Bacillus spp. Isolated from Miang as Potential Probiotics in Nile Tilapia Culture-In Vitro Research

Among 79 Bacillus spp. isolated from Miang, a fermented tea in north Thailand, 17 Bacillus strains were selected with probiotic potential in Nile tilapia culture based on the capabilities of bacteriocin production and associated antimicrobial activities against fish pathogens, Aeromonas hydrophila and Streptococcus agalactiae. However, only six isolates were selected for further extensive studies based on the strength of their antimicrobial activities and their tolerance against simulated gastrointestinal conditions. The molecular identification by 16S rRNA gene sequence analysis revealed that five isolates, K2.1, K6.1, K7.1, K15.4, and K22.6, were Bacillus tequilensis, and the isolate K29.2 was Bacillus siamensis. B. siamensis K29.2 showed complete susceptibility to antibiotics tested in this study, while B. tequilensis K 15.4 showed moderate resistance to some antibiotics; therefore, both strains were selected as potential probiotic bacteria. B. tequilensis K15.4 and B. siamensis K29.2 were capable of the production and secretion of extracellular protease and polysaccharide degrading enzymes, including cellulase, xylanase, and β-mannanase. The tannin tolerant test also demonstrated their ability to grow on selective agar plates and secrete cellulase and β-mannanase in the presence of hydrolyzable tannin. In addition, in vitro digestion of commercial fish substrate revealed that the extracellular enzymes produced by both strains efficiently reacted with feed protein and polysaccharides. Based on the results from this study, B. siamensis K29.2 was deemed to have the highest potential multifunctional probiotic qualities for application in Nile tilapia culture, while the antibiotic-resistant gene in B. tequilensis K15.4 must be clarified before field application.