Study On Digestive Product Characteristics And Immunomodulatory Effects Of A2 β-casein

One of the crucial components of cow milk,β-casein,has a significant nutritional value.There are several different types of β-casein,with A1 and A2 being the most prevalent.A2 milk is frequently referred to as milk that solely includes A2 β-casein,whereas A1 milk primarily contains A1 β-casein.The 67 th amino acid,which distinguishes the A1 and A2 versions,affects the structural and digestive properties of proteins,which naturally affects functional characteristics.In addition,β-casein,the primary casein found in breast milk,possesses physical and chemical properties,digestive qualities,and immunomodulatory effects,all of which are significant research topics.Yet,there is still debate regarding whether A1 and A2 β-casein have differing effects on health.and there is insufficient evidence.Therefore,the study of structural characteristics of A1 and A2 β-casein and its effects on body health provides an important theoretical basis for dairy products and functional foods.In this study,β-casein in A1 and A2 milk was extracted,purified and identified to determine the concentration and purity of A1 and A2 β-casein.Secondly,by constructing in vitro digestive system,the characteristics of digestive products of A1 and A2 β-casein and the activity of immune cells in vitro were compared.Finally,a mouse model of immunosuppression induced by cyclophosphamide(CTX)was constructed to explore the regulatory effects of A2 β-casein on immune activity and intestinal health in mice.This study provides a theoretical basis for the study of digestive characteristics and immune tolerance of A2 β-casein dairy products.The main research results are as follows:(1)High concentration and purity β-casein were isolated: Casein was separated from A1 milk and A2 milk by isoelectric point-precipitation method,and then purified and identified by anion exchange chromatography and polypropylene amyl gel electrophoresis.The results showed that the concentrations of A1 and A2 β-casein were 0.58 mg/m L and 0.64 mg/m L,respectively,and the purities were 92.45% and 94.21%,respectively.(2)A2 β-casein may be more conducive to gastrointestinal digestion and absorption: In vitro digestive system was constructed,and A1 and A2 β-casein were co-incubated with artificial digestive solution.The results showed that the digestibility of A2 β-casein in vitro increased gradually,reaching 94% at 4.0 h,while A1 β-casein reached 93.47% at 5.0 h.The particle size distribution of A2 β-casein was mainly concentrated in the range of 200-800 nm,and the particle size of β-casein decreased gradually with the increase of gastrointestinal digestion time.In the stage of intestinal digestion,the large protein particles were gradually decomposed into nano-sized substances <100 nm,while the particle size of A1 β-casein was still concentrated in the wide range of 100-500 nm.The results of free amino acid,degree of hydrolysis and secondary structure of protein showed that the content of essential amino acid increased gradually with the process of enzymatic hydrolysis,and the degree of hydrolysis increased rapidly at enteric digestion stage,and the content of α-helix and β-folded structure decreased,while the disordered structure increased.These results indicate that A2 β-casein may be more conducive to gastrointestinal digestion and absorption than A1 β-casein.(3)A2 β-casein can significantly regulate the activity of immune cells in vitro: CCK-8 method and neutral red method were used to evaluate the effects of β-casein digestion on splenic lymphocyte proliferation,macrophage phagocytosis and NK cell activity in mice.The results showed that type A2 β-casein digestive could promote splenic lymphocyte proliferation,significantly improve macrophage phagocytosis index and NK cell activity,while type A1 β-casein digestive showed weak immunomodulatory effect.Meanwhile,the results of β-casein digestives on the m RNA expression of four immune factors in spleen lymphocytes and macrophages showed that the two β-casein digestives could increase the m RNA expression levels of immune cytokines IL-6,IL-17 A and TNF-α,and decrease the m RNA expression levels of IL-10.Moreover,the regulation effect of A2 β-casein digestive was stronger than that of A1 β-casein.Therefore,based on the results of the studies on digestive properties and in vitro immune activity,A2 β-casein was selected for the follow-up study on immune function.(4)A2 β-casein can improve the immune function of immunosuppressed mice: CTX-induced immunosuppressive mouse model was constructed,and different doses of A2 β-casein diet were used to intervene.The results showed that A2 β-casein could improve the immune organ index,enhance the function of immune cells,alleviate the histopathological injury of spleen,significantly enhance the production of IL-17 A,TNF-α and IFN-γ in serum,decrease the production of IL-4,and significantly increase the contents of immunoglobulin Ig G,Ig M and Ig A in serum of mice.The values of serum biochemical indexes tended to healthy values,and the physiological conditions of mice were obviously improved.On the whole,A2 β-casein showed higher immune-boosting efficiency than A1 β-casein.At the same time,A2 β-casein can improve the gut microbiome disorder of immunosuppressed mice by regulating the relative abundance of beneficial and harmful bacteria in the intestinal tract,and significantly increase the contents of acetic acid,propionic acid,butyric acid and total short-chain fatty acids,so as to promote the balance of intestinal microecology.