any particle to perform as effective stabilizers for HIPEs is that it should keep its structure basically intact when adsorbed at the interface of droplets. The structure and properties of proteins considerably vary with the type of proteins, and environmental conditions. Among all the protein-based stabilizers for HIPEs, reported in the literature, alcohol-soluble proteins are one of the most investigated starting proteins for fabricating Pickering particles for stabilizing HIPEs, due to their insoluble nature in the aqueous phase (Huang et al., 2019 a). However, these proteins as Pickering stabilizers for HIPEs are generally effective at highly acidic pHs (Hu et al., 2016), or after the complexation with polysaccharides (Ma, Zou, McClements & Liu, 2020; Yuan, Hu, Zeng, Yin, Tang & Yang, 2016; Zeng et al., 2017; Zhou, Huang, Wu, Yin, Zhu, Tang & Yang, 2018; Zhou, Zeng, Yin, Tang, Yuan & Yang, 2018). For example, Ma et al. (2020) indicated the presence of increasing concentration of gum Arabic progressively improved the formation and gel strength of Pickering HIPEs stabilized by gliadin nanoparticles, and imparted a high stability to pH, ionic strength and heating to the HIPEs. In this work, the authors attributed the improvements to the surface coating of gliadin particle-stabilized droplets with an anionic polysaccharide, as well as the formation of a 3D-network through polymer bridging between different droplets. In fact, the improvements would be more likely due to the enhanced colloidal stability of gliadin particles in the 5

Journal Pre-proof system, as a result of increased intermolecular electrostatic and steric repulsions. It should be interestingly noted that under certain conditions, the complexes of whey protein isolate (and even sodium caseinate) with polysaccharides (e.g., alginate or pectin) could also perform as effective stabilizers for HIPEs (Wijaya et al., 2017, 2019), wherein the stabilization of HIPEs seemed to be more related to the steric stabilization of polysaccharides, rather than the proteins themselves. Thus, all the observations suggest the importance of highly hydrophilic layers surrounding the proteins or protein particles to the effective stabilization of HIPEs.

On the other hand, evidences are fast accumulating in recent years to indicate that HIPEs (gels) can be facily fabricated using many globular proteins as sole stabilizers (Xu, Liu & Tang, 2019; Xu, Tang & Binks, 2020; Xu et al., 2018). Ovalbumin and soy β-conglycinin (SC) in their native state are two globular proteins demonstrated to act as outstanding nanostabilizers for HIPEs (Xu et al., 2018, 2019). Ovalbumin did not suffer a distinct change in structural characteristics (quaternary, tertiary and secondary structure), upon adsorption at the interface in the HIPEs (and subsequent desorption from the interface by a freeze-thawing process) (Xu et al., 2018), confirming the Pickering stabilization. In the SC case, the Pickering stabilization was mainly associated with its dissociated subunits at the interface, wherein the tertiary and secondary conformations of the dissociated subunits were comparable to those of native SC (Xu et al., 2019). More interestingly, the HIPEs could be facily fabricated using a ‘one-pot’ shearing at very low concentrations in the aqueous phase (c), e.g., 0.2 wt%; the as-obtained HIPEs (gels) were extraordinarily stable against an extensive heating or long-term storage; a self-supporting gel network could be formed at low c values < 0.5 wt% 6

Journal Pre-proof (Xu et al., 2018, 2019). It should be noteworthy that both ovalbumin and SC are glycoproteins, which leads us to hypothesize that the presence of carbohydrate moieties might be one of key structural characteristics for globular proteins to perform as particles for stabilize Pickering HIPEs. Different from hard particles, e.g., silica particles, which undergo no stuctural change upon adsorption at the interface, protein (nano)particles would suffer a partial structural unfolding when adsorbed at the interface, thus sharing some common structural features with polymeric microgels (a kind of classic soft particles; Dickinson, 2015). From this viewpoint, protein (nano)particles can also be considered as a kind of soft (nano)particles. In a more recent work of ours, we successfully demonstrated that glycation with a carbohydrate (galactose) transformed bovine serum albumin (BSA; a nonglycoprotein) into a kind of outstanding soft particles for stabilizing HIPEs; and the ability of glycated BSA to stabilize HIPEs was even better than that of ovalbumin and SC (Xu et al., 2020). This transformation was largely attributed to the formation of a core-shell nanostructure with a hydrophilic galactose shell surrounding the protein core.

The incorporation of soy proteins in food formulations has become as one of topical subjects in the food colloid field, due to their health-benefiting effects (Tang, 2019). SC and soy glycinin (SG), usually in the 7S and 11S form, respectively, are two major storage globulins in the soy protein isolates. In general, SC exhibits a much better emulsification performance than SG, thus being considered as a better emulsifier (Tang, 2017). This is related to the fact that SC is a glycoprotein with a high structural stability (against aggregation), while SG is a non-glycated oligomeric protein with a plenty of disufide bonds and free sulfydryl groups, thus showing a high tendency to aggregate 7

Journal Pre-proof (Tang, 2017). Although native SC itself is an outstanding Pickering nanoparticle for stabilizing HIPEs (Xu et al., 2019), its emulsification performance as Pickering stabililizers for HIPEs could be further improved by an alcohol-induced aggregation (Peng, Xu, Li & Tang, 2020). In contrast, the potential of SG to act as Pickering stabilizers for HIPEs has been little investigated, despite a number of our previous works indicating that SG after a simple heating could perform as effective Pickering stabilizers for emulsions (Liu & Tang, 2016 a-c). Besides the heating, we also demonstrated that a glycation with soy soluble polysaccharide (SSPS; an acidic polysaccharide) greatly improved the emulsification performance and Pickering stabilization of SG (Peng, Xu, Liu & Tang, 2018).

Bases on the above considerations, the work was undertaken to investigate the potential of SG to act as stabilizers for HIPEs, as well as to confirm the importance of glycation to the improvement of the ability of SG as soft particles for stabilizing HIPEs. Following the same process of our previous finding (Peng et al., 2018), SG was glycated with SSPS for different incubation periods of 0-72 h, using a dry-heat process at 60 ºC. In the first part, the potential of untreated or glycated SG as sole stabilizers for HIPEs was evaluated, in terms of minimal c required for the formation of gel-like HIPEs (at ϕ = 0.8), rheological behavior and microstruture (and droplet size). Then, the stability of as-fabricated HIPEs (gels) upon a long-term storage, heating or freeze-thawing was characterized. Last, the structural changes of untreated or glycated SG, before or after the adsorption at the interface in the HIPEs, were characterized.

