Gut microbiota modulation and immunity enhancement by Bacillus amyloliquefaciens NL1.2: A fiber-degrading probiotic isolated from native Thai swine

Background and Aim: The pursuit of sustainable alternatives to antibiotic growth promoters has intensified interest in spore-forming probiotics with fiber-degrading capabilities. This study aimed to isolate, characterize, and evaluate the safety and functional properties of Bacillus spp. from native Thai swine, focusing on strains with probiotic potential and enzymatic activity for application in livestock nutrition. Materials and Methods: Spore-forming Bacillus isolates were obtained from fecal samples of backyard-raised native pigs. Isolates were screened for acid and bile tolerance, autoaggregation, hydrophobicity, biofilm formation, adhesion to Caco-2 cells, antimicrobial activity, and co-aggregation with pathogens. Enzyme production (cellulase, xylanase, and pectinase), hemolytic activity, and antibiotic susceptibility were also assessed. The most promising strain, Bacillus amyloliquefaciens NL1.2, was subjected to in vivo safety and efficacy evaluations in a mouse model, including assessments of toxicity, histopathology, secretory immunoglobulin A (IgA) levels, and gut microbiome modulation through full-length 16S ribosomal RNA sequencing. Results: B. amyloliquefaciens NL1.2 exhibited robust probiotic traits including high acid (115.05%) and bile (75.16%) tolerance, strong autoaggregation (65.99%), moderate hydrophobicity (34.13%), and effective adhesion (2.0%) to intestinal epithelial cells. It produced fiber-degrading enzymes (cellulase: 0.015 U/mL; xylanase: 0.522 U/mL; and pectinase: 0.374 U/mL) showed antimicrobial activity against Enterohemorrhagic Escherichia coli, Enteropathogenic E. coli, and Salmonella Typhimurium, and was non-hemolytic and antibiotic-sensitive. In vivo, NL1.2 induced no adverse effects and significantly elevated intestinal secretory IgA levels (p < 0.05). Microbiome analysis revealed enrichment of beneficial taxa (e.g., Bacteroidetes and Barnesiella) and reduction of potentially pathogenic taxa (e.g., Helicobacter and Deferribacteres). Conclusion: B. amyloliquefaciens NL1.2 is a safe, multifunctional probiotic with fiber-degrading, immunomodulatory, and gut microbiota-modulating properties. Its origin from native swine and broad functional attributes highlights its potential as a next-generation feed additive for sustainable animal production.