Study On The Allergenicity Reduction Of Soybean 7S Globulin And The Elimination Of Peptide Fractions Resistant To Enzymatic Hydrolysis

7S globulin(?-conglycinin)is one of the main allergic proteins in soybean.Thermal processing has a certain effect on reducing the allergenicity of soybean,but there is still some left after digestion.The residue peptides after hydrolysis in the gastrointestinaltract may be an important factor,but there are few studies on this aspect.In this paper,soybean 7S globulin was selected as the study object to investigate the influence of the different heating conditions,enzymatic hydrolysis of pepsin and trypsin respectively on its allergenicity and the relationship with structural changes.On this basis,the allergenicity and structure of the peptide fractions resistant to enzymatic hydrolysis were studied.Finally,how to prepare a kind of modified soy 7S globulin,which not only has functional properties of 7S globulin,but also has no peptide fractions resistant to enzymatic hydrolysis after continuous digestion in the gastrointestinal tract,was further explored.The research here aims to provide a basis for the development of hypoallergenic soybean products.Firstly,the influence of different heating conditions on the allergenicity of soybean 7S globulin and their relationship with structural changes was explored.According to SDS-PAGE,it was found that there was no significant change for ??,? and ? subunit of soybean 7S globulin after being heated at 70°C,80°C and 90°C for 10 min and 20 min,respectively.And the immunoblotting result showed 7S globulin still has strong allergenicity after heating treatment.The result of competitive ELISA showed that the highest allergenicity reduction ratio was 22.5% for heat-treated 7S globulin at 80°C,10 min.SEC-HPLC showed that the content of soluble aggregates was positively correlated with the allergenicity reduction ratio.It was observed that the tertiary structure of 7S globulin was destroyed by heating,and the secondary structure transformed into more disorder.The surface hydrophobicity was not highly correlated with the reduction ratio of allergenicity.In short,the changes of allergenicity reduction ratio of 7S globulin were related with the destroyed conformational epitopes.Secondly,the effects of trypsin and pepsin hydrolysis on the allergenicity and structure of soybean 7S globulin were investigated to study the allergenicity of peptide fraction resistant to peptic and tryptic hydrolysis.The ?? subunit rapidly degraded,? subunit gradually degraded and ? subunit showed higher digestive stability after trypsin hydrolysis of soybean7 S globulin.The allergenicity reduction ratio of trypsin hydrolysates was increased with the extension of hydrolysis time and the highest one was 41.08%.Heat pretreatment can promote hydrolysis of soybean 7S globulin by trypsin,the highest allergenicity reduction ratio of trypsin hydrolysates was 80%,which may due to the the peptide fractions resistant to enzymatic hydrolysis.For the direct hydrolysis with pepsin,the three subunits of soybean 7S globulin were hardly degraded and the reduction ratio of allergenicity was only 3.8%-4.5%.Heating pretreatment improved the 7S globulin hydrolysis with pepsin,and the highest allergenicity reduction ratio of pepsin hydrolysates was about 50%.And the combined results from SDS-PAGE and MS identification showed that both trypsin and pepsin hydrolysates had large molecular weight residual peptide fractions which derived from ? subunit of 7S globulin.On this basis,soybean 7S globulin was treated by sequentially peptic and tryptic hydrolysis with preheating.The results from SDS-PAGE showed that all the ??,? and ?subunit of soybean 7S globulin were hydrolyzed and the residue lanes were mainly the peptide fractions with the same molecular weight as shown above.Competitive ELISA results showed that the residue ratio of allergenicity was about 17.6% after continuous hydrolysis with pepsin and trypsin.Peptide fraction resistant to peptic and tryptic hydrolysis was separated by SEC-HPLC and their relationship with allergenicity were investigated.The results indicated that the peptide fraction resistant to hydrolysis accounted for 65% of allergenicity in sequentially peptic and tryptic hydrolysates and it was the main allergic component.The result of MS showed that the peptide was also derived from the ? subunit.Its C-terminal sequencing result was <VEKEEC> at sequence 194-202,and N-terminal sequencing results was <NPFLFGSNR> at sequence 63-68.Thus the sequence of the specified peptide was <VEKEECEEGEIPRPRPRPQHPEREPQQPGEKEEDEDEQPRPIPF PRPQPRQEEEHEQREEQEWPRKEEKRGEKGSEEEDEDEDEEQDERQFPFPRPPHQKEE RKQEEDEDEEQQRESEESEDSELRRHKNKNPFLFGSNR> at sequence 63-202 of ?subunit.The actual molecular weight determination result was 17755.621 Da.Finally,a method which can not only keep the functional properties of 7S globulin,but also prevent the production of the peptide fractions resistant to enzymatic hydrolysis in the gastrointestinal tract was studied.When the DH of Alcalase hydrolysate reached only 2.7%(0.5% enzyme addition,hydrolytic time 10 min),the emulsification and emulsion stability of soybean 7S globulin were significantly improved by 40.7% and 7.2%,respectively.At the same time,SDS-PAGE result showed that the peptide resistant to enzymatic hydrolysis mentioned above had completely disappeared.Competitive ELISA showed that the allergenicity decreased by nearly 100%.