Effect Of Steam-processing On The Content And Immunogenicity Of Purified Soybean Antigens

Immunological tests were conducted to study the immunological properties of isolated soybean antigens from raw and steam-processed soybeans. The main antigens which lead to the allergic responses in animals were determined. Glycinin and P-conglycinin were isolated from the raw and steam-processed soybeans respectively by immunological methods, and were assayed by polyacrylamide gel electrophoresis and Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . Then the purified antigens were injected subcutaneously into Kunming mice weighed 18~22g. The immune response between the antiserum and its corresponding antigen was examined by Agar-gel immunodiffusion. The content of specific antibody produced by the corresponding antigen in mice was tested by the test of specific serum antibody (IgG) titres.Immunological methods were utilized to separate and purify glycinin and P-conglycinin from raw and steam-processed soybeans respectively. Purified glycinin and P-conglycinin as well as their neighbouring fractions were obtained by gel filtration on a sephorose CL-6B column (4.2 × 130cm) , successively, the first and second peaks of 11S and those of 7S globulins of both raw and steam-processed soybeans. And the second peak of purified US and the first peak of purified 7S globulins achieved by these operations were designated glycinin and P-conglycinin respectively, which were the two major anyigetic proteins, assayed by electrophoresis.The results of the curse of the isolated proteins showed that the content of either glycinin or β -conglycinin decreased when steam-processed, which proved that steam-process could destroy the antigens in raw soybeans to some degree. The content of glycinin reduced obviously while that of P-conglycinin varied little after steam-processing, which was preliminarily proved by polyacrylamide gel electrophoresis.The results of SDS-PAGE showed that the major proteins in raw soybeans concentrated at the second peak of 11S and the first peak of 7S globulins,while the profile of the second peak of processed 7S globulin (lane8) has little difference with that of the raw 7S globulin, to which steam-process did little harm.All the Kunming mice aged 12~13 days were fed on man-made diet without soy proteins after weaning. After raised for 35 days, the little white mice were divided into nine groups randomly while reaching 18~22g, 5 every group, male and female atrandom. Each group was immunized one of the eight purified antigens from raw and steam-processed soybean, and the rest group was set as control. Boosting immunization was conducted every ten days after the initial immunity, the blood of a mouse was sampled and the serum was separated to assay the result price of the antibody before boosting the immunization. Another ten days passed the fourth immunization, when all the mice were killed to collect the blood, separated the serum, which could be used in Agar-gel immunodiffusion and ELISA.Agal-gel immunodiffusion were conducted with the sera of the control and the eight standard immune sera achieved by immunizing corresponding antigens to the mice. The results showed that antisera of glycinin and P -conglycinin showed no cross-reaction with P -conglycinin and glycinin antigens, respectively. The antisera of the second peak of US and the whole 7S fractions reacted to the corresponding antigen, which indicated that antibodies had been produced in animals when immunized such antigens. But after steam-processing at 120°C for 7.5min., the antisera of US and the first peak of 7S globulins had no response to the corresponding antigen, which instructed that such antigens had neither reactive competence nor imrnunocompetence. However, the second peak of 7S did have immunocompetence even when processed so that steam-process could not destroy 7S fraction completely. Therefore, glycinin and P -conglycinin did produce antibodies in mice, yet the concentration was not high.In the test of serum antibody (IgG) to the two specific antigens in mice using ELISA, the specific antibody (IgG ) trties of the 11S and the first peak of 7S globulin was high, but decreased dramatically after steam-processed. While that of the second peak of 7S globulin increased to a large extent. Therefore, it can be concluded that 7S globulin is the major heat-resisted antigen in soybeans.