Comparison of Viable and Heat‐Inactivated Lacticaseibacillus rhamnosus IDCC 3201: Anti‐Pathogenic, Anti‐Inflammatory, and Microbiota Modulating Effect

ABSTRACT Lacticaseibacillus rhamnosus is widely studied for strain‐specific antimicrobial and immunomodulatory properties, but comparative data for viable and heat‐inactivated preparations remain limited. This study compared the anti‐pathogenic activity of live L. rhamnosus IDCC 3201 and its commercially prepared heat‐inactivated formulation, RHT 3201, and examined their associated microbiota and metabolite profiles in a single‐donor ex vivo fecal fermentation model. Transcript‐level immunomodulatory activity was evaluated separately for RHT 3201 in LPS‐stimulated RAW 264.7 cells. Both preparations inhibited key intestinal pathogens, including Escherichia coli , Staphylococcus aureus , Enterococcus faecalis , and Salmonella Typhimurium, although live IDCC 3201 showed stronger inhibition against E. faecalis and S. Typhimurium . In LPS‐stimulated RAW 264.7 cells, RHT 3201 reduced IL‐1β, IL‐6, COX‐2, and TNF‐α mRNA expression by approximately 39%, 23%, 17%, and 16%, respectively, at 10 × 10 7 CFU/mL. At the species level in the ex vivo fermentation model, RHT 3201 was associated with a higher relative abundance of Bifidobacterium pseudocatenulatum and Lactobacillus ruminis , whereas L. rhamnosus was detected only in the IDCC 3201 group during the 24‐h culture period. Metabolite profiling also showed distinct product‐associated signatures between the two preparations, with higher lactic acid detected in RHT 3201. Overall, the two preparations showed comparable activity against some pathogens but distinct microbiota‐ and metabolite‐associated profiles in this single‐donor ex vivo system, while anti‐inflammatory activity was assessed only for RHT 3201 at the mRNA level. Additional multi‐donor, protein‐level, and in vivo studies are required to confirm the reproducibility and physiological relevance of these findings.