2. Materials and methods 8

Journal Pre-proof 2.1. Materials Soy glycinin (SG) was obtained from defatted soy flour (Shandong Yuwang Co. Ltd., Shandong province, China), according to the process of Nagano, Hirotsuka, Mori, Kohyama & Nishinari (1992), as described in our previous work (Peng et al., 2018). The SG sample contained about 95.5% of protein (dry basis), with a purity of about 90% as evaluated by a densitometeric analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Soy soluble polysaccharide (SSPS) was obtained from Fuji Oil Ltd. (Osaka, Japan). Soy soluble polysaccharide (SSPS) was purchased from Fuji Oil Ltd. (Osaka, Japan). Soy oil was purchased from a local supermarket in Guangzhou (China). MgSiO3-based adsorbent (Florisil® PR) with 100-200 mesh per particle and 289 m2/g surface area was purchased from Sigma-Aldrich LLC. (USA). All other chemicals were of analytical grade.

2.2. Glycation of SG with SSPS and preparation of glycated SG The SG glycated with SSPS at 60 oC after incubation periods of 0, 24 and 72 h was prepared according to the same dry-heat process as described in our prevous work (Peng et al., 2018). The degree of glycation for SG glycated after 0, 24 and 72 h of incubation periods, which was denoted as g-SG (0 h), g-SG (24 h) and g-SG (72 h), respectively, was determined to be about 1.2, 7.4 and 22.5%, using the o-phthaldialdehyde method (Li & Tang, 2013). The untreated SG (control) was denoted as n-SG.

2.3. Preparation of HIPEs 9

Journal Pre-proof The untreated or glycated SG dispersions at a protein concentration (c) of 1.0 wt% or below (if neccessary), containing a concentration of 0.02 wt% of sodium azide (for the inhibition of growth of microorganisms), were prepared by directly dispersing the freeze-dried SG samples in deionized water for 2 h, under stirred conditions. All the HIPEs at a specific oil fraction () of 0.8, or the emulsions at  values less than 0.7, were formed by directly homogenizing the mixtures of soy oil and SG dispersions, through a facile one-pot process, using a XHF-DY high-speed dispersing unit with a 10 mm head (Ningbo Scientz Biotechnology Co., China), at 5000 rpm for 1 min. The homogenization was carried out in serum bottles with inner diameter of 2 cm, with a total volume of 5 mL of emulsions.

2.4. Characterization of HIPEs When the vials containing the HIPEs were turned upside down and the HIPEs did not flow, they can be considered as a kind of gel-like HIPEs, or HIPE gels. The minimal protein concentration (cm) required for the formation of gel-like HIPEs was evaluated visually by observing the flow behavior of the HIPEs, formed at different c values of 1.0 wt% or less. The gel-like rheological behavior of the HIPEs stabilized by untreated or glycated (0-72 h) SG, at  = 0.8 and c = 0.8 or 1.0 wt%, before or after a storage of 30 days, was evaluated by dynamic oscillatory measurements, which was performed in a frequency sweep mode over frequency range 0.1-10 Hz, on a HAAKE RS600 Rheometer (HAAKE Co., Germany) with parallel plates (d = 27.83 mm) at 25 °C. To ensure that the frequency sweep experiments were conducted within the linear viscoelastic range, at a constant strain of 0.5%, a strain sweep experiment from 0.002 to 1.0% at a specific freuquency of 1.0 Hz was first 10

Journal Pre-proof performed. The frequency dependence of storage (G′) and loss (G″) moduli of all the test HIPEs was recorded.

The microstructure of the HIPEs formed under different conditions was observed using an optical microscope Phonix ph-100 with an Olympus DP70 camera (Shanghai, China). The droplet size data of these HIPEs (gels) were obtained using microscopic image analysis software (Nano Measurer 1.2, Fudan University, China) over more than 100 droplets per microscopic image, in terms of surface-average droplet size (d3,2). Each HIPE was diluted with water or 1.0 wt% SDS by 10-fold, prior to the optical microscopic observations. The percentage of adsorbed protein (AP%) at the interface in the HIPEs was determined according to the same process as described in our previous work (Xu et al., 2020). In brief, the HIPEs were incubated with 5 mM phosphate buffer (pH 7.0) at a relative weight ratio of 1:20 for 12 h, followed by a vortexing for 10 s and centrifugation at 1000 g for 5 min, in order to remove non-adsorbed proteins in the system. Then, the adsorbed proteins in the resulting creamed layers were obtained by a same freeze-thawing treatment as described for the freeze-thawing stability evaluation of the HIPEs (section 2.5). The AP% was calculated by the relative ratio of the amount of adsorbed proteins to the total protein amount (in the system).

2.5. Stability of HIPEs The coalescence stability of the HIPEs stabilized by untreated and glycated (24 and 72 h) SG, formed at  = 0.8 and c = 1.0 wt%, upon a long-term storage of 30 days at room temperature, or heating at 100 ºC for 15 min, was evaluated by visual observation, microstructural and rheological 11

Journal Pre-proof measurements. For the heating, all the sealed HIPEs were treated in boiling water for 15 min, followed by an immediate cooling in an ice bath for 15 min. The freeze-thaw stability of the above HIPEs was evaluated by visual observation and microstructural analysis, using a process of freezing at -20 °C for 24 h and subsequent thawing at 25 °C for 2 h. In general, all the HIPEs would completely destabilize to release the oil phase. The destabilized HIPEs were subjected to another homogenization (the same as the above), for the evaluation of the reversibility of destabilization and re-emulsification. If necessary, the freeze-thawing process could be repeated for more than 3 times.

The adsorbed SG (untreated or glycated) at the interface in the HIPEs was obtained after the first cycle of freeze-thawing, followed by a centrifugation at 8000 g for 20 min, for their structural characterization. All the obtained SG samples were redispersed in the aqueous phase (to an appropriate concentration for the characterization).

2.6. Dynamic adsorption at the oil-water interface The dynamic purified soy oil-water interfacial tension of different SG samples was monitored using an optical contact angle meter (OCA-20) with oscillating drop accessory ODG-20 (Dataphysics Instruments GmbH, Germany). The interface was formed with one drop of aqueous SG solution (0.05 wt%) in purified soy oil at 25 oC, using an injection syringe with a plastic tube. The interfacial tension (γ) was acquired by following the drop shape change according to the Young-Laplace equation of capillarity. Commerical soy oil was purified by mixing soy oil and MgSiO3-based adsorbent at a weight ratio of 3:1, and continuously stirred at room temperature for 3 h. After that, a 12

Journal Pre-proof centrifugation of 8000 g for 30 min was conducted at 4 oC, and the adsorbents were discarded. The process was repeated with two more times, to ensure that all the surface active agents in soy oil were removed.

