Evaluation of probiotic potential of Bacillus spp. isolated from the digestive tract of freshwater fish Labeo calbasu (Hamilton, 1822)

The probiotic use in aquaculture helps to improve the survival, growth and health of fish as it is a sustainable aquaculture practice. In the present study, Bacillus spp. were isolated from the digestive tract of freshwater fish, Labeo calbasu towards the assessment of their probiotic potential. Based on the preliminary screening on 64 isolates obtained from the digestive tract of L. calbasu, only the Bacillus strains FS1, FC3 and FC6 were selected by using Bacillus selective media (Himedia) for their probiotics characterization. The biosafety assay confirmed that the isolates were not pathogenic to the host fish. Further, the selected isolates Viz; FS1, FC3 and FC6 were evaluated for probiotic properties. These strains were able to survive in acidic and alkaline conditions, higher tolerance to bile salt, high surface hydrophobicity to solvents, and they were found to tolerate in gastric juice. All the three isolates have exhibited notable amylase, proteolytic, lipase activity and susceptibility to various antibiotics. Their antagonistic activity was assessed against the fish pathogens; Aeromonas hydrophila (KX756709), A. veronii (KX688046), Acinetobacter junii (KX756708), A. tandoii (KX775222) Acinetobacter sp. (KX775221), and Pseudomonas stutzeri (KX721473) that were isolated from the naturally infected fish, Dawkinsia filamentosa. Among the three isolates, the FC6 exhibited a good antagonistic activity against three fish pathogens Viz; Aeromonas hydrophila, Acinetobacter sp. and Acinetobacter tandoii; FS1 and FC3 strains did not show any activity against the pathogens. Time killing kinetics assay also revealed that pathogens treated sample attained stationary phase quickly when compared to the control. Only the FC6 isolate had exhibited positive results for biofilm formation assay. All the three isolates did not exhibit any haemolytic activity. The isolated species were identified and confirmed on the basis of their colony morphology, biochemical characteristics and 16S rRNA sequencing. The probiotic strains, FS1, FC3 and FC6 were identified to be Bacillus subtilis (KX756706), Bacillus cereus (KX756707) and Bacillus amyloliquefaciens (KX775224) respectively. Based on the presently observed characteristic features, it is concluded that the FC6 strain, Bacillus amyloliquefaciens found in the digestive tract of L. calbasu could form a potential probiotic candidate.