Genomic and physiological evaluation of Bacillus velezensis FLU-1 from largemouth bass (Micropterus salmoides) for probiotic development in aquaculture

Probiotics are increasingly recognized as viable alternatives to antibiotics in sustainable aquaculture. This study comprehensively evaluates Bacillus velezensis FLU-1, a novel strain isolated from largemouth bass, for its probiotic potential. Through in vitro and in vivo analyses, we assessed its functional traits, growth-promoting effects, and impacts on intestinal integrity, immunity, and antioxidant capacity. The in vitro analysis demonstrated that FLU-1 exhibits strong probiotic attributes, including production of key hydrolytic enzymes (protease, cellulase, amylase, and mannanase), tolerance to acidic conditions (pH 2–5), 0.3 % bile salts, and heat (90 °C; 65.77 % survival after 10 min), alongside strong auto-aggregation (71.2 % at 24 h) and hydrophobicity (76.95 %). The strain also met essential safety standards, as evidenced by its non-hemolytic nature and susceptibility to antibiotics. Whole-genome sequencing revealed a diverse set of genes involved in enzyme production, adhesion mechanisms, and the biosynthesis of vitamins and amino acids, further supporting its probiotic potential. A 56-day feeding trial demonstrated that dietary supplementation with FLU-1 significantly enhanced growth performance, intestinal morphology (elongated villi, deeper crypts), tight junction gene expression (e.g., occludin , claudin-2 , and zo-1 ), and digestive enzyme activities (protease, α-amylase, and lipase) of largemouth bass. Moreover, FLU-1 administration modulated the gut microbiota composition. Compared to the control group, fish fed FLU-1 exhibited markedly higher activities of antioxidant enzymes (SOD, CAT, and GSH) and reduced MDA levels. Immunological analysis showed that the strain fostered an anti-inflammatory profile by increasing the expression of cytokines such as TGF-β and IL-10 , while suppressing pro-inflammatory markers including TNF-α and IL-8 . Therefore, the host-adapted B. velezensis FLU-1 represents an effective probiotic for largemouth bass aquaculture. • A novel B. velezensis FLU-1 strain was isolated from largemouth bass. • FLU-1 exhibits promising in vitro probiotic traits. • FLU-1 genome harbors genes supporting gut colonization and health. • Dietary supplementation improves growth performance and intestinal health in largemouth bass.