2.7. Structural characterization of adsorbed SG The differences in structural characteristics between the adsorbed SG (untreated or glycated) and its non-adsorbed counterpart, e.g., subunit association and/or dissociation (structural change at the quaternary level), and tertinary and secondary conformation, were evaluated in terms of particle size distribution (PSD), intrinsic fluorescence and far-UV circular dichroism (CD) spectrum profiles. The PSD profles of the proteins were determined by dynamic light scattering (DLS) technique using a Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., Malvern, Worcestershire, UK) equipped with a 4 mW He-Ne laser (633 nm wavelength) at 25 °C. All the DLS determinations were performed at a constant c value of 0.1 wt% in 50 mM phosphate buffer (pH 7.0). The particle siz was calculated according to the Stokes-Einstein equation, based on the assumption that all the protein particles were spherical. The tertiary (and even quanternary) conformation of untreated or glycated SG, before or after the adsorption at the interface in the HIPEs, was assessed by intrinsic fluorescence spectroscopy using a fluorescence spectrophotometer (F-7000, Hitachi Co. Ltd, Japan). The intrinsic Trp emission fluorescence spectra of different proteins in 0.01 M phosphate buffer (pH 7.0; 0.02 wt%) were obtained, with an excitation wavelength at 280 nm and a scanning speed of 1200 nm/min.

13

Journal Pre-proof The differences in secondary structure of nonadsorbed and adsorbed SG were assessed by far- UV CD spectroscopy, using a ChirascanTM spectrometer (Applied Photophysics Ltd., UK). The far-UV CD spectra (180-260 nm) were obtained at a fixed c of 0.02 wt%. All the test protein solutions were filtered through Millipore PES hydrophilic membrane filters with a pore size of 0.22 μm, prior to the determination.

2.8. Particle morphology of untreated and glycated SG The particle morphology of untreated and glycated (72 h) SG in the solutions (0.025 wt%; at pH 7.0) was observed by transmission electron microscopy (TEM), using a JEM-2100 transmission electron microscope (JEOL Ltd., Japan) equipped with an XFlash® 5030T detector (Bruker Instruments Co. Ltd., Germany). The proteins were negatively stained with 1.0 wt% phosphotungstic acid, and dried with a vacuum drying oven (Shanhai Bluepard I Instruments Co. Ltd., China).

2.9. Statistics The significant difference (LSD) at p < 0.05 level between the means was analyzed using ANOVA with Duncan’s test using IBM SPSS Statistics 22.

3. Results and discussion 3.1. Formation and characterization of HIPEs (gels) 3.1.1. Formation of the HIPEs (gels) Our previous work demonstrated that both unglycated and SSPS-glycated SG (0-72 h) samples could 14

Journal Pre-proof be applied as effective Pickering stabilizers to form gel-like emulsions at ϕ = 0.6 and c = 1.0 wt%, using a microfluidization as the emulsification technique, and the glycation strengthened the Pickering stabilization of SG (Peng et al., 2018). In the current work, we further evaluated the potential of untreated and glycated (0, 24 and 72 h) SG to stabilize HIPEs at a constant ϕ value of 0.8, using a shearing as the homogenization technique, by monitoring the minimal protein concentration (cmin) required for the formation of a self-supporting gel network. The cmin for different SG samples was visually determined by observing the flow behavior of the HIPEs formed at varying c values, e.g., 0.2-1.0 wt%, as displayed in Figure 1. Interestingly, it can be observed that when the c was high enough, a kind of gel-like HIPEs could be formed, no matter what kind of SG samples was applied. However, the cmin distinctly varied with the type of applied SG samples. For the HIPE stabilized by untreated SG, the cmin was around 0.6 wt%, which is lower than that (0.8 wt%) by the glycated (0 h) SG (Figure 1). The poor ability of the untreated SG (relative to the glycated (0 h) counterpart) to stabilize HIPEs might be due to the protein aggregation of proteins occurring during the glycated sample preparation (Peng et al., 2018).

By comparison, the cmin for the HIPEs stabilized by the glycated (24 and 72 h) SG was much less than that by the glycated (0 h) SG (Figure 1), indicating that the glycation greatly improved the ability of SG to stabilize gel-like HIPEs. The glycated SG with longer incubation periods of glycation exhibited a higher ability to stabilize the gel-like HIPEs (Figure 1). The cmin for the HIPE by the glycated (72 h) SG is within the range 0.05-0.5 wt% as previously reported for the HIPE gels by two native glycoproteins (ovalbumin and β-conglycinin) (around 0.2 wt%; Xu et al., 2018, 2019), 15

Journal Pre-proof as well as Pickering HIPEs by microgel particles (Li et al., 2009), cellulose nanocrystals (Capron & Cathala, 2013; Chen et al., 2018), gelatin particles (Tan et al., 2014), or hydrophobically modified silica particles (Arditty, Schmitt, Giermanska-Kahn, & Leal-Calderon, 2004).

3.1.2. Rheological properties of the HIPEs (gels) The improvement of the ability of SG to stabilize the gel-like HIPEs by the glycation might be largely associated with the enhanced gel network formation in the HIPEs. To confirm this, we evaluated the rheological properties of the HIPEs (at ϕ = 0.8) stabilized by untreated and glycated (0, 24 and 72 h) SG at a comparable c value of 0.8 wt%, using a dynamic oscillatory measurement in a frequency sweep mode, as shown in Figure 2 a. As expected, all of the test HIPEs exhibited an elasticity-dominated viscoelastic property, e. g., slight frequency dependence (in the range 0.1-10 Hz), and remarkably greater storage modulus (G') in magnitude than the loss modulus (G'') (Figure 2 a), indicating the formation of gel network. Similar rheological phemona have been reported for the Pickering HIPEs or HIPE gels stabilized by ovalbumin (Xu et al., 2018), soy β-conglycinin (Xu et al., 2019), hydrophobically modified or protein-coated cellulose nanocrystals (Chen et al., 2018; Liu et al., 2018), or stach nanocrystals (Yang, Zheng, Zheng, Liu, Wang & Tang, 2018). The G' at a specific frequency of 1.0 Hz for all the test HIPEs is summaried in Figure 2 b. The G' for the HIPE stabilized by untreated SG was significantly higher than that by the glycated (0 h) SG (Figure 2 b), further confirming that the protein aggregation was unfavorable for the gel network formation. As the glycation increased from 0 to 72 h, the G' of the HIPEs or HIPE gels progressively and significantly increased (Figure 2 b). Together with the cmin data (Figure 1), the rheological 16

Journal Pre-proof observations clearly indicated that the glycation not only facilitated the initial gel network formation of the HIPEs, but also strengthened the stiffness of the formed gels, in a glycation degree dependent manner.

