Oligosaccharides With Defined Glycosidic Bonds Shape Gut Microbial Succession and Metabolism Via Bond‐Specific Microbial Responders

Functional oligosaccharides are important prebiotic ingredients, but the structure-function relationships and mechanisms by which defined glycosidic bonds shape microbial responses remain unclear. Five glucose disaccharides, trehalose (α-1,1), maltose (α-1,4), isomaltose (α-1,6), cellobiose (β-1,4), and gentiobiose (β-1,6), were used as minimal oligosaccharide models to isolate glycosidic bond effects. Absolute time-series profiling combined with Bayesian generalized Lotka-Volterra modeling identified bond-specific microbial responders, operationally defined as taxa with statistically supported substrate-associated growth advantages beyond endpoint dominance. α-Linked disaccharides mainly recruited Bifidobacterium pseudocatenulatum and Megamonas funiformis, cellobiose enriched Faecalibacterium prausnitzii, and gentiobiose enriched B. pseudocatenulatum. Monoculture assays confirmed direct cognate disaccharide utilization. Metaproteomics revealed linkage-matched modules: isomaltose responders upregulated GanO/ChvE and oligo-1,6-glucosidase; cellobiose responders expressed CebE/ChvE, ABC.MS.S, CelB, cellobiose phosphorylase, and β-glucosidases; whereas the molecular evidence for gentiobiose was based mainly on ABC.MS.S and general β-glucosidases. Metabolically, gentiobiose favored acetic acid accumulation, cellobiose yielded the highest butyric acid concentration, and isomaltose elevated trans-4-hydroxy-L-proline and 7,8-dihydroneopterin associated with redox and immune-related cofactor pathways. Guided by these ecological and molecular observations, microbial responder-centered synthetic microbial communities utilized cognate disaccharides, recapitulated glycosidic bond-specific ecological succession, showed greater net short-chain fatty acid (SCFA) accumulation than matched complex communities under equal initial substrate input in vitro, and elevated fecal SCFAs in mice, with cellobiose increasing butyric acid by 2.1-fold. These results support a mechanistically informed pathway linking glycosidic bond structure, microbial succession, and metabolic outputs, providing a basis for structure‑guided microbiome modulation.