| Polysaccharide and protein have been regarded as promising alternative emulsifiers as consumers are becoming increasingly interested in processed food products with Clean Labels.It has been proved that interactions between protein and polysaccharide could significantly improve their interfacial adsorption behaviours,thus extending the shelf life of emulsion-based food products.In comparison to traditional ways to prepare polysaccharide-protein complexes/conjugates,such as electrostatic interactions and Maillard reaction,enzymatic conjugation requires less strict reaction conditions and the resulted conjugates would remain stable against varied pH and ionic strength.However,the preparation of polysaccharide-protein conjugates using enzymatic reaction was not so successful.Besides that.there has not been any report on the interfacial adsorption mechanism of enzymatically-catalyzed polysaccharide and protein conjugates.In current research,corn fiber gum(CFG)was selected as a model polysaccharide,and external proteins were covalently crosslinked with CFG via enzymatic catalyzation to improve the emulsifying properties of CFG in oil-in-water emulsions.On one hand,a theoretical enzymatic catalyzed polysaccharide-protein conjugate preparation model as well as their interfacial adsorption mechanism were proposed.On the other hand,these models were applied to improve the emulsifying properties of protein-deficit CFG samples.Major conclusions were as follows:1.When catalyzed by horseradish peroxidase(HRP),CFG formed inter/intra-molecular conjugates with model protein,bovine serum albumin(BSA)via the covalent bonds between phenolic acids in CFG and tyrosine in BSA.There were unreacted CFG or BSA in the final enzyme-treated CFG/BSA conjugate products as well.The inter-molecular crosslinking between CFG and BSA was determined by UV-vis spectroscopy,Fourier transform infrared spectroscopy,and SDS-PAGE gel electrophoresis.The results of tyrosine content measured before and after enzymatic reaction showed that the yield of conjugates increased as the weight ratio of CFG/BSA increased.The optimum enzymatic reaction results were achieved when the weight ratio of CFG and BSA was 10:1.and the addition amount of HRP was 0.25 μg/mg BSA.2.CFG/BSA conjugates prepared under the optimum enzymatic conditions were applied as emulsifiers to stabilize oil-in-water emulsions.The average particle size and zeta potential of conjugate-stabilized emulsions remained relatively unchanged when the pH,ionic strength varied or when the emulsions went through freeze-thaw cycles.The satisfying emulsifying performance of the conjugates were attributed to their developing a considerably thick and complex interfacial layer on the oil-water surface.The layer thickness of the adsorbed conjugates were higher than that of untreated CFG/BSA mixtures,which was investigated by the mono-dispersed polystyrene latex particles.When adsorbed on the surface of emulsion droplets,the proteinaceous fraction in CFG and BSA would gather on the oil-water interface with the polysaccharide chains extending into the watcr phase.Meanwhile,the polysaccharide chains would crosslink with BSA,forming an intertwined complex interfacial structure.This unique interfacial structure enable CFG and BSA molecules to stay intact on the surface even after being hydrolyzed by hemicellulase.Confocal laser scanning microscopy results showed that CFG and BSA molecules originally dispersed in the water phase moved and adsorbed onto the oil-water interface when peroxidase was presented in the emulsions.3.To investigate the practical application of this model CFG-BSA enzymatic reaction,sodium caseinate(NaCAS)was selected to prepare conjugates with CFG via the oxidation of transglutaminase(TGase).Results showed that CFG was able to form intermolecular conjugates with NaCAS as well.On top of that,CFG was partially hydrolyzed to further optimize the enzymatic reactions.Hydrolyzed CFG had a higher crosslinking degree with NaCAS compared with unhydrolyzed CFG. |
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