| Soybean is a high-quality plant protein resource and also one of the eight major food allergens. Glycation is widely used to modify protein allergens. Therefore, it is significant that studing on the soybean protein allergen modification using glycation.The anti-glycinin and anti-β-conglycinin polyclonal antibody were successfully prepared to optimize the conditions for indirect ELISA in the paper. Results showed that the optimal concentration of the coating glycinin antigen and β-conglycinin antigen were 0.025μg/mL and 0.3 μg/mL, coated at 4 °C overnight. Homemade polyclonal antibody anti-glycinin and anti-β-conglycinin, horseradish peroxidase conjugated goat anti-rabbit IgG all were 1:3200 and 1:10000 respectively. The best antigen and antibody reaction condition was 37 °C coated for 1 h. For glycinin and β-conglycinin, the coefficients of variation of intra-plate error and inter-plates error were 3.82%, 2.19% and 10.22%, 9.61% separately within the permissible range and detecting the linear range were 0.01 μg/mL~0.05μg/mL and 0.1 μg/mL~2.0 μg/mL respectively. The repetitive and sensitivity of the method were measured and could used to test in the antigenicity of soy allergies.The antigenicity of glycated products which used glucose, lactose and dextran as donors at different conditions was measured by JMP software. Results showed that the antigenicity of glycated products decreased overall with the reaction time increased. At the conditions of temperature at 60 °C, mass ratio of protein and sugar 3:1, the reaction time at 9 d, the antigenicity inhibition rate of β-conglycinin and glycinin in soy protein isolate-dextran conjugates reduced 30% and 20% respectively. The antigenicity inhibition rate of β-conglycinin and glycinin in soy protein isolate-lactose conjugates reduced 50% and 30% respectively at the conditions of temperature at 60 °C, mass ratio of protein and sugar 4:1 and the reaction time at 84 h. At the conditions of temperature at 55 °C, mass ratio of protein and sugar 3:1, the reaction time at 72 h, the antigenicity inhibition rate of β-conglycinin and glycinin in soy protein isolate- glucose conjugates reduced 70% and 25% respectively. Among them, glucose as the glycosyl donor had the best modification effect.The degree of Maillard reaction of soy protein isolate-glucose, soy protein isolate-lactose and soy protein isolate-dextran conjugates under optimized conditions was measured. The degree of glycation was researched from different angles. The found that color deepened and the free amino group content reduced indicated soy protein isolate with different saccharides occurred at different degrees of reaction. SDS-PAGE indicated the occurrence of Maillard reaction and accompanied with generation macromolecules.The UV spectrum showed that the introduction of the sugar chain in protein was easy to expand protein peptide chain so that the interior amino acids exposed. When the auxochromes were conjugated with carbonyl group in sugar chains lead to blue shift of the absorption bands. The increased fluorescence intensity of glycated conjugates indicated that more hydrophobic region of protein was exposed and made a more compact tertiary structure. FTIR indicated that the protein structure of glycated conjugates was unfolded as the introduced surger chains. The reduced β-corner and random coil structure content impacted the epitope of β-conglycinin antigen in soybean protein, and then reduced the antigenicity of soybean protein. These showed that the relationship between the degree of glycation and the reduced degree of antigenicity is an unlinear and the key effect relies on the structure changes caused by the binding site between protein and saccharide. |
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