Potential gut adherent probiotic bacteria isolated from rohu, Labeo rohita (Actinopterygii: Cypriniformes: Cyprinidae): Characterisation, exo-enzyme production, pathogen inhibition, cell surface hydrophobicity, and bio-film formation

Background. Previous reports emphasized exo-enzyme production and pathogen inhibition as major criteria to select putative probiotics in carps. However, adhesion ability to the gut epithelium could be one of the decisive factors. The presently reported study was aimed to determine the probiotic potential of autochthonous bacteria isolated from gastrointestinal (GI) tract of rohu, Labeo rohita (Hamilton, 1822). Apart from characterization of functional probiotic attributes and bio-safety, the presently reported study utilized in vitro model system for preliminary selection of potentially adherent strains. Materials and methods. Altogether, 126 exo-enzyme producing bacteria were isolated from the proximal (PI) and distal (DI) segment of the GI tract and evaluated for enzyme-producing ability (viz., amylase, protease, lipase, cellulase, phytase, xylanase). Pathogen inhibition was tested by cross-streaking and double-layer method. Based on the cumulative results, isolates LR3H1A and LR3F3P were selected and identified by phenotypic and 16S rRNA partial gene sequence analyses. Both the strains were tested for their ability to grow in fish mucus, tolerance to fish bile, and bio-safety. Cell surface characteristics of the strains were analysed by aggregation assays and bio-film forming ability was determined through adherence to glass and polystyrene surfaces. Results. Seven strains (PI-4, DI-3) were primarily selected as efficient exo-enzyme producers, of which 3 strains (PI-2, DI-1) were found to be antagonistic against ≥1 of the 6 tested fish pathogens. Partial characterization of the cell-free supernatant revealed that the antagonistic compound was proteinaceous, showing its maximum efficacy at 30°C and pH 7. Isolates LR3H1A and LR3F3P were identified as Bacillus subtilis subsp. spizizenii (KF623286) and Bacillus tequilensis (KF623287), respectively. Both the strains grew well in fish mucus, tolerated diluted bile juice, and showed evidence of bio-safety. Both the strains were categorized as a moderate bio-film producer, although LR3F3P was noticed to possess stronger cell surface hydrophobicity, auto‐aggregation and co‐aggregation capacity than LR3H1A. Conclusion. Owing to better colonization potential, presently reported study indicated B. tequilensis LR3F3P as a putative probiotic for feed application.