Postbiotic cell-free supernatant of Enterococcus faecium L6 enhances growth performance, intestinal homeostasis and disease resistance in largemouth bass (Micropterus salmoides)

E. faecium L6 harbors biosynthetic gene clusters encoding diverse bioactive metabolites. • Dietary CFS supplementation improved growth performance by enhancing antioxidant capacity, innate immunity, and intestinal barrier integrity in largemouth bass. • CFS reshaped the intestinal microenvironment by enriching beneficial bacteria and functional metabolites. • CFS significantly increased survival following A. veronii challenge through modulation of inflammatory responses. Probiotics are widely used to promote fish health. However, the application of live probiotics is limited by poor stability and potential safety concerns. Postbiotics have drawn increasing attention because they may provide beneficial effects without the limitations associated with live bacteria. This study investigated the effects of probiotic (live Enterococcus faecium L6, LEF) and postbiotics (cell-free supernatant, CFS, and heat-killed E. faecium L6, HKEF) on growth performance, intestinal health, and disease resistance in Micropterus salmoides ( M. salmoides ). A 56-day feeding trial was conducted, followed by an Aeromonas veronii ( A. veronii ) challenge test. Growth parameters, intestinal morphology, antioxidant and non-specific immune enzyme activities, inflammatory cytokine expression, intestinal microbiota composition, and metabolomic profiles were analyzed. Both LEF and CFS significantly improved final body weight, weight gain rate, and specific growth rate while decreasing the feed conversion ratio, whereas HKEF had no significant effect. LEF and CFS improved intestinal barrier integrity by increasing villus height and the villus height:crypt depth ratio, and by up-regulating ZO -1, Claudin -1, and Occludin . Antioxidant (SOD, CAT) and non-specific immune (ACP, AKP, LZM) activities were also significantly elevated in the LEF and CFS groups. After A. veronii challenge, survival rates were significantly higher in the LEF (55.0%) and CFS (60.0%) groups compared with the PBS control (30.0%). The improved resistance was associated with reduced intestinal pathogen loads and a balanced inflammatory response, characterized by down-regulated TNF-α and IL-1β and up-regulated IL-10 . Furthermore, CFS reshaped the intestinal microbiota and increased functional metabolites. In conclusion, E. faecium L6-derived CFS conferred growth-promoting and disease-protective effects comparable to, or stronger than, its live probiotic counterpart, supporting its potential as a stable and effective postbiotic strategy for enhancing fish health. Download: Download high-res image (96KB) Download: Download full-size image