Preparation Of Hypoallergenic Ovalbumin By Pulsed Electric Field Combined With Phosphorylation Modification

Ovalbumin(OVA),as a natural biological macromolecule,has biological characteristics such as antihypertensive,antibacterial,antioxidant and immunomodulatory activity,and has great potential in the application of medicine.In addition,OVA is a highquality and low-cost protein with good amino acid ratio,easy absorption by the human body,and good processing characteristics,widely used in the food processing industry.However,the strong sensitization of OVA poses a great threat to allergic populations,seriously affecting its application in the food and drug industry.Therefore,solving the problem of OVA sensitization is urgent and has received increasing attention from researchers.In this study,high purity OVA was prepared by isoelectric point precipitation,ammonium sulfate precipitation and anion exchange chromatography.With this as the research object,an indirect competitive enzyme-linked immunosorbent assay(ELISA)was established to detect the Antigenicity of egg allergen OVA by preparing OVA polyclonal antibodies.And OVA was modified by pulsed electric field(PEF)combined with phosphorylation reaction.The changes of OVA structure,Antigenicity and antigen binding ability before and after modification were studied.The results showed that:The purity of OVA obtained through isoelectric point precipitation,ammonium sulfate precipitation,and anion exchange chromatography for protein separation and purification is over 99%.The operation is simple and the preparation cost is low,which meets the OVA needs of subsequent research and provides new ideas and methods for the large-scale preparation of high-purity OVA in the laboratory.Immune experiments were conducted on Japanese rabbits using intravenous injection to obtain OVA antiserum.The titer of the antiserum was measured using indirect ELISA,while the specificity of the serum was detected using direct and Western blot assays.The results showed that the OVA antiserum obtained through animal experiments had a high efficacy and price,with a maximum of 1:25000;Immunoblotting experiments showed that the antiserum had strong specificity with OVA;The linear regression equation for OVA allergy detection method established with the logarithm of antigen concentration as the xaxis and percentage absorbance as the y-axis is y=-19.097x+79.361,R2=0.9913,and the optimal detection range is 0.25-32 μ G/m L,indicating that this method meets the requirements for OVA allergy testing.This provides a good method for detecting OVA allergenicity and a basis for monitoring changes in OVA allergenicity during processing.The molecular weight of OVA meridian pulse electric field(PEF)increases after phosphorylation modification;OVA molecular structure expansion,α-The content of spiral and disordered structures increases,the UV absorbance increases,and the intrinsic fluorescence intensity decreases;The introduction of phosphate groups and the exposure of hydrophobic groups within molecules enhance their surface hydrophobicity.The ELISA test results showed that the antigen binding ability of modified OVA was significantly reduced.It can be concluded that the introduction of phosphate groups to modify the amino acid residues of OVA masked its Linear List binding to Ig G and Ig E,and the changes in its secondary and tertiary structures destroyed the conformational epitopes on the surface of OVA molecules.Therefore,PEF binding phosphorylation significantly reduced the sensitization of OVA,indicating that PEF binding phosphorylation modification has the potential to develop hypoallergenic OVA.