3.1.3. Microstructure of the HIPE gels To unveil the underlying mechanism for the improvement of the HIPE gel formation by the glycation, the microstructure of the HIPEs stabilized by untreated and glycated (0, 24 and 72 h) SG was evaluated by optical microscopy. Figure 3 shows the representative optical micrographs of these fresh HIPE gels, formed at varying c values of 0.3-1.0 wt%. The evolution of d3,2 of these HIPE gels as a function of c in the range 0.3-1.0 wt% is summarized and displayed in Figure 4 a. At a specific c of 0.8 wt%, most of the droplets in the HIPE gel stabilized by untreated SG were interconneted each other with deformed facets between neighoring droplets, and the extent of interconnetion was more distinct in the HIPE gels by the glycated (24 or 72 h) SG (Figure 3 a). In contrast, most of droplets in the HIPE gel by the glycated (0 h) SG were present in a relatively undeformed state (Figure 3 a). On the other hand, it can also be observed that at c = 0.8 wt%, the d3,2 of these HIPE gels significantly decreased in the order: glycated (0 h) SG > untreated SG > glycated (24 h) SG > glycated (72 h) SG (Figure 4 a), indicating the improvement of emulsification performance by the glycation (with 24-72 h period incubation). A similar result has been reported for the emulsions formed at ϕ = 0.3 and c = 1.0 wt%, using microfluidization as the emulsification process (Peng et al., 2018). The observations are basically consistent with their G' data (Figure 2 b), reflecting that in the current case, the elasticity of these HIPE gels might be dominated by the number density of 17

Journal Pre-proof deformed facets of droplets and the droplet Laplace pressure (which is the ratio of interfacial tension to droplet size) (Lee, Chan & Mohraz, 2012). In addition, the formation of bridging droplets in Pickering emulsions with two droplets sharing a same particle monolayer, as proposed by Horozov & Binks (2006) and confirmed in the HIPE gels by ovalbumin and β-conglycinin (Xu et al., 2018, 2019), might also contribute to the elasticity of these HIPE gels. Thus, the improvement of gel network formation in the HIPEs by the glycation would mainly come from two contributions: improved emulsification performance and facilitated formation of bridged emulsions.

For any set of HIPE gels stabilized by the same SG sample, it is generally observed that their d3,2 progressively decreased with increasing the c from their cmin up to 1.0 wt% (Figure 4 a; with representative optical micrograms for the HIPE gels stabilized by the glycated (72 h) SG shown in Figure 3 b). This is one of classic features observed for Pickering emulsions stabilized by inorganic or organic particles, e.g., hydrophobized fumed silica (Frelichowska, Bolzinger & Chevalier, 2010), polysaccaride nanocrystals and modified starch (Rayner, Timgren, Sjӧӧ & Dejmek,, 2012; Tzoumaki, Moschakis, Kiosseoglou & Biliaderis, 2011), and even heat-induced soy protein isolate nanoparticles (Liu & Tang, 2013).

3.1.4. Percentage of adsorbed proteins at interface The percentage of adsorbed proteins at the interface (AP%) in the HIPE gels stabilized by untreated and glycated (24 and 72 h) samples, formed at c = 1.0 wt% and ϕ = 0.8, was determined, as summarized in Figure 4 b. The AP% of the HIPE gel stabilized by untreated SG, in the current case, 18

Journal Pre-proof was around 10%, which is considerably lower than that (54.5%) observed for the emulsion stabilized by untreated SG at the same c but lower ϕ (0.3), using the microfluidization as the emulsification process (Peng et al., 2018). The difference might be largely due to the difference in emulsification efficiency between two different emulsification processes. In the microfluidization case, more proteins are expected to be adsorbed at the interface with larger interfacial areas, created during the emulsification. In addition, surface hydrophobic modification, as well as structural dissociation (of large particles) by the microfludization might be also favorable for the adsorption of SG at the interface during the emulsification (Shen & Tang, 2012). The glycation remarkably increased the AP% in these HIPE gels, in a glycation incubation period dependent way (Figure 4 b). A similar phenomenon has been previously observed for the emulsions stabilized by untreated and glycated (24 and 72 h) SG, produced by the microfluidization (Peng et al., 2018). It should be noteworthy that the AP% data of these HIPEs are well in agreement with the their inverse droplet diameter (1/d3,2) (Figure 4 a, b), indicating that in the protein-rich regime (like 1.0 wt% in the case), the adsorption of untreated or glycated SG at the interface was determined by their emulsification performance. The enhanced conformation flexibility at the subunit level of SG by the glycation with SSPS (Peng et al., 2018) might largely account for the remarkably higher AP% in the glycated SG stabilized HIPEs (as compared to that by untreated SG).

3.2. Stability against long-term storage and heating The stability of the HIPEs (ϕ = 0.8) stabilized by untreated and glycated (24 and 72 h) SG, formed at a constant c value of 1.0 wt%, against a long-term storage of 30 days, as well as heating in boiling 19

Journal Pre-proof water for 15 min, was evaluated and compared, in terms of changes in their visual appearance and microstructure. The results showed that all the test HIPEs did not suffer a distinct change in visual appearance and droplet size (Figures 5 and 6 a), indicating a high coalescence stability against a long-term storage (30 days) or heating (100 ºC). Similar phenomena have been previously reported for the Pickering HIPE gels stabilized by ovalbumin and β-conglycinin (Xu et al., 2018, 2019), indicating that regardless of glycation or not, SG could perform as effective stabilizers for the HIPEs or HIPE gels.

However, it can be still obseved that after the heating (followed by a fast cooling), the G' (at a specific frenquency of 1.0 Hz) of the HIPE by the untreated SG remarkably increased (Figure 6 b), indicating the strengthening of gel network. Considering that in the HIPE gel stabilized by untreated SG, the AP% was very low (Figure 4 b), the remarkably increased stiffness could be largely attributed to the increased attractive interactions between adsorbed proteins and heat-denatured proteins in the aqueous phase. This is consistent with the fact that at pH 7.0 and low ionic strengths, the denaturation temperature of SG (at low c concentrations, e.g., < 1.0 wt%) is far below 100 ºC (Tang, 2017). Considerable increases in G' have been similarly observed for the HIPE gels stabilized by ovalbumin at c values of 0.8-2.0 wt% (Xu et et al., 2018). The strengthening of gel network by the heating can also be observed for the HIPE gel stabilized by the glycated (24 h) SG (Figure 6 b), reflecting that in this case, the attractive interactions between adsorbed proteins and non-adsorbed proteins still distinctly increased, as the result of the heating. In contrast, the G' of the HIPE gel stabilized by the glycated (72 h) SG was almost unchanged by the heating (Figure 6 b), reflecting 20

Journal Pre-proof that in this case, the glycated SG molecules, adsorbed or unadsorbed, were extremely heat stable. The observations clearly indicated that the glycation resulted in a progressive increase in coalescence stability and interfacial stabilization of the resultant HIPEs upon heating, in a glycation degree dependent way. The high heat stability seems to be one of common features for the HIPEs (gels) stabilized by glycated globular proteins (Xu et al., 2019, 2020).

