High-Internal-Phase Pickering Emulsions Stabilized Solely by Peanut-Protein-Isolate Microgel Particles with Multiple Potential Applications

: High-internal-phase Pickering emulsions have var-ious applications in materials science. However, the biocom-patibility and biodegradability of inorganic or synthetic stabilizers limit their applications. Herein, we describe high-internal-phase Pickering emulsions with 87% edible oil or 88% n-hexane in water stabilized by peanut-protein-isolate microgel particles. These dispersed phase fractions are the highest in all known food-grade Pickering emulsions. The protein-based microgel particles are in different aggregate states depending on the pH value. The emulsions can be utilized for multiple potential applications simply by changing the internal-phase composition. A substitute for partially hydrogenated vegetable oils is obtained when the internal phase is an edible oil. If the internal phase is n-hexane, the emulsion can be used as a template to produce porous materials, which are advantageous for tissue engineering.

High-internal-phase emulsions (HIPEs) are commonly referred to as superconcentrated emulsions with am inimum internal-phase volume fraction of 0.74. [1] Their wide applicability in pharmaceutical formulations,t issue engineering, and food products has received considerable attentions. [2] HIPEs contain an internal phase,acontinuous phase,a nd necessary stabilizers to keep the system kinetically stable.I n most cases,H IPEs are stabilized by large amounts of surfactants (5-50 wt %). [3] Some surfactants,such as carboxymethylcellulose and polysorbate-80, however, are subject to concerns,which may limit the application of HIPEs. [4] Besides the conventional emulsions mentioned above,so-called Pickering emulsions [5] stabilized by solid particles are usually more stable.Many surface-active particles adsorb irreversibly to the oil-water interface,thereby lowering the total free energy.In addition, alayer of adsorbed particles envelops droplets,thus endowing the emulsion with high stability to coalescence and Ostwald ripening. [6] High-internal-phase Pickering emulsions (HIPPEs) have been used as templates for the preparation of porous materials,inwhich the particles form the walls,which can result in av ariety of applications. [1,7] In general, many Pickering emulsions used currently have ar elatively low dispersed phase fraction. [8] Nevertheless, HIPPEs have also been reported. Akartuna et al. [9] prepared HIPPEs with internal-phase volume fractions between 72 % and 78 %, but the systems required high particle concentrations (35 vol %). Zhou et al. [10] produced both methyl myristate-in-water and soybean oil-in-water(o/w) emulsions with high (80 %) internal-phase fractions by using hydroxyapatite nanoparticles as emulsifiers and to further prepare porous materials;h owever,t he route seems to be tedious and timeconsuming (more than 7days). Arditty et al. [11] reported HIPPEs with silane-modified silica particles that reached an internal-phase content of 90 %. Ikem et al. [7b] and Li et al. [7c] also reported that by the use of inorganic particles or synthetic microgel particles as emulsifiers,s table HIPPEs with an internal-phase content above 90 %could be prepared. Such HIPPEs are ag ood template for producing highly porous materials.U nfortunately the biosecurity,b iocompatibility and biodegradability of these particles greatly limit their pharmaceutical applications. [12] On the other hand, Pickering emulsions are also important in the food industry, [13] which have ag reat demand for natural rather than chemosynthetic particles.
There is ag rowing interest in natural food-grade Pickering emulsions owing to their long shelf life and their ability to deliver functionally active substances, [13,14] and af ew studies have been concerned with HIPPEs.T an et al. [15] prepared HIPPEs stabilized by gelatin particles with an internal-phase of 80 %. Zeng et al. [16] reported an antioxidant HIPPE stabilized by gliadin/chitosan hybrid particles with an internal phase up to 83 %. However,n one of the particles in the aforementioned studies were fabricated without using organic solvents (acetone,e thyl alcohol, or glutaraldehyde) or other reagents.T he incorporation of organic solvents or residual reagents is ab ig concern when claims are made that the HIPPEs can be applied in the food industry.C apron and Cathala [6a] also reported high-internal-phase emulsions stabilized by cellulose nanocrystals,but the oil used was not edible, thus impeding application in the food industry.T he cleanlabel HIPPEs applicable in the food industry should be composed of food-grade materials,i ncluding both oils and emulsifiers.HIPPEs composed of food-grade particles,water, and edible oil have amargarine-like appearance and can be of similar composition to margarine formulations, [17] which makes food-grade HIPPEs good substitutes for margarine [16] containing partially hydrogenated oils (PHOs). Serious food safety hazards mean that PHOs have been forbidden by the FDAtobeused in food production in the USA after 2018. [18] Herein, we report high-internal-phase emulsions stabilized solely by natural peanut-protein-isolate (PPI) microgel particles,inwhich the fraction of the internal phase,either an edible oil or n-hexane,c an be as high as 0.87 and 0.88, respectively.T he PPI microgel particles adsorb at the oilwater interface to form ac lose-packed layer around the droplets,and some excess PPI microgel particles dispersed in the continuous phase form an etwork, both leading to stabilization of the emulsion against to coalescence.T he prepared emulsions could have multiple potential applications simply by changing the composition of the internal phase.Whenthe internal phase is an edible oil, it is possible to obtain am aterial with high viscosity similar to that of margarine.I ft he internal phase is n-hexane,ap rotein-based porous material can be obtained after evaporating the oil and water in air.The 3D structure of this material can be tailored by simply changing the pH value of the aqueous phase.Other applications are also possible with the present HIPPEs.
