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.