3.3. Reversibility of destabilization (against freeze-thawing) and re-emulsification The freeze-thaw stability of the HIPE gels stabilized by untreated or glycated (24 and 72 h) SG was also evaluated. As expected, all the test HIPE gels were very prone to a freeze-thaw treatment (freezing at -20 oC for 24 h, then followed by thawing at 25 oC for 2 h) (Figure 7). After the freeze-thaw treatment, all the HIPE gels completely destabilized to release the oil phase. However, when another homogenization was applied, a kind of stable HIPE gels could be reformed (Figure 7). In this case, it can be still observed that after the 1st cycle of freeze-thaw treatment, the droplet size of the HIPE gels distinctly increased, but the extent of increases was lower in the glycated SG case than in the untreated SG (Figure 7), indicating that the glycation improved the reversibility of the destabilization/re-emulsification of these HIPEs. This can be further corrobrated by the freeze-thaw experiments with the cycle number increasing up to 3 (Figure 7). For example, for the HIPE gel stabilized by untreated SG, the destabilized HIPE gel could not be restored back to a homogenous and gel state any more, after 3 cycles of freeze-thaw treatment (Figure 7). Whereas, in the case of the HIPE gel by the glycated (72 h) SG, the cycle number of freeze-thaw treatment could be repeated up to 3 times, though in this case, it could be still observed that the droplet sizes of the re-formed 21

Journal Pre-proof HIPE gels progressively increased with the number of freeze-thaw cycle (Figure 7). The observations clearly indicated that the glycation significantly improved the reversibility of destabilization (upon freeze-thawing) and re-emulsification, which might be largely associated with the enhanced structural stability of SG (or its subunits) by the glycation.

3.4. Dynamic adsorption of glycated SG at oil-water interface The emulsification performance of proteins is generally associated with their interfacial behavior, e.g., diffusion from bulk to the interface, structural unfolding/orientation at the interface, and rearrangement of unfolded molecules at the interface. We evaluated the evolution of interfacial tension (γ) for unglycated and glycated (24 and 72 h) SG at the oil-water interface, with time increasing up to 180 min, as shown in Figure 8. It can be observed that there was no distinct difference in the changing pattern of γ between all the test SG samples (Figure 8). This seems to be contrasting from the data that the glycated (24 or 72 h) SG had significantly lower surface hydrophobicity than unglyated SG (Peng et al., 2018), since the proteins with lower surface hydrophobicity would show slower rate of initial adsorption at the interface. Herein, it should be to note that besides the surface characteristics, the decreasing pattern of γ of proteins is also dependent on their ease of orientation and even structural unfolding and rearrangement at the interface. Thus, the insignificant difference in changing pattern of γ between different glycated SG samples just indirectly confirmed the importance of enhanced conformation flexibility at the quaternary level to the improvement of emulsification efficiency of SG by the glycation (Liang & Tang, 2013; Peng et al., 2018). 22

Journal Pre-proof

3.5. Structural integrity and subunit dissociation of untreated glycated SG at the interface The emulsification performance of oligomeric globulins is highly dependent on their conformational flexibility at the quaternary level, or ease of structural unfolding/dissociation at the interface (Liang & Tang, 2013; Tang, 2017). Besides the subunit dissociation, the dissociated subunits of oligomeric globulins at the interface may further undergo a structural change. In the current work, we evaluated and compared the structural changes of untreated and glycated (24 and 72 h) SG, at quaternary, tertiary and secondary levels, before and after adsorption at the interface during the HIPE formation, using DLS (protein aggregation or subunit dissociation), intrinsic fluorescence (tertiary and/or quaternary conformation) and far-UV CD (secondary structure) spectral techniques, as displayed in Figure 9. All the adsorbed SG samples were obtained by freeze-thawing the corresponding HIPE gels, as applied in our previous works (Xu et al., 2018, 2019 and 2020). The DLS profile of untreated SG (initial) exhibited a monodal particle size distribution (PSD) peak centered at around 9.7 nm (Figure 9 a), which is well in accordance with the contour size of hexameric SG (8-10 nm; Marcone, 1999), indicating that the untreated SG was mainly composed of the 11S-form SG. Upon the glycation with SSPS at increasing incubation periods (24 and 72 h), the PSD peak of SG gradually shifted to a greater size (up to 13.5 nm; Figure 9 a), reflecting the gradual increase in hydrodynamic diameter of SG molecules, as a result of introduction of hydrophilic SSPS moieties (Peng et al., 2018).

On the other hand, it can be well observed that the untreated or glycated (24 h) SG did not suffer a 23

Journal Pre-proof distinct change in their PSD profile, upon adsorption at the interface (and subsequently desorption via freeze-thawing), while in the glycated (72 h) SG case, a shift of the major PSD peak towards a smaller size (at around 4.2 nm) occurred (Figure 9 a). The observations clearly indicated that the untreated SG molecules exhibited a relatively high structural integrity with strong intermolecular attractive interactions that could resist the disruption upon adsorption at the interface (and a further freeze-thawing process). The whole structural integrity of SG was almost unaffected by the glycation of 24 h incubation period, but became much susceptible to the adsorption at the interface, when the glycation incubation period was prolonged up to 72 h (Figure 9 a). The observations thus suggested that the glycation with SSPS (after a prolonged incubation period, e.g., 72 h) remarkably improved the ease of the 11S-form SG molecules to unfold or dissociate at the interface, or conformational fexibility at the quaternary level. Our previous work indicated that the glycation led to a gradual dissociation of the 11S-form SG at the subunit ([AB]; A= acidic polypeptide, B=basic polypeptide) level (Peng et al., 2018). Thus, the new PSD profile in the glyated (72 h) SG case could be largely attributed to the dissociated [AB] subunits of glycated SG.

All the test SG samples exhibited a relatively structural stability at the tertiary and secondary levels upon adsorption at the interface, as evidenced by insignificant changes in the intrinsic Trp fluorescence and far-UV CD spectra between the initial and adsorbed SG samples (Figure 9 b, c). However, it can be still noticable that the glycation with SSPS, or introduction of SSPS moieties, increased the structural stability of SG at the tertiary level, since upon the adsorption at the interface, the extent of decreases in quantum yield of fluorescence was less in the glycated SG case than in the 24

Journal Pre-proof untreated counterpart (Figure 9 b). Especially in the glyated (72 h) SG case, the tertiary and secondary structures of the were almost unaffected by the adsorption at the interface (and subsequent desorption from the interface) (Figure 9 b, c), though the adsorption led to a significant dissociation of subunits (Figure 9 a).

