Approaches And Molecular Mechanisms For Globular Proteins To Stabilizing High Internal Phase Emulsions As Soft Particles

Because of the developing science and technology and increasing demands of human,various advanced materials are booming everywhere,and Pickering high internal phase emulsions(HIPEs)have also been applied in numerous high-tech industries.Compared with inorganic particles and synthetic organic particles,biocompatible protein-based Pickering stabilizers more highly satisfy the requirements for ecofriendly and healthy products.Considering that most protein-based particles are fabricated through complicated physical,chemical and/or biological processings,it is a better choice to search for a natural globular protein with stable conformation,and directly use it as a Pickering stabilizer.But the classic emulsifying mechanism for globular proteins is that they will be cracked and crosslink each other at the oil-water interface and establish protein layers wrapping oil droplets.As known,this theory was acquired based on protein with high surface hydrophobicity.Unusually,this study started with a protein with high surface hydrophilicity,glycoprotein,to find a novel emulsifying mechanism for globular proteins and form high-performance Pickering HIPEs.First,a monomeric glycoprotein,ovalbumin(OVA),and a trimeric glycoprotein,?-conglycinin(?-CG),were proved to be natural Pickering stabilizers,which could high-efficiently stabilize oil-in-water(O/W)HIPEs just by one-step shearing homogenization.Unlike OVA,?-CG would be first disaggregated into three glyco-subunits,??,? and ? at the oil-water interface.Both OVA and ?-CG subunits were soft particles with high structural stability.The strong intermolecular cohesion made them unfolded extremely limitedly under the synergistic effect of the interfacial tension and hydrophobic interaction between hydrophobic moieties and oil phase.OVA and ?-CG could not only package high-volume oil phase,but also stabilize HIPEs with gel-like self-supporting and remoldable ability.When the oil phase fraction(?)was fixed at 0.8,the minimal protein concentration(c)for these two glycoproteins to form HIPE gel was as low as 0.2 wt.%.And the rheological behavior,microstructure and dispersibility of the formed HIPEs could be feasibly regulated by varying c.At lower c,the gel network structures of OVA-and ?-CG-stabilized HIPEs highly depended on bridging flocculation between oil droplets which were difficult to be separated.However,at higher c,the depleting flocculated oil droplets within HIPEs were facilely dispersed.Besides,these formed HIPEs possessed high storage stability,thermal stability,freeze-thaw sensitivity and attractive temperature responsiveness.Given the outstanding properties and the corresponding structure-function relationships of these glycoproteins and the formed Pickering HIPEs,this study indeed created a new situation in colloidal science and preliminarily revealed the importance of carbohydrate chains for globular proteins to act as Pickering stabilizersThen,to sufficiently verify the critical role of carbohydrate chains stated above,this study used galactose(Gal)to transform native bovine serum albumin(nBSA)into glycated BSA(gBSA)through Maillard reaction.The hydrated carbohydrate chains on each gBSA generated a dense and thick aqueous layer closely surrounding BSA particle with a more compact molecular structure induced by glycosylation.Therefore,similar with natural OVA or ?-CG subunits,gBSA was a soft particle with core-shell structure.Obviously different from nBSA,gBSA exhibited higher structural stability,steric hindrance between particles and efficiency to stabilizing Pickering HIPEs.In addition,gBSA-stabilized HIPEs owned high coalescence stability,freeze-thaw sensitivity and distinguished temperature responsiveness.So,the presence of carbohydrate chains is exactly enough but not necessary condition for globular proteins to be Pickering stabilizers.To certify the significance of carbohydrate chains,the unreacted Cal was removed using dialysis.In view of the case that free sugar molecules did exist,this study developed a new strategy to converting nonglycoproteins into core-shell soft particles,i.e.,adding polyols into the aqueous phase.The bund polyols built a thick and dense “shell” on the surface of protein,making the molecular structure of “core” protein more compact and stable.And the unbound polyols could synergistically stabilize the oil-water interface,indirectly enhancing the emulsifying efficiency of nonglycoproteins.While the concentration of polyols was high to some extent,lots of unbound polyols formed large micelles or even networks,which could much increase the adsorption rate and desorption energy of nonglycoproteins to oil-water interface.This new approach to fabricating Pickering stabilizers and the relevant emulsifying mechanisms were not only applicable to nonglycoproteins,but also suitable for glycoproteins and even other inorganic or organic particles.Last,using OVA as a glycoprotein model,this study first demonstrated that adjusting pH could regulate the emulsifying mechanism of glycoproteins.In the neutral(pH 7.0)solution,glycoproteins were soft Pickering stabilizers.But basic(pH 11.0)environment could destroy the original structures of glycoproteins,transform them into incompact particles with irregular and inconstant shape,and make them highly unfold and crosslink each other to be a thin film at the oil-water interface.When dispersed in the acid(pH 3.0)solution,due to the limited unfolding and exposed hydrophobic moieties,glycoproteins aggregated tightly and produced larger spherical particles.The aggregate particle also possessed a hydrated shell generated by carbohydrate chains and a compact protein core,which offering strong intramolecular cohesion and structural stability.Compared with glycoproteins at pH 7.0,these aggregate particles exhibited higher desorption energy to interface,could stabilize larger interface area and establish more bridging flocculation structures,which completely reflected the classic size effect of Pickering stabilizers.Besides,the Pickering HIPEs stabilized by glycoproteins at pH 3.0 with higher coalescence stability and firmer gel network,were ideal alternatives for traditional mayonnaise.Summarily,this study innovatively revealed a new emulsifying molecular mechanism for globular proteins,and provide ideas and strategies for the developments and applications of novel Pickering HIPEs.