Background: The infant gut microbiota establishes during a critical window of opportunity when metabolic and immune functions are highly susceptible to environmental changes, such as diet. Human milk oligosaccharides (HMOs) for instance are suggested to be beneficial for infant health and gut microbiota. Infant formulas supplemented with the HMOs 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) reduce infant morbidity and medication use and promote beneficial bacteria in the infant gut ecosystem. To further improve infant formula and achieve closer proximity to human milk composition, more complex HMO mixtures could be added. However, we currently lack knowledge about their effects on infants’ gut ecosystems. Method: We assessed the effect of lactose, 2′-FL, 2′-FL + LNnT, and a mixture of six HMOs (HMO6: consisting of 2′-FL, LNnT, difucosyllactose, lacto-N-tetraose, 3′- and 6′-sialyllactose) on infant gut microbiota and intestinal barrier integrity using a combination of in vitro models to mimic the microbial ecosystem (baby M-SHIME^®) and the intestinal epithelium (Caco-2/HT29-MTX co-culture). Results: All the tested products had bifidogenic potential and increased SCFA levels; however, only the HMOs’ fermented media protected against inflammatory intestinal barrier disruption. 2′-FL/LNnT and HMO6 promoted the highest diversification of OTUs within the Bifidobactericeae family, whereas beneficial butyrate-producers were specifically enriched by HMO6. Conclusion: These results suggest that increased complexity in HMO mixture composition may benefit the infant gut ecosystem, promoting different bifidobacterial communities and protecting the gut barrier against pro-inflammatory imbalances.

Because of the recognized health benefits of breast milk, it is recommended as the sole nutrition source during the first 6 months of life. Among the bioactive components are human milk oligosaccharides (HMOs) that exert part of their activity via the gut microbiota. Here, we investigated the gut microbiota fermentation of HMO 2’fucosyllactose (2’-FL), using two in vitro models (48 h fecal incubations and the long-term mucosal simulator of the human intestinal microbial ecosystem [M-SHIME^®]) with fecal samples from 3-month-old breastfed (BF) infants as well as 2–3 year old toddlers. The short-term model allowed the screening of five donors for each group and provided supportive data for the M-SHIME^® study. A key finding was the strong and immediate increase in the relative abundance of Bifidobacteriaceae following 2’-FL fermentation by both the BF infant and toddler microbiota in the M-SHIME^®. At the metabolic level, while decreasing branched-chain fatty acids, 2’-FL strongly increased acetate production together with increases in the health-related propionate and butyrate whilst gas production only mildly increased. Notably, consistently lower gas production was observed with 2’-FL fermentation as compared to lactose, suggesting that reduced discomfort during the dynamic microbiome establishment in early life may be an advantage along with the bifidogenic effect observed.