Simulating Static Gastrointestinal Digestion Of Milk Proteins In Vitro And Allergenicity Evaluation Of Transported Milk Digestive Peptides

The incidence of cow's milk allergy is rising in the worldwide,especially in infants and young children.More than 30 proteins in cow's milk are potential allergens,including casein,beta-lactoglobulin and alpha-lactalbumin,which are regarded as main allergens.Cow's milk allergy involves the process of digestion and absorption in the gastrointestinal tract.A series of immune responses were initiated,since some digested peptides with allergen epitopes are presented to the immune cells.Therefore,the change of immune cells after the allergen peptides after digestion were investigated,which hope to provide valuable information to explore the underlying mechanisms of cow's milk allergy.In this study,four kinds of commercially available milk including infant milk powder,ultra-high temperature sterilized milk,pasteurized milk and skim milk powder were used to evaluate the digestive stability and IgE binding ability of milk protein in the gastrointestinal tract of infants and adults by establishing an in vitro gastrointestinal digestive model.The intestinal transport absorption of bovine milk protein in gastrointestinal was performed with Ussing Chamber,and the antigen peptides entering the intestinal mucosal were analyzed.A Brown Norway rat sensitization model was used to evaluate the level of specific antibodies and cytokines in serum and the differentiation of different T cell subsets in spleen.The differentiation of DCs and T cells in MLN of sensitized rats were performed to test the allergenicity of the antigen peptides.The main methods and results of this study as follows:1.Based on the in vitro simulating gastrointestinal digestion model of infants and adults,the effects of time and pH on digestive stability and IgE binding ability of milk protein were investigated.The results showed that?-lactoglobulin has the highest digestive stability,while casein is most sensitive during digestion.The small molecular fragments increased continuously upon the extension of gastric digestion,and the peptides with molecular weights of about 19 kDa and 5 kDa were accumulated.In the intestinal digestion stage,the digestion-resistant peptide fragments were further degraded with the prolong of intestinal digestion and forming smaller peptides with a molecular weight of about 3.5 kDa.The different pH values?1.0,2.0,3.0 and 4.0?had a great influence on the digestion stability of milk protein in gastrointestinal digestive juices of adults,and the degree of influence is greater than that of infants.With the increase of pH in simulated gastric fluid,the larger fragments the stronger allergnicity.Among them,the gastrointestinal fluid of infants with a pH of 3.0 and the gastrointestinal fluid of an adult with a pH of 2.0 have the strongest ability to reduce the IgE binding capacity of milk protein.2.The four above dairy product were used to sensitize Brown Norway rat.The effects of allergenicity of cow milk protein was evaluated by humoral immunity and cellular immunity.The results showed that milk proteins increased the level of IgG1and Th2-related specific antibody IgG and IgG2a in rat sera.Moreover,milk proteins also increased the level of Th2 related cytokines?IL-4 and IL-13?,decreased the level of Th1 related cytokines?IFN-?and TNF-??.Histological analysis indicated that milk protein could disrupt the integrity of rat intestinal tissue.Additionally,there were nine antigenic peptides that can enter the intestinal mucosal system,including(¹²⁵TPEVDDEALEK¹³⁵,¹²⁵TPEVDDEALEKFDK¹³⁸,⁷⁷WENGECAQK⁸⁵,¹⁰⁸VLVLDTDYK¹¹⁶,¹⁰⁸VLVLDTDYKK¹1717 and ¹⁵⁸ALPMHIR¹⁶⁴ from?-lactoglobulin;⁹⁹EDVPSER¹0505 and ¹⁴⁰EGIHAQQK¹5757 from?S1-casein;⁸⁶ITVDDK⁹11 from?S2-casein.3.The spleen and MLN from rat were isolated respecitvely,and the effects of four dairy products on immune cells were determined by flow cytometry.The result showed milk protein promoted the expression of CD4⁺T lymphocytes and Th2 cells in rat spleen cells and MLN,and weaken the expression of Th1 cells and Tregs.Besides,milk protein facilitated the maturation of DCs in the intestinal MLN of rats.Obviously,cow's milk protein enhances the antigen-presenting ability of dendritic cells,resulting in the migration of immunity to the Th2 direction.