Prebiotic Structural Diversity Shapes Gut Microbial Diversity, Community Composition, and Metabolic Activity In Vitro

Prebiotics are selectively utilized substrates that modulate gut microbiota and host health, yet different prebiotic structures may elicit distinct ecological and metabolic responses. In this study, we investigated the effects of five structurally diverse prebiotics-isomaltooligosaccharides (IMO), arabinogalactans (AG), pectin, inulin, and stachyose-on human gut microbiota via a 24 h in vitro anaerobic culture with healthy donors' gut microbiota. Microbial community dynamics were profiled by 16S rRNA gene sequencing, and short-chain fatty acids (SCFAs) production was analyzed. All treatments resulted in decreased α-diversity compared with baseline, with pectin most effectively preserving microbial richness and evenness, whereas stachyose led to the greatest reduction. Community composition and functional profiles shifted in a substrate-specific manner, with AG promoting <i>Bacteroidaceae</i>, IMO stimulating <i>Lachnospiraceae</i> and <i>Faecalibacterium</i>, and pectin supporting balanced microbial structures and SCFA production. Pectin, IMO, and inulin enhanced butyrate levels, whereas AG and pectin promoted propionate formation. These findings demonstrate that prebiotic structural differences strongly shape gut microbial ecology and metabolism, providing a mechanistic basis for rationally selecting and combining prebiotics to beneficially modulate the gut microbiota.