Research On Glycation To Soybean Protein Allergenicity And Modification Mechanism

Soybean is a high-quality plant protein resource for all ages,but it is also one of the eight major food allergens.About 0.3%-0.4%of people suffer from soybean allergies in the word.Soybean allergy has affected infants’ health severely.Glycinin and β-conglycinin are considered as the major allergens in soybean.This paper reduces the allergenicity and antigenicity of soybean by glycation,and it may provide a new approach for the development of hypoallergenic soybean.The ELISA method of detecting antigenicity and allergenicity was established firstly.The specific IgE antibodies for two soybean protein allergens of thirty one soy patients’ sera were detected by indirect competitive ELISA method.Twenty five patients’ sera displayed strong IgE-binding to soybean proteins compared with the negative control sera.Twenty five patients’ sera were selected to constitute a sera pool which was used in the allerginicity determination.The conditions of indirect competitive ELISA were optimized.As for the determination of antigenicity,the best concentration of coating antigen for glycinin andβ-conglycinin were 0.025 and 0.3 μg/mL respectively and the optimum working dilutions of the rabbit antiserum both were 1:10000.As for the determination of allergenicity,the best concentration of coating antigen for glycinin and β-conglycinin both were 10 μg/mL and the optimum working dilutions of patients’ antiserum both were 1:2.The antigenicity and allergenicity of glycated products which used xylose,lactose and dextran as donors at 55℃,relative humidity 79%was measured.The changes laws of the antigenicity and allergenicity of glycinin and β-conglycinin during the glycosylation were investigated.Results showed that the antigenicity and allergenicity of glycated products decreased overall along with the reaction time increased.At the conditions of temperature at 55℃,mass ratio of protein and sugar 3:1,the reaction time at 10 h,the antigenicity inhibition rate of glycinin and β-conglycinin in soy protein isolate-xylose conjugates reduced 44.35%and 62.53%respectively.The allergenicity inhibition rate reduced 33.87%and 46.37%respectively.The antigenicity inhibition rate of glycinin and β-conglycinin in soy protein isolate-lactose conjugates reduced 32.37%and 52.05%respectively at the conditions of temperature at 60℃,mass ratio of protein and sugar 4:1 and the reaction time at 96 h.The allergenicity inhibition rate reduced 18.97%and 16.67%respectively.At the conditions of temperature at 55℃,mass ratio of protein and sugar 3:1,the reaction time at 11 d and 10 d,the antigenicity inhibition rate of glycinin and β-conglycinin in soy protein isolate-dextran conjugates reduced 17.88%and 35.06%respectively.The allergenicity inhibition rate reduced 15.83%and 46.57%respectively at the same condition.Western blotting showed that the antigenicity of soybean protein was decreased by glycosylation.The degree of glycosylation reaction and molecular weight of two allergenic proteins were analysed by the degree of glycation and SDS-PAGE.The relationship between the structure and allergenicity of the glycated products was analyzed by CD spectrum,FTIR and the UV absorption spectra.The degree of glycation increased with the increasing reaction time.SDS-PAGE indicated the occurrence of Maillard reaction and accompanied with generation macromolecules.The UV spectrum showed that soy protein banded with carbohydrate,and the introduction of the different sugar chains in protein made the protein peptide chain expand or collapse,wherein the aromatic amino acid absorption of ultraviolet intensity had changed;FTIR show that the secondary structure of soy protein:α-helix,β-turns,β-sheet and random coil ocurred mutual conversion and the ordered molecules decreased after glycosylation;CD spectrum also proved that the spatial structure of soybean protein had been changed after glycosylated modification.The changes in the structure of soy protein may shield or mask the antigen epitopes,changing its antigenicity and allergenicity.In addition,The allergenic properties of glycosylation products were evaluated by the animal immunology experiments.Results showed that the introduction of the sugar chain in soy protein could reduce the content of IgE antibodies produced by BALB/c mice and inhibit the skin allergic reactions.