Screening of bacteriocin producing Bacillus spp. isolated from Miang for application as probiotic in nile tilapia culture

Abstract

Seventy-nine Bacillus strains isolated from Miang samples were screened for potential probiotics for applying in aquaculture. Only six strains of Bacillus spp. (K2.1, K6.1, K7.1, K15.4 and K22.6, and K29.2) were revealed the antimicrobial ability against Gram-negative Aeromonas hydrophila and Gram-positive Streptococcus agalactiae. Through the molecular identification by 16S rRNA gene sequence and phylogenetic analysis, five isolates were identified to be Bacillus tequilensis (K2.1, K6.1, K7.1, K15.4, and K22.6) and the remaining isolate K29.2 was Bacillus siamensis. All selected isolates were susceptible to some tested antibiotics via agar well diffusion assay, except for B. tequilensis K6.1 and B. tequilensis 15.4, which were slightly resistant to streptomycin. In addition, they were all resistant to 0.3% (w/v) bile salt with a reasonable survival rate against extreme pH and secreted extracellular proteolytic enzymes. B. tequilensis K15.4 and B. siamensis K29.2 were selected to study for tannin tolerance and polysaccharides degrading enzyme production owing to their high tolerance to the gastric simulated and 0.3% (w/v) bile salt conditions. The time course study across 24 h on submerged fermentation using the specific enzyme inducer of each 0.5% (w/v) of carboxymethyl cellulose (CMC), birch wood xylan and locust bean gum (LBG), independently, revealed their ability to secret cellulase, xylanase and β-mannanase with the overall enzyme activity of B. tequilensis K15.4 higher than B. siamensis K29.2. The tannin tolerance analysis showed that two selected strains exhibited the the ability to grow and secrete cellulase and β-mannanase in the presence of hydrolysable tannin. Therefore, these characteristics supported their potential to be applied as multifunctional probiotic strains in Nile tilapia cultivation. The growth dynamics study of B. tequilensis K15.4 and B. siamensis K29.2 showed the optimum bacteriocin-producing time at 24 h cultivation at 37C, and the final pHs are 8.13 and 8.3, respectively. Antimicrobial activity reduction of culture supernatants after incubation with proteinase K reaffirmed their proteinaceous nature of bacteriocin from both strains. Purification of bacteriocins from both selected strains by 80% ammonium sulfate precipitation and gel filtration using 26/60 Sephacryl S100HR column revealed that the native molecular weight of the bacteriocin from B. tequilensis K15.4 and B. siamensis K29.2 were estimated to be 23 and 25 kDa, respectively. However, the exact elucidation of the authentic bacteriocins from both selected bacillus strains have to be clarified by more efficient SDS-PAGE or other analytical techniques. The purified bacteriocins from both two selected B. tequilensis K15.4 and B. siamensis K29.2 demonstrated broad pH tolerance and thermostability, demonstrating bactericidal inhibition against A. hydrophila cell growth, and retaining antimicrobial activity over a storage time of at least four months in a lyophilized form.