Abstract：In order to evaluate the probiotic potential of dominant bacterial strains isolated from intestine of grass carp Ctenopharyngodon idellus using Aeromonas hydrophila as an indicator bacterium through the Oxford cup method. A strain XYCC11 with significant antagonistic activity was screened out by morphological characterization, physiochemical tests, and molecular analysis including 16S rRNA and housekeeping gene sequencing and its aquaculture application as probiotics and safety was systematically investigated through artificial infection tests, hemolytic assays, along with evaluation of antibacterial capacity, temperature/pH tolerance, and adhesion capability. Furthermore, the effects of XYCC11 supplementation on growth performance, antioxidant capacity, non-specific immunity, and liver function in grass carp were investigated. Results showed that XYCC11 as Bacillus spizizenii by integrated identification exhibited strong and stable antagonism against A.hydrophila among 12 intestinal isolates. Hemolytic tests confirmed its non-pathogenicity. Protease K treatment eliminated its antibacterial activity, indicating proteinaceous antimicrobial substances. The cell-free supernatant of XYCC11 showed inhibitory effects against four major pathogenic bacteria including A. hydrophila. The strain displayed remarkable tolerance to extreme pH and high temperature, with superior adhesion capability compared to non-intestinal isolates. The grass carp fed the XYCC11-supplemented diets for 60 days had significantly elevated weight gain rate and specific growth rate (P<0.05) and reduced food conversion ratio (P<0.05). There were elevated activities of alkaline phosphatase and acid phosphatase (P<0.05), and decrease in activities of aspartate aminotransferase and alanine aminotransferase (P<0.05), and malondialdehyde content in low-dose and high-dose groups (P<0.05). The significantly enhanced serum superoxide dismutase and lysozyme activities were observed in high-dose group at 60 days (P<0.05). These findings suggest that B. spizizenii XYCC11 represents a safe and effective probiotic candidate for grass carp, warranting further development as aquatic probiotics preparation.
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