Although the whole structural integrity of the untreated SG was greater than that of the glycated (72 h) SG, the much higher surface hydrophobicity and lower magnitude of ζ-potential of the former (Peng et al., 2018) would make it more susceptible to protein aggregation in the solution, or at the interface (than the latter). This was confirmed by the particle morphological observation of untreated and glycated (72 h) SG by TEM (Figure 10). The TEM observations indicated that most of the proteins in the untreated SG case were irregular in morphology, with sizes of approximately 40-50 nm, while the glycated (72 h) SG sample showed a uniform spherical morphology with sizes of about 20 nm (Figure 10). Herein, it is worth mentioning that all the samples for the TEM observations must be in a dry state. Thus, the observations clearly confirmed that the glycation with SSPS remarkably inhibited the protein aggregation of SG molecules during the drying. Due to this, we can reasonably suggest that the greater stability (against heating or freeze-thawing) of the HIPEs stabilized by glycated SG would be largely ascribed to the inhibited protein denaturation and aggregation at the interface.

4. Conclusions Although untreated SG exhibited a potential to stabilize HIPEs, the glycation with SSPS 25

Journal Pre-proof progressively improved its emulsification performance, gel network strength and stability (against heating or freeze-thawing) of the resultant HIPEs, in a glycation incubation period dependent manner. The gel-like HIPEs could be formed using extensively glycated (72 h) SG at a protein concentration in the aqueous phase as low as 0.3 wt%. All the HIPEs stabilized by SG, untreated or glycated, exhibited good stability against a long-term storage. The improved emulsfication performance of SG by the glycation was largely associated with the enhanced adsorption of the proteins at the interface, as well as facilitated subunit dissociation. Although the untreated SG did not suffer distinct structural changes at the quaternary, tertiary and/or secondary levels, after adsorption at the oil-water interface during the HIPE formation, it tended to aggregate in the solution, or at the interface, due to its high surface hydrophobicity and low ζ-potential in magnitude. In contrast, most of the glycated (e.g., 72 h) SG molecules would dissociate into [AB] subunits when adsorbed at the interface, but the presence of hydrophilic SSPS moieties ensured the high structural integrity of dissociated subunits. The findings would be of importance not only for the use of plant 11S globulins as emulsifiers or stabilizers to prepare emulsion or HIPE formulations, but also for eludicating the importance of glycation with carbohydrates to the development of food grade protein-based Pickering stabilizers.

Acknowledgements This work was supported by the National Natural Science Foundation of China, under projects No. 21872057 and 31771917, Guangzhou Natural Science Foundation under project (201904010143) and GDHVPS (2017).

26

Journal Pre-proof References Arditty, S., Schmitt, V., Giermanska-Kahn, J., & Leal-Calderon, F. (2004). Materials based on solid-stabilized emulsions. Journal of Colloid and Interface Science, 275, 659-664. Capron, I.. & Cathala, B. (2013). Surfactant-free high internal phase emulsions stabilized by cellulose nanocrystals. Biomacromolecules, 14, 291-296. Chen, Q. H., Zheng, J., Xu, Y. T., Liu, F., Yin, S. W., & Tang, C. H. (2018). Surface modification improves fabrication of Pickering high internal phase emulsions stabilized by cellulose nanocrystals. Food Hydrocolloids, 75, 125–130. Dickinson, E. (2015). Microgels – An alternative colloidal ingredient for staiblization of food emulsions. Trends in Food Science & Technology, 43, 178-188. Frelichowska, J., Bolzinger, M.-A., & Chevalier, Y. (2010). Effects of solid particle content on properties of o/w Pickering emulsions. Journal of Colloid and Interface Science, 2010, 351, 348-356. Horozov, T., & Binks, B. P. (2006). Particle-stabilized emulsions: a bilayer or a bridging monolayer? Angewandte Chemie International Edition, 45 (5), 773-776. Hu, Y. Q., Yin, S. W., Zhu, J. H., Qi, J. Q., Guo, J., Wu, L. Y., Tang, C. H., & Yang, X. Q. (2016). Fabrication and characterization of novel Pickering emulsions and Pickering high internal phase emulsions stabilized by gliadin colloidal particles. Food Hydrocolloids, 61, 300-310. Huang, X. N., Zhou, F. Z.,Yang, T., Yin, S. W., Tang, C. H., & Yang, X. Q. (2019 b). Fabrication and characterization of Pickering high internal phase emulsions (HIPEs) stabilized by chitosan-caseinophopeptides nanocomplexes as oral delivery vehicles. Food Hydrocolloids, 93, 27

Journal Pre-proof 34-45. Huang, X. N., Zhu, J. J., Xi, Y. K., Yin, S. W., Ngai, T., & Yang, X. Q. (2019 a). Protein-based Pickering high internal phase emulsions as nutraceutical vehicles of and the template for advanced materials: A perspective paper. Journal of Agricultural and Food Chemistry, 67, 9719-9726. Jiao, B., Shi, A., Wang, Q., & Binks, B. P. (2018). High-internal-phase Pickering emulsions stabilized solely by peanut-protein-isolate microgel particles with multiple Potential applications. Angewandte Chemie International Edition, 57, 9274-9278. Lee, M., Chan, H. K., & Mohraz, A. (2012). Characteristics of Pickering emulsion gels formed by droplet bridging. Langmuir, 28 (6), 3085-3091. Li, X. R., & Tang, C. H. (2013). Influence of glycation on microencapsulating properties of soy protein isolate-lactose blends. Journal of the Science of Food and Agriculture, 93, 2715-2722. Li, Z., Ming, T., Wang, J.. & Ngai, T. (2009). High internal phase emulsions stabilized solely by microgel particles. Angewandte Chemie International Edition, 48, 8490-8493. Li, Z., Xiao, M., Wang, J., & Ngai, T. (2013). Pure protein scaffolds from Pickering high internal phase emulsion template. Macromolecules Rapid Communications, 34, 169-174. Liang, H. N., & Tang, C. H. (2013). Emulsifying and interfacial properties of vicilins: Role of conformational flexibility at quaternary and/or tertiary levels.

Journal of Agricultural and

Food Chemistry, 61(46), 11140-11150. 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, 8888-8898. 28

Journal Pre-proof Liu, F., & Tang, C. H. (2016 a). Soy glycinin as food-grade Pickering stabilizers: Part. I. Structural characteristics, emulsifying properties, and interfacial adsorption/arrangement at interface. Food Hydrocolloids, 60, 606-619. Liu, F., & Tang, C. H. (2016 b). Soy glycinin as food-grade Pickering stabilizers: Part. II. Improvement of emulsification and interfacial adsorption by electrostatic screening. Food Hydrocolloids, 60, 620-630. Liu, F., & Tang, C. H. (2016 c). Soy glycinin as food-grade Pickering stabilizers:

Part. III.

