Controlling Allergenicity Of Ovomucoid Based On Epitope Processing

Objective:To study relationship between epitopes and allergenicity of hypoallergenic ovomucoid products through food-grade processing technology for providingin order to provide a theoretical guidance for the development of hypoallergenic or desensitized egg products.Methods:1.Purification of ovomucoid:Egg white was separated from the yolk,and the interfering proteins such as ovomucin were removed through pre-processing.The pre-processed egg white solution was then applied to CM-Sepharose FF cation exchange chromatography.Ovomucoid was eluted prior to ovalbumin?which has large amount and would require longer time for elution?so the latter would not interfere with ovomucoid purification.Highly purified ovomucoid was obtained,which was identified by electrophoresis and mass spectrometry,and protein purity was analyzed by the grayscale analysis.Ovomucoid with a purity higher than 90%was collected.2.Unfolding and alkylation of ovomucoid:Glutathione reduction method and cysteine reduction method were both used to destroy its intramolecular disulfide bonds.According to the electrophoresis spectrum of each target band,the conformation change tests,the IgG binding and IgE reactivity results,the optimal reducing conditions to reduce immunoreactivity was chosen,and unfolded ovomucoid was prepared for subsequent experiments.After the disulfide bond of ovomucoid was destroyed by reduction,the disulfide bond was likely to refold due to change of the reducing conditions or the dilution of the reducing agent.Therefore,alkylation was perfomed before detection,and spare reactants were removed by speedvac to improve accuracy.3.Optimization of Maillard reaction conditions for ovomucoid with low immunoreactivity:heating temperature,heating time,types of reducing sugars,different reducing methods,and the ratio of lysine residues to reducing sugars were important factors affecting Maillard reaction.The factors that affect the IgG binding ability of ovomucoid was determined through single-factor experiments,and then assess whether optimization should be performed.The products with the lowest immunoreactivity would be used in animal model test.4.Evaluation of immunoreactivity of glycated ovomucoid:Glycation was performed on ovomucoid before and after unfolding,and rabbit serum IgG binding capacity and egg allergic patients serum IgE binding ability were detected by indirect EILSA or competitive inhibition ELISA.The glycation products with the lowest immunoreactivity were selected.Their secondary and tertiary structure characteristics were analyzed by circular dichroism,ultraviolet spectrum and fluorescence spectrum,respectively.And by comparing with alkylated ovomucoid,it was determined that ovomucoid refolding had occured.By comparing with untreated ovomucoid,conformation analysis was to assess whether the disulfide bond had re-formed in its original state.5.Animal experiments evaluating allergenicity of ovomucoid product:Immunize mice with the ovomucoid product with the lowest IgE binding capacity,and observe the allergic symptoms of the mice;detect the levels of IgE,IgG,IgG1,and IgG2a in the serum;detect changes in level of spleen cytokines?IL-4,IL-13IL-17A and IFN-??.Results:1.Isolation and purification of ovomucoid:After a complete separation process?2 h?,14.33 mg of ovomucoid could be collected with a yield of 45.8%and a purity of94.1%.It had similar immune activity with ovomucoid standard.2.Optimal reducing conditions:When glutathione?1 mg/ml?was selected as the reducing agent,both the binding ability to bothrabbit serum IgG and human serum IgE decreased the most.3.Structural changes of unfolded ovomucoid:secondary structure changes,mainly in the form of?-helix and random coils,and fluorescence spectrum results showed changes in hydrophobicity.4.Optimal glycation condition:The lowest IgG binding capacity was reached when glycated with maltose at 90?for 8 h,at glucose/lysine ratio of 10:1.Results showed that the IC₅₀0 value of G-M-OVM increased from 0.3741?g/ml to 112.6?g/ml after glycation,and its IgE binding capacity was also significantly reduced compared to unprocessed ovomucoid?OVM??P<0.05?5.Refolding of ovomucoid:Ovomucoid did not completely refold at the original position.6.Evaluation of allergenicity by animal model:The levels IgE,IgG,IgG1,and IgG2a were lower than those of mice immunized with unprocessed ovomucoid?P<0.05?.Compared with unprocessed ovomucoid,the levels of IL-4,IL-17A and IFN-?decreased,and the difference was statistically significant?P<0.05?;The level of IL-13 was only slightly decreased,with no significant difference.Conclusion:1.A simple and convenient chromatographic method has been developed,which can be used to isolate high-purity ovomucoid in a short time?within 2 h?.It has similar immune activity to commercial ovomucoid standards and can be used for laboratory research.2.Hypoallergenic ovomucoid is produced by unfolding,glycation and refolding process.