Dose-Dependent Commercial Probiotic Intervention Modulates Gut Microbiota and Fecal Metabolome Associated with Improved Intestinal Function in a Loperamide-Induced Constipation Rat Model.

Therapeutic probiotic formulations are promising microbiome-directed interventions for constipation, but their dose-dependent mechanisms remain unclear. This study investigated whether probiotic treatment induces dose-dependent gut microbiota and fecal metabolome remodeling in a loperamide-induced rat model of constipation and identified microbiome-metabolome association signatures linked to intestinal recovery. Male SPF Sprague-Dawley rats were assigned to Control, Model, low-dose probiotic (1 × 10⁹ CFU/day), high-dose probiotic (1 × 1010 CFU/day), and PFK groups. Constipation was induced with loperamide (10 mg/kg/day) for 14 days, followed by 14 days of intervention. Intestinal function was assessed by body weight, food intake, fecal output, fecal water content, and intestinal transit rate. Gut microbiota was profiled by 16 S rRNA sequencing, and fecal untargeted LC-MS/MS metabolomics was performed in 36 samples. Differential metabolites were identified using VIP > 1 and p < 0.05, followed by microbiome-metabolome correlation analysis. Given the exploratory nature and limited group sizes, these metabolites were considered candidate features rather than statistically validated biomarkers. High-dose probiotic intervention significantly improved intestinal function and reshaped the gut microbiota, enriching short-chain fatty acid-associated genera including Blautia, Fusicatenibacter, and Muribaculaceae-related taxa. Despite reduced alpha diversity, functional recovery was accompanied by selective microbial restructuring. Metabolomics identified 5,257 annotated features, with high-dose probiotics producing the strongest remodeling (1,006 differential metabolites vs. Model). Reversal analysis identified 68 constipation-associated metabolites shifted in the opposite direction after treatment. Microbiome-metabolome correlation analysis identified associations between key genera and lipid-, organic acid-, and indole-related metabolites, highlighting potential immunometabolic interactions. High-dose probiotic intervention was associated with coordinated changes in the gut microbiota and fecal metabolome together with improved intestinal function, supporting further investigation of dose optimization for microbiome-based constipation therapies.