Ulcerative colitis (UC), a debilitating inflammatory bowel disease, is characterized by intestinal inflammation, barrier dysfunction, and dysbiosis. Current treatment options are limited. Dietary interventions, particularly prebiotics, probiotics, and synbiotics, offer a promising approach due to their ability to modify the gut microbiota, modulate immune responses, and enhance barrier function. Galactooligosaccharides (GOS) and *Limosilactobacillus reuteri* are known prebiotic and probiotic agents, respectively, with documented benefits for microbial balance and intestinal barrier integrity. Previous research indicated that GOS enhances intestinal barrier function by selectively enriching certain lactobacilli, including *L. reuteri*. The synergistic mechanisms of GOS and *L. reuteri* in mitigating inflammation and enhancing barrier function, however, remain largely uncharacterized. This study aims to investigate the mechanisms underlying the potential therapeutic effects of a synbiotic comprised of GOS and *L. reuteri* in treating UC, focusing on identifying the specific intestinal bacteria and metabolites involved in this process.

The etiology and pathogenesis of UC remain poorly understood, but emerging evidence strongly implicates intestinal inflammation, barrier dysfunction, and dysbiosis. Prebiotics, probiotics, and synbiotics have demonstrated promise in alleviating UC symptoms by modifying the gut microbiota, modulating the immune response, and strengthening the intestinal barrier. GOS and *L. reuteri* individually have shown beneficial effects on the gut microbiome and intestinal barrier function. Prior work from the authors showed that GOS selectively enriches certain lactobacilli, including *L. reuteri*, in the gut. *L. reuteri* can effectively utilize GOS as a carbon source, making this pair a suitable candidate for synbiotic development. However, the combined effect of GOS and *L. reuteri* and the underlying mechanisms of their synergy in combating UC inflammation remain unexplored. Understanding the specific bacterial species and metabolites regulated by this synbiotic is crucial for developing effective therapeutic interventions.

This study employed a multifaceted approach involving murine models of DSS-induced colitis, fecal microbiota transplantation (FMT), and in vitro experiments using bacterial cultures. The study design involved administering GOS and *L. reuteri* separately and in combination to mice, followed by DSS treatment to induce colitis. Body weight changes, colon length, histological damage scores, and levels of inflammatory cytokines were assessed. The study also utilized 16S rRNA gene sequencing and shotgun metagenomic sequencing to analyze changes in gut microbiota composition. Targeted metabolomics was employed to identify differentially abundant metabolites associated with the synbiotic treatment. To test the efficacy of the synbiotic-altered microbiota, FMT experiments were conducted using microbiota-depleted recipient mice. The role of *Bacteroides acidifaciens* was investigated directly by administering this bacterium alone to mice and evaluating its effects on colitis. In vitro bacterial cultures were used to study C15:0 synthesis and its regulation. The effect of C15:0 on intestinal inflammation was investigated in cell cultures. RNA sequencing (RNA-seq) was performed to investigate the molecular mechanisms of action of C15:0. Finally, the clinical relevance of the findings was assessed by comparing *B. acidifaciens* and C15:0 levels in fecal samples from human UC patients and LPS-challenged piglets. The study also analyzed publically available datasets on C15:0 levels in UC patients.

The combination of GOS and *L. reuteri* was significantly more effective in ameliorating intestinal inflammation and barrier dysfunction compared to either component alone. The synbiotic treatment resulted in a marked enrichment of *Bacteroides acidifaciens* in the gut microbiota. This enrichment correlated with increased levels of pentadecanoic acid (C15:0), an odd-chain fatty acid. Both *B. acidifaciens* and C15:0 independently showed protective effects against DSS-induced colitis, reducing inflammation and improving barrier function. In vitro experiments revealed that *B. acidifaciens* synthesizes C15:0, particularly when co-cultured with both GOS and *L. reuteri*. Mechanistically, C15:0 suppressed NF-κB activation, a key inflammatory signaling pathway, through the involvement of FATP4. Importantly, lower levels of both *B. acidifaciens* and C15:0 were observed in fecal samples from UC patients and LPS-challenged piglets, confirming the clinical relevance of the findings. Analysis of multiple public datasets consistently demonstrated reduced C15:0 levels in serum, feces, and colonic biopsies from UC patients compared to healthy controls.

The study's findings demonstrate a novel synergistic mechanism by which a synbiotic of GOS and *L. reuteri* protects against intestinal inflammation and barrier dysfunction. The enrichment of *B. acidifaciens* and subsequent production of C15:0 are critical components of this protective effect. The observation that both *B. acidifaciens* and C15:0 independently mitigate colitis underscores their potential as therapeutic agents. C15:0's mechanism of action involves suppression of NF-κB activation via FATP4. This pathway is highly relevant to intestinal inflammation, and this study's findings enhance the understanding of the role of fatty acid metabolism in the gut and its impact on immune responses. The consistent finding of reduced *B. acidifaciens* and C15:0 in UC patients across multiple datasets strengthens the clinical relevance of the study's conclusions. This research points toward a new strategy for UC and potentially other inflammatory bowel diseases using dietary supplements to modulate the gut microbiota and restore beneficial metabolites.

This study elucidates a previously unknown mechanism linking a synbiotic combination of GOS and *L. reuteri* to gut health via *B. acidifaciens* and C15:0 production. This research strongly suggests the synbiotic, *B. acidifaciens*, and C15:0 have potential therapeutic value for UC and other inflammatory bowel diseases. Further research should focus on optimizing the synbiotic formulation, identifying additional beneficial bacterial strains, and conducting human clinical trials to validate the findings and determine the optimal dosage and administration strategies.

The study primarily used murine models of colitis, which may not fully recapitulate the complexities of human UC. Although the results were corroborated with human and porcine data, further clinical studies are necessary to confirm the efficacy and safety of the synbiotic, *B. acidifaciens*, and C15:0 in human patients. The specific mechanisms underlying the interaction between GOS, *L. reuteri*, and *B. acidifaciens* require further investigation. The study focused on a specific strain of *B. acidifaciens*, and additional strains need to be examined to understand the extent of strain-specific effects.