Fabrication of gel-like emulsions and their potential as sustained-release delivery systems for β-carotene. Food Hydrocolloids, 56, 434-444. Liu, F., Zheng, J., Huang, C. H., Tang, C. H., & Ou, S. Y.. (2018). Pickering high internal phase emulsions stabilized by protein-covered cellulose nanocrystals. Food Hydrocolloids, 82, 96-105. Ma, L., Zou, L., McClements, D. J., & Liu, W. (2020). One-step preparation of high internal phase emulsions using natural edible Pickering stabilizers: Gliadin nanoparticles/gum Arabic. Food Hydrocolloids, 100, 105381. Marcone, M. F. (1999). Biochemical and biophysical properties of plant storage proteins: a current understanding with emphasis on 11S seed globulins. Food Research International, 32, 79-92. Nagano,T., Hirotsuka, M., Mori, H., Kohyama, K., & Nishinari, K. (1992). Dynamic viscoelastic study on the gelation of 7S globulin from soybeans. Journal of Agricultural and Food Chemistry, 40 , 941-944. Patel, A. R. (2018). Funtional and engineered from edible materials for emerging applications in 29

Journal Pre-proof designing the food of the future. Advanced Funtional Materials, 1806809. Patel, A. R., Rodriguez, Y., Lesaffer, A., & Dewettinck, K. (2014). High internal phase emulsion gels (HIPE-gels) prepared using food-grade components. RSC Advances, 4, 18136-18140. Peng, L. P., Xu, Y. T., Li, X. T., & Tang, C. H. (2020). Improving the emulsification of soy β-conglycinin by alcohol-induced aggregation. Food Hydrocolloids, 98, 105307. Peng, X. Q., Xu, Y. T., Liu, T. X., & Tang, C. H. (2018). Molecular mechanism for improving emulsification efficiency of soy glycinin by glycation with soy soluble polysaccharide. Journal of Agricultural and Food Chemistry, 66, 12316−12326.. Perrin, E., Bizot, H., Cathala, B., & Capron, I. (2014). Chitin nanocrystals for Pickering high internal phase emulsions. Biomacromolecules, 15, 3766-3771. Rayner, M., Timgren, A., Sjӧӧ, M., & Dejmek, P. (2012). Quinoa starch granules: a candidate for stabilising food-grade Pickering emulsions. Journal of the Science of Food and Agriculture, 92, 1841-1847. Shen, L., & Tang C. H. (2012). Microfluidization as a potential technique to modify surface properties of soy protein isolate. Food Research International, 48, 108-118. Tan, H., Sun, G., Lin, W., Mu, C., & Ngai, T. (2014). Gelatin Particle-Stabilized High Internal Phase Emulsions as Nutraceutical Containers. ACS Applied Materials and Interfaces, 6, 13977-13984. Tang, C. H. (2017). Emulsifying properties of soy proteins: A critical review with emphasis on the role of conformational flexibility. Critical Reviews in Food Science and Nutrition, 57(12), 2636-2679. Tang, C. H. (2019). Nanostructured soy proteins: Fabrication and applications as delivery systems 30

Journal Pre-proof for bioactives (a review). Food Hydrocolloids, 91, 92-116. Tzoumaki, M. V., Moschakis, T., Kiosseoglou, V., & Biliaderis, C. G. (2011). Oil-in-water emulsions stabilized by chitin nanocrystal particles. Food Hydrocolloids, 25, 1521-1529. Wijaya, W., Sun, Q.-Q., Vermeir, L., Dewettinck, K., Patel, A. R., & van der Meeren, P. (2019). pH and protein to polysaccharide ratio control the structural properties and viscoelastic network of HIPE-templated biopolymeric oleogels. Food Structure, 21, 100112. Wijaya, W., Van der Meeren, P., Wijaya, C. H., & Patel, A. R. (2017). High internal phase emulsions stabilized solely by whey protein isolate-low methoxyl pectin complexes: Effect of pH and polymer concentration. Food and Function, 8, 584-594. Xu, Y. T., Liu, T. X., & Tang, C. H. (2019). Novel Pickering high internal phase emulsion gels stabilized solely by soy β-conglycinin. Food Hydrocolloids, 80, 21-30. Xu, Y. T., Tang, C. H., & B. P. Binks (2020). High internal phase emulsions stabilized solely by a globular protein glycated to form soft particles. Food Hydrocolloids, 98, 105254. Xu, Y. T., Tang, C. H., Liu, T. X., & Liu, R. H. (2018). Ovalbumin as an outstanding Pickering nano-stabilizer for high internal phase emulsions. Journal of Agricultural and Food Chemistry, 66 (33), 8795-8804. Yang, T., Li, X. T., & Tang, C. H. (2020). Novel edible Pickering high-internal-phase-emulsion gels efficiently stabilized by unique polysaccharide-protein hybrid nanoparticles from Okara. Food Hydrocolloids, 98, 105285. Yang, T., Zheng, J., Zheng, B. S., Liu, F., Wang, S., & Tang, C. H.. (2018). High internal phase emulsions stabilized by starch nanocrystals. Food Hydrocolloids, 82, 230-238. 31

Journal Pre-proof Yuan, D. B., Hu, Y. Q., Zeng, T., Yin, S. W., Tang, C. H., & Yang, X. Q. (2017). Development of stable Pickering emulsions/oil powders and Pickering HIPEs stabilized by gliadin/chitosan complex particles. Food & Function, 8, 2220-2230. Zamani, S., Malchione, N., Selig, M. J., & Abbaspourrad, A. (2018). Formation of shelf stable Pickering high internal phase emulsions (HIPE) through the inclusion of whey protein microgels. Food & Function, 9, 982-990. Zeng, T., Wu, Z. L., Zhou, J. Y., Yin, S. W., Tang, C. H., Wu, L. Y., & Yang, X. Q. (2017). Development of antioxidant Pickering high internal phase emulsions (HIPEs) stabilized by protein/polysaccharide hybrid particles as potential alternative for PHOs. Food Chemistry, 231, 122-130. Zhou, F. Z., Huang, X. N., Wu, Z. L., Yin, S. W., Zhu, J. H., Tang, C. H., & Yang, X. Q. (2018). Fabrication of zein/pectin hybrid particle-stabilized Pickering high internal phase emulsions with robust and ordered interface architecture. Journal of Agricultural and Food Chemistry, 66, 11113-11123. Zhou, F. Z., Zeng, T., Yin, S. W., Tang, C. H., Yuan, D. B., & Yang, X. Q. (2018). Development of antioxidant gliadin particle stabilized Pickering high internal phase emulsions (HIPEs) as oral delivery systems and the in vitro digestion fate. Food & Function, 9, 959-970.

