The Cross-linking Of Ovalbumin Catalyzed By Polyphenol Oxidase And The Corresponding Effect On Its Structure And Function

Ovalbumin?OVA?is the most abundant protein in egg white.OVA is a kind of high quality protein,and easy to be prepared.As a model protein,OVA was widely used in the research work on protein function,structure and food allergy.Moreover,OVA has functional properties in food processing,such as gel,emulsifiability and foaming.However,OVA is described as one of the major allergens in egg white.Therefore,reducing allergenicity and promoting function of OVA by food processing will benefit egg allergy sufferers and promote eggs'utilizations.In this study,the native OVA?N-OVA?firstly was denatured at different themal temperature and time,or treated by chemical reagents,respectively.Then it was cross-linked by polyphenol oxidase?PPO?.The methods of UV spectrum and sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?were used to optimize the cross-linking conditions.And the structural changes of the OVA samples were characterized by spectroscopic methods.Their gastrointestinal digestibilitits in vitro were evaluated by SDS-PAGE.Moreover,the potential allergenicity was assessed by competitive ELISA and in KU812 cell model in vitro.The major methods,results and conclusions of the study are as follows.1.According to the results of SDS-PAGE and UV spectrum,the conditions for cross-linking the denatured OVA by PPO was optimized as followes,OVA was at a concentration of 1.0 mg m L^-1,pH 7.0,and the PPO acitity was 1000 U mL^-1,including two kinds of denature conditions:at 100°C for 5 min as thermal denature,while 0.5%SDS at 50°C for 1 h as chemical denature.The mixture of cross-lingking were at 50?for 8 h.2.The structure of OVA was characterized by high performance size exclusion chromatography,circular dichroism spectroscopy and ANS fluorescence spectroscopy.The secondary structure of other OVA samples were all changed compared with N-OVA.Thermal polymerization of heat-treated OVA?H-OVA?existed with increased molecular weight and surface hydrophobicity.The thermal and SDS denatured-assisted cross-linking of OVA?CL-H-OVA and CL-SDS-OVA?aggregated at different degrees,and the surface hydrophobicity reduced but their surface hydrophobicity still higher than that of N-OVA.3.The digestibility of OVA was assessed by simulating gastrointestinal digestion in vitro.It was shown that N-OVA was not easily digested by gastrointestinal,and denaturation and denaturation assisted cross-linking can significantly improve the digestion characteristics of OVA.4.The competitive ELISA was used to detect the IgG and IgE binding abilities of OVA samples and their gastrointestinal products.Among the OVA samples,the IgG and IgE binding abilities of CL-H-OVA and its gastrointestinal products were the lowest.5.Basophil cells?KU812?were used out for allergenicity evaluation.Bioactive mediators,such as beta-HEX,histamine,TNF-?and IL-6,were detected from activated KU812 cells.The results showed that highest contents of bioactive mediators were induced by N-OVA group,while,CL-H-OVA group had the the lowest sensitization.6.The functional properties of OVA samples were tested.It was found that foamability,emulsifying activity and emulsifying stability of denatured-assisted cross-linking OVA were enhanced,but their foaming stability was not significant changes compared to N-OVA.The viscoelasticity of denatured-assisted cross-linking OVA was increased.The superoxide free radical scavenging ability and ferric ion reducing antioxidant capacity of CL-H-OVA and CL-SDS-OVA were significantly improved.