• Functional properties of polysaccharides depend on their structural features. • IR spectroscopy is widely used in polysaccharide structural analysis. • Classical applications of IR spectroscopy in polysaccharide are reviewed. • IR integrating techniques can considerably expand its application scope.

Intake of dietary fiber is associated with a reduced risk of inflammatory bowel disease. β-Glucan (BG), a bioactive dietary fiber, has potential health-promoting effects on intestinal functions; however, the underlying mechanism remains unclear. Here, we explore the role of BG in ameliorating colitis by modulating key bacteria and metabolites, confirmed by multiple validation experiments and loss-of-function studies, and reveal a novel bacterial cross-feeding interaction. BG intervention ameliorates colitis and reverses Lactobacillus reduction in colitic mice, and Lactobacillus abundance was significantly negatively correlated with the severity of colitis. It was confirmed by further studies that Lactobacillus johnsonii was the most significantly enriched Lactobacillus spp. Multi-omics analysis revealed that L. johnsonii produced abundant indole-3-lactic acid (ILA) leading to the activation of aryl hydrocarbon receptor (AhR) responsible for the mitigation of colitis. Interestingly, L. johnsonii cannot utilize BG but requires a cross-feeding with Bacteroides uniformis, which degrades BG and produces nicotinamide (NAM) to promote the growth of L. johnsonii. A proof-of-concept study confirmed that BG increases L. johnsonii and B. uniformis abundance and ILA levels in healthy individuals. These findings demonstrate the mechanism by which BG ameliorates colitis via L. johnsonii–ILA–AhR axis and reveal the important cross-feeding interaction between L. johnsonii and B. uniformis.