A comprehensive evaluation of the impact of cow's milk containing different beta-casein profiles on gut health of aging mice

Introduction. Aging is often characterised by nutritional deficiencies and functional alterations of the digestive and immune system. Cow's milk containing A2 beta-casein has been reported to be better tolerated in subjects with unspecific milk intolerance, in comparison to milk containing A1 beta-casein. The aim of this study was to evaluate whether diet supplementation with cow's milk containing different beta-casein profiles could impact on gut health in an animal model of aging mice.Materials and methods. Twenty-four Balb-c mice (20 months old) were randomly allotted to three nutritionally balanced, isocaloric and isoproteic diets. Mice were fed for 30 days a control diet (CTRL, a modified version of the AIN-93M diet, where casein was replaced with a casein-like aminoacid mix) or a diet with cow's milk containing either the A1/A2 beta-casein (STD) or the A2/A2 beta-casein (TEST). At day 30, intestine, liver, spleen, serum and fecal samples were collected and analysed. Lymphocyte subpopulations, dipeptidyl peptidase-4 and myeloperoxidase activities were evaluated in jejunum, cytokine secretion was assessed in colon, IgG levels were quantified in serum, while gut morphology was evaluated in duodenum, ileum, spleen and liver through morphometric measurements of villus height (Vh), crypt depth (Cd) and villus height to crypt depth ratio (Vh/Cd). Histopathological alterations were scored using a semiquantitative scoring system. Fecal SCFA content was assessed by HPLC and microbiota composition was assessed by NGS analysis. Data were analysed by univariate and multivariate statistics.Results and discussion. A higher percentage of T-helper and B lymphocytes associated to decreased numbers of T-cytotoxic lymphocytes were observed in mice supplemented with TEST milk. Multivariate analysis by unsupervised principal component analysis (PCA) highlighted a rough separation of the groups. Multivariate linear discriminant analysis (LDA) supervised model allowed to identify the main parameters accounting for group discrimination. The A2/A2 beta-casein TEST group showed higher content of fecal SCFA (in particular, acetate/butyrate), of CD3CD4 (T-helper) and CD45CD19 intestinal B lymphocytes, and higher jejunal Vh/Cd. The A1/A2 beta-casein group showed higher intestinal TCR?d lymphocytes. The control diet showed increased jejunal Cd, increased CD3CD8 (T cytotoxic) and increased TCRaß. Data suggest that gut health in A2/A2 beta-casein-fed mice is improved in terms of better gut morphology in the jejunum, increased percentage of T-helper and B lymphocytes and higher concentration of favourable microbial fermentation products.Beta diversity analysis highlighted specific, significant variations of faecal microbiota composition in TEST milk-supplemented mice with respect to the other groups. Comparison of taxonomical assignments among groups by Linear discriminant analysis Effect Size (LEfSe) identified Deferribacteriaceae and Desulfovibrionaceae as the most discriminant families for the TEST group.Conclusions. Taken together, results suggest a positive role of milk containing A2 beta-casein when consumed by aged mice as supplementation, both in terms of increased percentages of T-helper and B lymphocytes and of higher concentration of beneficial microbiota-derived fermentation products.