Abstract
BACKGROUND AND AIM: The giant freshwater prawn (Macrobrachium rosenbergii) is an economically valuable aquaculture species, yet its production often faces challenges related to poor growth and disease outbreaks caused by intensive farming practices and excessive antibiotic use. Probiotics offer a sustainable alternative for improving growth, immunity, and pond health, but their efficacy and safety are highly strain specific. This study aimed to evaluate the probiotic potential of Bacillus pseudomycoides, Bacillus safensis, and Bacillus infantis isolated from prawn farms in Kalasin Province, Thailand, focusing on digestive enzyme activities, hemolytic safety, and antibiotic susceptibility. MATERIALS AND METHODS: Three Bacillus strains previously identified by 16S ribosomal RNA sequencing were characterized using standard in vitro assays. Amylase, protease, and lipase activities were assessed using the halo/colony (H/C) ratio and enzyme unit measurements. Hemolytic patterns were examined on Columbia agar supplemented with 5% sheep blood, and antibiotic susceptibility was tested by the disk diffusion method against seven antibiotics following Clinical and Laboratory Standards Institute (2024) guidelines. All assays were performed in triplicate, and data were analyzed using one-way analysis of variance (p < 0.05). RESULTS: All strains exhibited strong amylolytic (H/C ratio 2.05-2.27) and proteolytic (H/C ratio 1.75-1.96) activities, while lipase activity was undetectable. Hemolysis testing revealed γ-hemolysis for all strains, confirming non-hemolytic and non-pathogenic properties. Antibiotic susceptibility profiles indicated broad sensitivity to penicillin G, tetracycline, streptomycin, erythromycin, and chloramphenicol. Moderate susceptibility to vancomycin was observed in B. pseudomycoides and B. safensis, while B. infantis remained fully susceptible. The results suggest a strong digestive enzyme potential and an acceptable safety profile among all isolates. CONCLUSION: The evaluated Bacillus strains exhibit favorable probiotic attributes, including high amylase and protease activity, non-hemolytic safety, and broad antibiotic susceptibility, which support their suitability for probiotic application in M. rosenbergii culture. These native isolates may serve as sustainable, locally adapted alternatives to antibiotics, contributing to improved feed efficiency, growth, and disease resistance in freshwater prawn aquaculture. Further in vivo validation and genomic safety analyses are recommended to confirm their efficacy under commercial conditions.