Context-dependent functionality of 2'-fucosyllactose with Bifidobacterium breve M-16 V and short-chain galacto-/long-chain fructo-oligosaccharides in infant gut fermentation.

Human milk oligosaccharides, particularly 2'-fucosyllactose (2'-FL), are important functional components involved in infant gut microbiota development and immune-related functions. However, the efficacy of 2'-FL may be limited when applied alone, especially in microbiotas with insufficient 2'-FL-utilizing bacteria. This study investigated how the functional performance of 2'-FL changed when incorporated into a short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (scGOS/lcFOS)-supported system containing Bifidobacterium breve M-16 V. A continuous in vitro fermentation system was inoculated with fecal microbiota from four donors selected from 22 infant volunteers. Five treatments were compared: 2'-FL, GF (scGOS/lcFOS), GFB (GF + B. breve), GFF (GF + 2'-FL), and GFFB (GF + 2'-FL + B. breve). 2'-FL alone showed limited bifidogenic activity and weak support for microbial growth, whereas GFFB increased Bifidobacterium to 205.62% of the day-10 stabilization baseline (P < 0.01) and reduced Pseudomonadota to 14% of baseline (P < 0.001). GFFB also increased acetate to 140% of the 2'-FL group (P < 0.01), increased propionate to 110% of baseline (P < 0.05), and enriched tryptophan-derived indoles. Immunomodulatory activity was evaluated using cell-free fermentation supernatants rather than direct ingredient exposure. GFFB-derived supernatants showed the strongest attenuation of LPS-induced inflammatory responses in RAW 264.7 macrophages, reducing IL-1β, IL-6, nitric oxide, and TNF-α to 26.70%, 57.50%, 29.84%, and 35.91% of the LPS control, respectively (all P < 0.001). These findings indicate that the functional expression of 2'-FL was broadened in an scGOS/lcFOS-supported microbial context containing B. breve M-16 V.