Soybean is rich in protein,oil and bioactive components,but it is also a major allergic food.It is well known that soybean allergic proteins enter the body along with other nutritional components in the food when people consume soybean products.Many studies have reported that supplementation of bioactive substances or their conjugated with allergic proteins could be a new strategy to reduce the allergenicity.Soy isoflavones,as anti-allergic components,are in the same system as soybean allergic protein,and they may form conjugates during processing.However,it is still a scientific issue to be solved whether soy isoflavones affect the allergenicity of proteins in a binding state(bound to allergic proteins)or a free state.In this study,the conjugates and mixtures of soy active isoflavones(genistein Gen,daidzein Dai,glycitein Gly)with soybean allergic proteins(7S and 11S)were used as the research objects.Firstly,the allergenicity of the conjugates and mixtures were preliminarily evaluated at the cellular level.Then,in vitro gastrointestinal digestion of infants and children was simulated to explore the digestive stability of conjugates and mixtures,and the allergenicity of the digestive products were tested at the cellular level.Finally,a BALB/c mouse sensitization model was established to analyze the allergenicity of conjugates and mixtures at animal level.This study comprehensively illustrated the allergic reaction of the conjugates and mixtures at cellular and animal levels.Accordingly,the mechanism of soy isoflavones(conjugated with protein or free)on the allergenicity of7S or 11S protein were revealed,which provided theoretical guidance for scientific understanding the allergenicity of allergic proteins in soybean matrix.The main methods,results and conclusions of the study are described as follows:(1)Six alkaline conjugates of soybean allergic protein-active isoflavones and their corresponding mixtures were prepared.Models of KU812 cell degranulation induced by 7S or 11S protein was established,and the effect of soy isoflavones on allergenicity of soybean protein was preliminarily explored.By detecting the bioactive mediators,it was found that the effects of conjugated and free isoflavones on 7S/11S inducedβ-HEX,histamine and IL-6 release were inconsistent,but they could inhibit release of IL-4.Furthermore,7S-Gly and 11S-Dai with slightly lower allergenicity were selected from the conjugates for subsequent studies.Moreover,the expressions of signaling proteins were detected by flow cytometry after KU812 cells activated degranulation.The results showed that the induction of cell degranulation by soybean protein was regulated by Lyn,MAPK,and NF-κB signaling pathways.Both conjugated and free isoflavones can significantly inhibit the expression of JNK protein.(2)The digestive stability of conjugates and mixtures were explored by simulating gastrointestinal digestion of infants and children in vitro,and the allergenicity of digestive products were assessed using the KU812 cell degranulation model.Tricine-SDS-PAGE showed that the conjugated isoflavones did not affect the digestibility of11S protein,but could promote the digestion ofαsubunit in 7S protein;free isoflavones had little effect on the digestibility of 7S and 11S proteins.Additionally,the digestion products of the native-protein,conjugates and mixtures retained strong allergenicity,which resulted in the release of higher levels ofβ-Hex,histamine,IL-6 and IL-4 after degranulation of KU812 with little overall difference,indicating that isoflavones has no significant effect on the allergenicity of digestive soybean protein.(3)A BALB/c mouse sensitization model was constructed using 7S-Gly and 11S-Dai and their corresponding mixtures,and the allergenicity of the conjugates and mixtures were comprehensively evaluated through humoral immunity and cellular immunity.The results showed that the levels of specific antibodies(IgE,IgG,IgG1 and IgG2a)and mouse mast cell protease 1(mMCP-1)in the serum of mice in the native-protein and conjugate groups were higher than those of the mixture groups.In addition,it was observed that there was no significant difference in the proportion of DC cells in the spleen of mice in each group.Both conjugated and free isoflavones induced a shift in the Th1/Th2 balance toward Th1 and promoted an increase in the proportion of Tregs.According to various sensitization indicators,free isoflavones can alleviate systemic allergic responses induced by 7S and 11S proteins,whereas conjugated isoflavones could only attenuate cellular immune responses.(4)The intestinal tissues and cecal contents of mice were isolated,and the changes in intestinal tissues were observed by HE staining.D-lactate and diamine oxidase in serum were detected using commercial ELISA kits.The differentiation of immune cells in mouse MLN were identified by flow cytometry,and the gut microbiota in the cecum contents of mice was also detected.The results showed that the conjugated and free isoflavones could alleviate intestinal injury induced by allergy,promote the differentiation of Tregs and regulate the balance of Th1/Th2 in MLN to reduce the allergenicity of 7S or 11S proteins.Besides,free isoflavones could also inhibit DC differentiation in MLN and alleviate the imbalance of Th17/Tregs induced by 7S protein.Soy isoflavones could also reversed the allergy-induced gut microbiota imbalance by up-regulating the ratio of F/B,increasing the abundance of Clostridiales and reducing the abundance of Bacteroidales,while the conjugated isoflavones have better effects than the free isoflavones. |