Thep reparation of peanut-protein-isolate microgel particles from PPI molecules involves two steps:i )transglutaminase (TG) enzyme cross-linking and ii)gel crushing.I nt he first step,T G-catalyzed acyl-transfer reactions between gcarboxamide groups or protein-bound glutaminyl residues of peptides and primary amines of lysine promote the formation of an entire PPI hydrogel. [19] Thea bove-mentioned hydrogel was crushed into microgel particles by using ar otor-stator homogenizer followed by ah igh-pressure homogenizer.
Thep rotein-based microgel particles were amphoteric and pH-sensitive.T he isoelectric point of the PPI microgel particles (where the overall charge on the surface of the particles was zero owing to the equal positive charges provided by amino groups and negative charges provided by carboxylic groups) and the PPI suspension (without TG enzyme cross-linking) was around pH 4.0-4.5 (see Figure S1 in the Supporting Information). Below the isoelectric point, PPI microgel particles are positively charged owing to the protonation of amino groups.A bove it, they are negatively charged owing to the deprotonation of carboxylic groups.This charge variation results from the balance between the charges of carboxylic and amino groups of the peanut protein as afunction ofpH value.
Thea verage diameter of PPI microgel particles varied between 200 and 300 nm (Figure 1a). Visible aggregates in water were formed at pH 3.0 and pH 4.5, thus precluding determination of their size by light scattering. Optical microscopy images showed that the size of the aggregates in aqueous solution was around 10-30 mma tp H3.0 and 10-60 mma tp H4.5 (see Figure S2). When the pH value was increased above the isoelectric point, the hydrophilicity of the PPI increased, which resulted in swelling of the PPI microgel particles (Figure 1a). Thet hree-phase contact angle of aw ater droplet on as ubstrate composed of PPI microgel particles in oil reached the highest value (1568 8 for edible oil and 1328 8 for n-hexane) at pH 4.5 (around the isoelectric point), implying the PPI microgel particles were at their most hydrophobic (Figure 1b). This contact angle decreased either side of the isoelectric point, meaning that the hydrophobicity of the PPI microgel particles decreased at pH values both below and beyond the isoelectric point.
Thed etailed morphology of PPI microgel particles was characterized by cryo-SEM (Figure 1d). Thep articles were spherical with diameters of 40-150 nm, but could be in different aggregate states,a nd the extent of aggregation of PPI microgel particles depended on the pH value.T he relatively large aggregates observed at ap Hvalue of around 4.5 are associated with zero zeta potential, at which particles are most hydrophobic owing to areduction in the electrostatic repulsion between them and to van der Waals attraction. As the pH value was lowered to 3.0, PPI microgel particles became positively charged, which resulted in stretching of the aggregates.A ss een in the cryo-SEM image,t he aggregates were composed of spherical particles,which aligned in strips. Above the isoelectric point, the aggregates were also linear in shape.Some ellipsoidal structures (major diameter:ca. 1 mm) could also be found. At pH 9.0, all particles were fused, and no individual particles could be found. TEM results were in accordance with the findings above (see Figure S3). However, the PPI microgel particles were smaller because they shrinked during the drying step in the preparation of the TEM sample.
Emulsions stabilized by peanut-protein-isolate microgel particles were prepared by mixing the oil phase (cold-pressed peanut oil or n-hexane) and aqueous particle dispersion at different pH values.E mulsification was carried out using ar otor-stator homogenizer at 3000 rpm for 30 s. When commercial cold-pressed peanut oil was used as the internal phase,astable HIPPE with an internal-phase fraction of 0.85 could only be obtained at pH 3.0 and 9.0 (Figure 2a,b), but not at pH 4.5 or pH 7.0. Both emulsions had ac ream-like, homogeneous appearance.F rom the cryo-SEM images,w e can see that the oil droplets were trapped by a3 Ds tructure formed by PPI microgel particles in the continuous phase (Figure 2c,f), which also provided steric hindrance to the coalescence of droplets.AtpH3.0, the droplet size was larger (5-50 mm; Figure 2c,e) than that at pH 9.0 (1-20 mm; Figure 2d,f)as PPI microgel particles in the aqueous phase before emulsification were aggregated (see Figure S3 a). Thus, aggregates of large "particle" size stabilized larger droplets. Thes maller droplets formed during emulsification were subject to limited coalescence after the agitation stopped. [11] On the other hand, the diameter of the PPI microgel particles at pH 9.0 was around 270 nm (Figure 1a), and al arger interfacial area could be coated by them, thus leading to asmaller droplet size.
