Galactooligosaccharides drive dose-dependent improvements in child gut microbiome, metabolome and barrier function ex vivo

Background Galactooligosaccharides (GOS) are well-established prebiotic fibers, yet dose-response relationships and functional effects on children's microbiota remain incompletely characterized. Methods Using the predictive ex vivo SIFR ® technology platform (systemic intestinal fermentation research), this study assessed the dose-dependent effects of GOS (1.25, 2.5 and 5 g/day equivalents) on the gut microbiome of children from two different age groups (3–6 years and 6–12 years, n = 6 × 2). Shifts in microbial composition were evaluated via shallow shotgun metagenomic sequencing coupled with flow cytometry, metabolite production was evaluated through GC-FID and LC-MS/MS, while effects on epithelial barrier integrity were studied by measuring transepithelial electrical resistance (TEER) in a Caco-2/THP-1 co-culture model. Results GOS induced pronounced, dose-dependent bifidogenic effects and significantly increased other beneficial microbial taxa, including butyrate-producing Lachnospiraceae and Ruminococcaceae members, in both age groups. These compositional shifts were accompanied by elevated SCFA levels (acetate, propionate, butyrate), with significant effects already observed at the lowest dose (1.25 g/day), while gas formation increased proportionally less, resulting in a lower gas-to-SCFA ratio. Untargeted metabolomics revealed stimulation of additional microbial metabolites, including N -acetylated amino acids, agmatine and polyamines along with tryptophan-derived indoles and p -hydroxyphenyllactic acid, many of which were already significantly increased at lower GOS doses. Importantly, fermented GOS markedly supported gut barrier integrity, a hallmark of gut health, reflected by increased TEER values under both normal and LPS-induced inflammatory conditions, with significant improvements evident from the lowest dose onward. Overall, the effects on microbiome composition, metabolite production and gut barrier integrity were highly comparable between younger and older children. Conclusions Ex vivo GOS supplementation drove consistent, dose-dependent improvements in the child gut microbiome, microbial metabolic activity and gut barrier function. The efficacy already observed at low doses highlights GOS as a promising nutritional strategy to potentially support gut health in children aged 3–12 years.