Fermented soymilk (FSM4) has attracted much attention due to its nutritional and health characteristics. Exploring FSM4 products to alleviate diarrhea can ensure their effectiveness as a therapeutic food for alleviating gastrointestinal disorders. However, the relationship between gut microbiota and gut metabolite production remains unknown during diarrheal episodes. Therefore, the diarrhea-alleviating role and mechanisms of FSM4 in diarrhea rats were investigated via biochemical, gut microbiota, and serum metabolite analyses. The findings showed that consuming FSM4 improved diarrhea symptoms and reduced systemic inflammation better than non-fermented soymilk (NFSM). It is worth noting that FSM4 promoted the diversity, richness, structure, and composition of gut microbiota. It increased the ability to reduce inflammation associated with harmful bacteria (Anaerofilum, Flavonifractor, Bilophila, Anaerostipes, [Ruminococcus]_torques_group, Clostridium_sensu_stricto_1, Turicibacter, Ruminococcus_1, Ruminiclostridium_6, Prevotellaceae_NK3B31_group and Fusicatenibacter), while stimulating the growth of healthy species (Lactobacillus, Ruminococcaceae_UCG-014, Oscillibacter, [Eubacterium]_coprostanoligenes_group, Negativibacillus, and Erysipelotrichaceae_UCG-003). Moreover, metabolomics analysis showed that lipid metabolites such as lysophosphatidylethanolamine (LysoPE) and sphingolipids were upregulated in the NG group, closely related to pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, and IFN-γ) and the aforementioned pathogenic bacteria. Notably, in treatment groups, especially FSM4, the accumulation of L-ornithine, aspartic acid, ursocholic acid, 18-oxooleate, and cyclopentanethiol was increased, which was robustly associated with the anti-inflammatory factor IL-10 and beneficial bacteria mentioned above. Therefore, it can be inferred that the amino acids, bile acid, 18-oxooleate, and cyclopentanethiol produced in the FSM4 group can serve as metabolic biomarkers, which synergistically act with the gut microbiota to help alleviate inflammation for diarrhea remission. Overall, FSM4 may provide a new alternative, as an anti-inflammatory diet, to alleviate diarrhea.

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