LC-MS and 1H-NMR Untargeted Metabolomic Profiling of Probiotic Yogurt Consumption in C56BL6/N Mice

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Abstract Objectives Probiotic yogurt consumption is associated with gut health, including nutrient production and immune regulation, largely via modulation of the gut microbiome. However, metabolites that mediate host-microbiome interactions are largely unknown. We hypothesize consumption of probiotic yogurt results in distinct metabolic signatures in colon, including decreased levels of branched-chain amino acids, triglycerides, and bile acids relative to probiotic-free yogurt, milk, and water controls. We further hypothesize these differences are driven by increased abundances of Lactobacilli and Bifidobacteria following probiotic yogurt consumption. Methods A juvenile mouse model (3 weeks old, n = 32 male, 32 female) was fed AIN-93G diet for 3 weeks, followed by 3 weeks of daily gavage of 150 µL probiotic yogurt (PY), heat-inactivated yogurt (HY), milk (M), or water (W). Animals were cohoused in the initial phase and singly housed in the gavage phase. Polar and nonpolar colon extracts were analyzed with ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) and 1H NMR metabolomics. Features detected by1H-NMR and C18 LC-MS in positive mode were compared, pairwise, between diet groups. Results Metabolite classes detected via UHPLC-HRMS included bile acids, amino acids and phospholipids. NMR yielded the following number of features with an absolute log2-fold change > 2 and unadjusted P-value < 0.05 between dietary groups: 74 (PY v HY), 17 (PY v M), 57 (PY v C), 79 (HY v M), 14 (HY v C), and 47 (M v C). C18 LC-MS in positive mode yielded the following numbers of metabolites with the same cutoffs used in NMR: 220 (PY v HY), 39 (PY v M), 196 (PY v C), 80 (HY v M), 58 (HY v C), and 61 (M v C). Conclusions As hypothesized, probiotic yogurt consumption for 3 weeks significantly altered the colon metabolome relative to controls. Further metabolite identification, pathway enrichment analysis, and next generation microbiome sequencing analyses will aid in identifying specific pathways altered upon PY consumption and correlated with alterations in colonic microbial taxa abundances. Ultimately, this work will help us to better understand how PY consumption influences gastrointestinal health in children. Funding Sources Dannon Gut Microbiome, Yogurt and Probiotics Fellowship Grant awarded to HC. Sample analyses partially supported by an NIH Award.