Characterization Of Protein Structure And Cytological Evaluation Of The Allergenicity Of Soymilk Fermented By Lactic Acid

Soymilk is a healthy drink with high nutritional value.It is rich in high-quality protein,iron,unsaturated fatty acids,vitamins,dietary fiber and isoflavones.It is cheap and suitable for most people in the world,especially those who are lactose intolerant population or allergic to milk.However,soybean is one of the eight major allergens,which severely limits the consumption of soymilk.Currently,lactic acid bacteria fermentation has been used to prepare fermented soymilk.Studies have shown that microbial fermentation can affect the allergenicity of soy protein.Therefore,it is of positive social value to explore the effect of fermentation on the allergenicity of soymilk protein.In this study,Lactobacillus brevis and Lactobacillus.sp were mixed to liquid fermentation of soymilk,and the effect of fermentation on the structure and allergenicity of soy protein were analyzed.This study included the following three aspects: Firstly,the changes of structure,epitopes and physicochemical characteristics of fermented soymilk were analyzed;Secondly,in vitro gastrointestinal digestion was simulated to explore the effect of fermentation on the digestion stability of soymilk protein,and to assess the IgG-binding and IgE-binding capability of digestion products.Thirdly,the allergenicity of fermented soymilk protein and its digestion products in gastrointestinal tract were evaluated by human basophil KU812 degranulation model.The main methods,results and conclusions of this study are as follows:1.The fermented soymilk was prepared according to optimal fermentation conditions: inoculation quantity of 4%,the inoculation ratio of Lactobacillus brevis and Lactobacillus.sp of 3:2,temperature of 33 °C,fermentation time of 24 h.The proteins in fermented soymilk were extracted,and characterized by circular dichroism,ultraviolet absorption spectra,ANS fluorescent probe method and Ellman spectrophotometry.LC-MS was used to identify the small molecular peptides in fermentation products.Simultaneously,the effects of fermentation on the IgG-binding ability of soymilk protein were analyzed by immunoblotting.And the degree of hydrolysis,emulsifying property and foaming property of the fermented soymilk protein were measured.The results showed that the spatial structure of fermented soymilk protein was loose compare with soymilk protein,and five human IgE binding epitopes were destroyed by fermentation,including glycinin G2 subunits K57-P111,S219-N233,E169-S215,S249-Q271 and ?-conglycinin ? subunit N232-M383.In addition,the IgG-binding capacity of soymilk protein was significantly reduced.The hydrolysis degree of fermented soymilk protein was 20.96%,the emulsifying property,emulsion stability and foam stability were significantly reduced,and the foaming property was significantly increased.2.The soymilk was digested by in vitro simulation of gastrointestinal tract,and the effects of fermentation on the digestion stability of soymilk protein were analyzed by Tricine-SDS-PAGE,RP-HPLC and the degree of hydrolysis.In addition,the IgG-binding and IgE-binding capability of digestion products were examined by competitive inhibition ELISA.It was observed that the digestibility of ?-glycinin and glycinin was significantly improved,and the IgG-binding and IgE-binding capability of digestion products of fermented soymilk decreased significantly,which indicated that the fermentation could reduce the potential allergenicity of soymilk protein.3.A human basophil KU812 cell sensitization model was established,and the potential allergenicity of fermented soymilk protein and gastrointestinal digestive products was evaluated by detecting inflammatory mediators and cytokines released by cell degranulation.It showed that ?-HEX,IL-4 and intracellular calcium concentration in KU812 cells stimulated by fermented soymilk protein were lower than that those in the soymilk group,and histamine and TFN-? in fermented soymilk also decreased,but IL-6 was elevated significantly.For gastric digestion products,the level of ?-HEX,IL-6,TFN-?,histamine,IL-4 and intracellular calcium produced by the fermented soymilk group cells were significantly lower than those in soymilk group.For gastrointestinal digestion products,except for histamine,the levels of ?-HEX,IL-6,and TFN-? produced by the fermented soymilk group cells were significantly decreased,and the IL-4 level and intracellular calcium concentrations also decreased.In summary,fermentation has the potential to reduce the allergenicity of soymilk protein.