Theo ther big difference is that the droplets were deformed at pH 3 ( Figure 2c,e) but mostly spherical at pH 9 (Figure 2d,f). This difference should have an impact on the consistencya nd viscosity of the emulsions.T he deformation may due to the fact that when the number of PPI microgel particles is insufficient to coat all the droplets,s ome PPI microgel particles may be shared by adjacent droplets in abridging monolayer conformation. [20] This behavior leads to flocculation of the emulsion droplets without coalescence. HIPPEs with 85 %( w/w) edible oil as the internal phase cannot be obtained at pH 4.5 and 7.0, which made the HIPPEs pH-responsive.T he pH response of the emulsions may be divided into two scenarios.A tp H3.0, the striplike aggregates can attach to the interface and form aviscoelastic film to trap the oil droplets.AtpH4.5, the aggregates are the most hydrophobic (Figure 1b), and the oil/water (o/w) interface tends to bend towards the water phase.B esides,t he big aggregates are too large to fully cover the huge specific surface area of an o/w HIPPE, so aw ater-in-oil (o/w) emulsion (with 15 %i nternal phase) with al ower specific surface area was obtained (see Figure S4). At pH 7, an o/w emulsion with ahigh internal-phase fraction could be formed, but with excess coalesced oil surrounding the HIPPE (see Figure S4). At pH 9, the particles were swollen, deformed, and fused into an elastic interfacial film, which covered the oil droplets. [15,21] Theu pper limit of the internal-phase fraction within the emulsion was also established at pH 9.0. AHIPPE containing an oil-phase fraction of 0.87 could be reached. To the best of our knowledge,this is the highest reported value in all known food-grade Pickering emulsions.
PPI microgel particles at pH 3.0 and 9.0 were also used to stabilize HIPPEs with 85 % n-hexane as the internal phase. Theappearances of these emulsions are shown in Figure 3a,b. Them acroscopic shapes can be readily tailored. TheP PI microgel particle-stabilized emulsions at pH 3.0 and 9.0 were also characterized by cryo-SEM and CLSM (Figure 3c-f). In both emulsions,ad ense layer of the PPI microgel particles can be observed (see insets in Figure 3c,d). Thel ayer of particles surrounding the deformed droplets ensures the emulsion is mechanically stable. [7b] Theoil and water of both emulsions were allowed to evaporate overnight in air. Evaporation resulted in al arge number of pores in which the oil was located, with the adsorbed and excess PPI microgel particles forming the walls. [7c] From SEM images (Figure 3g,h), we can see that porous materials were obtained from both emulsions.The pore size is alittle smaller than the precursor droplet size (see Figure S5), since PPI microgel particles were fused and formed uniform walls during solvent evaporation processing (see Figure S6). Furthermore,t he thickness of the cell wall could be tailored by changing the concentration of the PPI microgel particles (see Figure S7). At low particle concentrations,the wall of the porous material shrank and was fragile (see Figure S7 a,b,0.83 and 1.17 wt %), and the pore structures almost collapsed. By increasing the concentration of particles,p orous materials with relatively uniform walls could be obtained (see Figure S7 a,b,1 .50 and 1.83 wt %). Theupper limit of the internal-phase volume was also tested with n-hexane.P PI microgel particles at pH 9.0 can stabilize aHIPPE with 88 %internal phase.These porous materials should have great potential as catalyst supports,i n encapsulation applications,a nd as scaffolds for tissue engineering.W ith the great advantages of an atural product, biotoxicity would not be aproblem in applications compared to previous studies. [7c,d] In summary,w eh ave presented an ew natural proteinbased material (peanut-protein-isolate microgel particles) for stabilizing o/w Pickering emulsions.T he internal-phase fraction can reach 87 %f or an edible oil and 88 %f or n-hexane. Edible materials as substitutes for PHOs can be obtained when the internal phase is an edible oil. Natural, nontoxic porous materials can be produced by using these Pickering emulsions as at emplate.W eb elieve that the present emulsions can be used in av ariety of areas,e specially those with high biosafety requirements,s uch as food and tissue engineering.