Unravelling functional Bacillus strains from Ngari: A step toward sustainable probiotic applications in food systems

Two Gram-positive, facultatively anaerobic, motile, rod-shaped Bacillus isolates—designated FPIN1 and FPIN2—were obtained from Ngari, a traditional fermented fish product of Manipur, India, during the exploration of indigenous functional probiotic microorganisms. The isolates were subjected to comprehensive safety and probiotic evaluations, including assessments of hemolytic and DNase activities, antibiotic susceptibility, acid and bile salt tolerance, phenol and NaCl tolerance, temperature adaptability, auto-aggregation, and adhesion to epithelial cells. Molecular identification was performed through 16S rRNA gene sequencing and phylogenetic analysis. Both isolates exhibited non-hemolytic and DNase-negative phenotypes, confirming their non-pathogenic nature. Antibiotic susceptibility profiling revealed sensitivity to a wide spectrum of clinically important antibiotics, minimizing potential risks of antibiotic resistance transfer. Strains FPIN1 and FPIN2 demonstrated exceptional physiological resilience, showing optimal growth at 37 °C, strong acid tolerance (47–52% survival at pH 2), and resistance to 2% bile salts (~ 60% viability). The isolates further exhibited substantial tolerance to osmotic (at 8% NaCl) and phenolic stress (at 0.4% phenol), maintaining viability exceeding 18% and 60%, respectively. Auto-aggregation potential surpassed 80% after 5 h, and adhesion to epithelial cells exceeded 60%, underscoring strong colonization capability. Statistical analyses (one-way ANOVA and Kruskal–Wallis, p < 0.05) confirmed significant inter-strain variability, identifying FPIN1 and FPIN2 as the most promising candidates. 16S rRNA gene sequencing revealed the close relatedness of FPIN1 and FPIN2 to Bacillus sp. clade, with > 99% sequence similarity and strong bootstrap support (> 90%) in phylogenetic analysis. The isolates exhibited superior probiotic attributes—acid and bile tolerance, antibiotic sensitivity, strong adhesion, and high auto-aggregation—alongside non-virulent profiles. These findings signify the presence of safe, indigenous Bacillus strains with high functional potential from traditional fermented foods of Northeast India. Their robust stress tolerance, combined with the utilization of naturally occurring microbes from local resources, aligns with the principles of sustainable food biotechnology by reducing dependence on imported starter cultures and supporting eco-friendly, community-driven fermentation systems. Further genomic and in vivo validations are required to confirm safety and substantiate their potential for sustainable probiotic applications in functional food production.