32

Journal Pre-proof Figure captions. Figure 1. Visual observation for the formation of gel-like HIPEs stabilized by untreated and glycated (24 and 72 h) SG, at ϕ = 0.8 and varying protein concentrations of 0.2-0.8 wt%. The minimal protein concentration (cm) below which the HIPEs began to flow was obtained visually by placing the samples upside down. Figure 2. a) Typical frequency dependent profiles of moduli (including storage modulus, G' or loss modulus, G'') of fresh HIPEs (at ϕ = 0.8) stabilized by untreated and glycated (0-72 h) SG at a protein concentration of 0.8 wt%. b) The G' at a specific frequency of 1.0 Hz for the corresponding HIPEs stabilized by untreated and glycated (0-72 h) SG. Each datum is the means and standard deviation (n = 3). Different characters (a-d) on the top of columns represent significant difference at p < 0.05 level. Figure 3. Optical micrographs of different fresh HIPE gels (ϕ = 0.8) stabilized by untreated SG, or SG glycated with SSPS for 0, 24 and 72 h, at varying protein concentrations of 0.3-1.0 wt%. Panel a: representative optical micographs of the fresh HIPE gels stabilized by different SG samples, at a specific c value of 0.8 wt%. Panel b: representative optical micrographs of the HIPE gels stabilized by the glycated (72 h) SG, at varying c values of 0.3-1.0 wt%. The bars represent 100 μm in scale. Figure 4. a) The surface-average diameter (d3,2) values of droplets in different fresh HIPEs (ϕ = 0.8) stabilized by untreated SG, or SG glycated with SSPS for 0, 24 and 72 h, as a function of protein concentration (c; in the range 0.3-1.0 wt%). Each datum is the means and standard deviation (n = 3), which was obtained from individual optical micrographs performed on separate samples. b) Percentage of adsorbed proteins at interface (AP%) of the fresh HIPE gels (ϕ = 0.8) stabilized by 33

Journal Pre-proof untreated and glycated (24 and 72 h) SG samples, at a specific c value of 1.0 wt%. Each datum is the means and standard deviation (n = 3). Different characters (a-c) represent significant difference at p < 0.05 level. Figure 5. a) The morphology and optical micrographs of the HIPEs stabilized by untreated SG (n-SG) and glycated (24 and 72 h) SG, fresh or after a storage of 30 days at room temperature. Bars: 50 μm in scale. b) The surface-average diameter (d3,2) values of droplets in the correspondings HIPEs, fresh or after a storage of 30 days. Data reported are the means ± S. D. (n =3). All the HIPEs were formed at  = 0.8 and c = 1.0 wt%. Figure 6. a) Visual observation and optical micrographs of the HIPEs (ϕ = 0.8) stabilized by untreated SG, or SG glycated with SSPS for 24 and 72 h, at a c value of 1.0 wt%, without or with heating in boiling water for 15 min. The bars: 100 μm in scale. b) The storage modulus (G') at a specific frequency of 1.0 Hz for the corresponding unheated or heated HIPEs. Each datum is the means and standard deviation (n = 3). Figure 7. Visual observation and optical micrographs of the HIPE gels stabilized by untreated SG, or SG glycated with SSPS for 24 and 72 h, at a c value of 1.0 wt%, before or after different cycles (1-3) of freeze-thaw treatment. Scale bars, 100 µm. All the HIPE gels were frozen at -20 oC for 24 h, and then thawed at 25 oC for 2 h. In all cases, a same emulsification process was performed using a one-pot shearing at 5000 rpm for 1 min at room temperature. Figure 8. Evolution of interfacial tension (γ) for SG glycated with SSPS for different period times of 0, 24 and 72 h, at oil-water interface, as a function of incubation time (up to 180 min). All the experiments were performed at a constant c value of 0.05 wt%. 34

Journal Pre-proof Figure 9. The hydrodynamic size distribution profiles (a), intrinsic fluorescence (b) and far-UV CD (c) spectra of untreated or glycated (24 and 72 h) SG, initial or adsorbed at the interface at 25 °C. The adsorbed SG in the HIPEs, formed at ϕ = 0.8 and c = 1.0 wt%, was obtained via emulsion breaking by freeze-thawing (-40 °C, 24 h→25 °C, 2 h), followed by sequential centifugation and redispersion in water. Figure 10. Representative TEM images of untreated and glycated (72 h) SG in the solutions or dispersions (c = 0.02 wt%) at pH 7.0. Scale bars: 100 nm in scale.

35

Journal Pre-proof Figure 1.

36

Journal Pre-proof Figure 2.

a

b

1000

800

a

G' (Pa)

moduli (Pa)

600

100

400

b

c d

200 n-SG g-SG (24 h)

10

0.1

1

g-SG (0 h)) g-SG (72 h)

10

n-SG

frequency (Hz)

37

g-SG (0 h)

g-SG (24 h)

g-SG (72 h)

Journal Pre-proof Figure 3.

38

Journal Pre-proof Figure 4.

b

a

adsorbed protein (%)

d3,2(m)

a

60

100 80 60 40

n-SG g-SG(0 h) g-SG(24 h) g-SG(72 h)

20 0.2

0.4

0.6

0.8

c (%,w/v)

50 40 30

10 0

1.0

b

20

c

untreated

24 h

Glycated

39

72 h

Journal Pre-proof Figure 5.

b

60

fresh 1 month

d3,2 (m)

45 30 15 0

40

n-SG

g-SG(24h)

g-SG(72h)

Journal Pre-proof Figure 6. b

1000

unheated Heated

G' (Pa)

800 600 400 200

untreated

24 h

72 h

Glycated

41

Journal Pre-proof Figure 7.

42

Journal Pre-proof Figure 8.

interfacial tension (mN/m)

30

g-SG (0 h) g-SG (24 h) g-SG (72 h)

25

20

15 0

2000

4000

6000

time (s)

43

8000

10000

Journal Pre-proof Figure 9.

volume fraction (%)

9.7

b

initial adsorbed

fluorescnence intensity (A.U.)

a

n-SG 9.9

g-SG (24 h) 4.2

13.5

g-SG (72 h) 10

100

size (nm)

1000

c

333

g-SG (24 h)

g-SG (96 h)

300

10000

320

340

initial adsorbed

n-SG

g-SG (24 h)

g-SG (72 h)

190

200

210

360

wavelength (nm)

2 -1 [q]（ deg.cm .dmol （

1

initial adsorbed

n-SG

220

230

wavelength (nm)

44

240

250

380

400

Journal Pre-proof Figure 10.

45

Journal Pre-proof The authors declare no competing financial interest.

Journal Pre-proof Ze-Zhou Hao and Xiu-Qing Peng have performed the experiments and data analyses, and written the original version of manuscript. Dr. Chuan-He Tang designed the research and supervised the project, has written and edited the manuscript, and had primary responsibility for all the contents. All the authors have read and approved the final manuscript.

Journal Pre-proof Highlights:  Soy glycinin (SG) exhibits a potential to act as stabilizers for high internal phase emulsions (HIPEs).  The glycation with soy polysaccharide improved the performance of SG to stabilize the HIPEs.  The HIPEs by glycated SG exhibited better coalescence stability than those by unglycated SG.  The glycation facilitated the subunit dissociation of adsorbed SG, but increased the structural integrity of its subunits.