Isolation, Phenotyping and Antimicrobial Activity of Bacillus spp. from Fermented Zamné, a Fermented Condiment from Senegalia macrostachya Seeds

Abstract

Background: Fermented Zamné is a traditional condiment produced from Senegalia macrostachya seeds. Aims: This study aims to isolate and characterise predominant Bacillus-related bacteria from fermented Zamné and to evaluate their antimicrobial activity against selected indicator microorganisms. Methods: A total of 65 aerobic mesophilic bacterial isolates obtained from four independent fermentations were characterised using morphological and biochemical methods. Forty-four isolates were spore-forming, and 29 isolates displaying morphological characteristics consistent with Bacillus-related bacteria were further characterised and identified using the API 50 CHB system. The antimicrobial activity of the isolates was evaluated by an agar well diffusion assay against 13 indicator microorganisms, including Gram-positive and Gram-negative bacteria, yeasts, and moulds. Results: The isolates were assigned to five taxonomic groups: Bacillus subtilis/Bacillus amyloliquefaciens group (n = 17), Bacillus cereus (n = 3), Brevibacillus laterosporus (n = 5), Bacillus licheniformis (n = 3), and Bacillus megaterium (n = 1). Antimicrobial activity was detected only against five Gram-negative bacteria: Salmonella infantis, Salmonella Enteritidis, Pseudomonas aeruginosa, Proteus vulgaris, and Yersinia enterocolitica. Six isolates exhibited activity against at least one indicator microorganism. Among them, Brevibacillus laterosporus E4S1-5 inhibited all five susceptible Gram-negative bacteria, with inhibition zones ranging from 6.97 ± 0.06 to 13 ± 0.5 mm, while the isolate E4S2-2 (B. subtilis/ B. amyloliquefaciens group) displayed the broadest inhibitory spectrum of 14.5 ± 0.5 mm. Isolates E3S1-4 (Bacillus cereus), E3S3-4 and E1S2-5 (Brevibacillus laterosporus), and E4S2-1 (B. subtilis/B. amyloliquefaciens group) also exhibited variable antimicrobial activity. Conclusion: These findings indicate that fermented Zamné harbours diverse Bacillus-related bacteria with antimicrobial potential. Selected Bacillus isolates with optimal antimicrobial properties may warrant further investigation as potential protective cultures for controlled Zamné fermentation. Molecular identification, safety assessment, and characterisation of the antimicrobial compounds produced by the active isolates are required before their technological application.