The Effect Of Ultra-high Pressure Combined With Thermal Processing On The Antigenicity Of Soybean Globulin And The Location Of A2 Damaged Epitope

Soybean plant protein has high nutritional value and has been widely used in the food industry,such as making flour food,baking food,children’s food and health food.In daily life,soybean or soybean products are often consumed,which has a positive impact on human growth and development.However,soybean has also been listed as one of the fourteen major food allergens recognized by the Food and Agriculture Organization of the United Nations.A large number of studies have shown that processing can significantly reduce the antigenicity of allergen proteins in soybean,accurately locate the antigenic epitopes damaged by processing,and guide the safe production of soybean globulin processed foods,which is of great significance to the soybean allergic population.In this paper,soybean globulin was extracted from defatted soybean powder by alkali dissolution and acid precipitation.The effect of ultra high pressure combined with thermal processing on the antigenicity and structure of soybean globulin was studied.SDS-PAGE,exogenous fluorescence,free sulfhydryl and Fourier transform infrared spectroscopy were used to detect the effect of processing on the structure of soybean globulin.Enzyme-linked immunosorbent assay（ELISA）and Western blotting were used to detect the effect of processing on the antigenicity of soybean globulin.In vitro digestion experiments were used to evaluate the digestion stability of processed soybean globulin.The results showed that compared with the unprocessed samples,the disulfide bond of the processed soybean globulin was broken and the molecular weight changed.With the gradual increase of temperature,the acidic peptide chain band gradually became shallower and disappeared.The content of hydrophobic groups,free thiols,α-helix,β-turn and random coil in protein samples increased,while the content ofβ-sheet decreased.When the temperature reached130°C,the inhibition rate of protein antigen reached 37%,50%lower than that of untreated protein antigen,and the inhibition rate of antigen reached the lowest.Processing can significantly reduce the antigenicity of soybean globulin by in vitro simulated digestion.According to the biological information technology,the possible antigen epitope in the A2 peptide chain was predicted.The adjacent fragments of the A2 peptide chain were overlapped by about 135 bases,and they were divided into three fragments:A2-1,A2-2,and A2-3.The primers were designed using primer 5 software,and the gene gel after PCR amplification of the full length of A2 and its overlapping segments was recovered.The cloning vector was constructed by connecting it with the T-vector and transformed into E.coli.The successfully transformed positive bacteria were expanded and cultured to extract the plasmid.After enzyme digestion,it was connected with the arm of the T7 phage vector,and the E.coli was infected after in vitro packaging to obtain the positive recombinant phage.The phage proteins of A2 peptide chain and its overlapping fragment genes were obtained after expanded culture.Under the processing conditions with the strongest destruction antigen,excessive hot-processed and combined-processed soybean globulins were fully mixed and absorbed with polyclonal antiserum to obtain the specific antibodies against the two processing destruction antigen epitopes.The dominant antigenic region of the processed antigenic epitope was A2-3 fragment,and its amino acid sequence was²⁰¹SGFAPEFLKEAFGVNMQIVRNLQGENEEEDSGAIVTVKGGLRVTAPAMRKPQQE EDDDDEEEQPQCVETDKGCQRQSK²⁷⁸,The A2-3 fragment was overlapped by 15 bases and divided into three fragments:A2-3-A,A2-3-B,and A2-3-C.The antigenic dominant region of processing damage was screened again,and finally the antigenic dominant region of processing damage was accurately located as the A2-3-B fragment.The amino acid sequenceofthisfragmentwas²³³AIVTVKGGLRVTAPAMRKPQQEEDDDDEEEQPQCVE²⁶⁸.The secondary structure of A2-3-B fragment was predicted by online analysis software.The fragment contained 11.11%α-helix,25%β-folding and 63.89%random coil.About six amino acids overlapped with each other were divided into three peptides.The results of Dot-Blot and ELISA showed that the polyclonal antibody reacted with peptide 3 but did not react with peptide 1 and peptide 2,indicating that peptide 1 and peptide 2 did not contain linear antigen binding sites with soybean globulin polyclonal antibody.Peptide 3 contained antigen linear epitopes,and neither of the two processed damaged antibodies reacted with the three peptides,indicating that the processing did not destroy the linear epitopes binding with the three peptides.The A2-3-B fragment expressed by phage could react with the processed specific antibodies,indicating that the fragment contained the processed damaged conformational epitopes,and the corresponding linear antigen epitopes were not destroyed,in response to the serum of allergic patients,and the allergenicity of peptide 3 was strong.The amino acid sequence of peptide 3 fragment was ²⁵³QEEDDDDEEEQPQCVE²⁶⁸,and the site-directed mutagenesis of peptide 3 was carried out by alanine substitution technique.Dot-Blot and ELISA tests showed that when the amino acid at position 2,3,4,6 and 9 was mutated,the mutated peptide did not specifically bind to the soybean globulin polyclonal antiserum.D259,E260,E261,Q263 and C266 might be the key amino acids that significantly affected the antigenicity of peptide 3.