Interindividual Variability in Raffinose Fermentation by Human Gut Microbiota and Metabolomic Signature of a High-Efficiency In Vitro Degrader, Limosilactobacillus reuteri subsp. reuteri

Background/Objectives: Raffinose is a prebiotic trisaccharide fermented by human gut microbiota, yet the strain-level determinants of its degradation and associated metabolic outputs remain poorly characterized. Methods: In the present study, anaerobic fermentation, 16S rRNA gene amplicon high-throughput sequencing, culturomics, and metabolomics were conducted to address this question. Results: In vitro fermentation of raffinose was performed using fecal samples from 16 healthy donors, and substantial interindividual variation was observed in substrate consumption, microbiota composition, and short-chain fatty acid production. Through culturomics, 204 bacterial strains spanning 18 species were isolated, among which Limosilactobacillus reuteri subsp. reuteri exhibited the highest raffinose-degrading efficiency. Untargeted metabolomics comparing L. reuteri subsp. reuteri cultured on raffinose versus glucose revealed a distinct metabolic shift, with 290 metabolites significantly upregulated, including the putatively identified fructooligosaccharide 1-kestose—a compound with documented prebiotic and anti-inflammatory properties. KEGG enrichment further highlighted coordinated remodeling of nucleotide and amino acid metabolism. Conclusions: Collectively, this study provides a strain-level framework for understanding how raffinose shapes the gut microbiota and identifies L. reuteri subsp. reuteri as a key metabolic hub linking prebiotic consumption to the production of bioactive metabolites.