Characterization and In Vitro Probiotic Assessment of Alkalihalobacillus wakoensis as New Potent Probiotic Candidate

Bacillus spp. probiotics have been shown to be promising due to their resilience in the gut. This study evaluated the in vitro probiotic potential of Alkalihalobacillus wakoensis PTCC 1596, using Bacillus clausii ATCC 700160 as a control. In vitro probiotic characteristic assays were conducted to compare A. wakoensis and B. clausii (control) in terms of acid and bile tolerance, enzymatic activity, antimicrobial potential, antibiotic susceptibility, biofilm formation, hydrophobicity, autoaggregation, enterotoxin gene presence, and cytotoxicity. A. wakoensis spores demonstrated high survival (95.6% ± 0.9%) at pH 2.5 for 3 h and tolerance to 0.5% oxgall bile salts. The strain produced catalase, protease, and nattokinase, and exhibited moderate cell surface hydrophobicity (47.1% ± 3.12% to xylene) and autoaggregation (7.0% ± 1.2% after 1 h). It showed sensitivity to several antibiotics (e.g., chloramphenicol, gentamicin) and lacked detectable plasmids and tested enterotoxin genes ( hblA / hblC / hblD , nheA / nheB , bceT , and cytK ). Cytotoxicity assays using HepG‐2 cells indicated non‐toxicity, with supernatants potentially enhancing cell viability. While exhibiting lower biofilm formation than B. clausii , A. wakoensis displayed promising traits. These findings suggest A. wakoensis PTCC 1596 is a potential probiotic candidate warranting further investigation, particularly given its alkaliphilic nature.