Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5-N-glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants.

Bovine milk oligosaccharides (BMOs), like their analogues in human milk, have important prebiotic functions. Environmental factors have previously been linked to variation in BMO structures, and thus to test the hypothesis that the bovine diet may lead to these changes in relative BMO abundances, a rapid capillary electrophoresis (CE)-based work flow was developed to profile the BMOs extracted from the milk of cows fed distinctly different diets. Over the first week of lactation, few significant differences were observed between the different diet groups, with the dominant changes being clearly linked to lactation period. CE analyses indicated the presence of ten unusually anionic BMOs, which were predicted to be phosphorylated and sulfated species. Nine unique sulfated BMOs were detected by high-resolution accurate mass spectrometry, none of which have been previously described in bovine milk. The biosynthesis of these was in direct competition with 3'-sialyllactose, the most abundant BMO in bovine milk.