Characterization and In Vitro Probiotic Assessment of <i>Alkalihalobacillus wakoensis</i> as New Potent Probiotic Candidate

<i>Bacillus</i> spp. probiotics have been shown to be promising due to their resilience in the gut. This study evaluated the in vitro probiotic potential of <i>Alkalihalobacillus wakoensis</i> PTCC 1596, using <i>Bacillus clausii</i> ATCC 700160 as a control. In vitro probiotic characteristic assays were conducted to compare <i>A. wakoensis</i> and <i>B. clausii</i> (control) in terms of acid and bile tolerance, enzymatic activity, antimicrobial potential, antibiotic susceptibility, biofilm formation, hydrophobicity, autoaggregation, enterotoxin gene presence, and cytotoxicity. <i>A. wakoensis</i> 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 (<i>hblA</i>/<i>hblC</i>/<i>hblD</i>, <i>nheA</i>/<i>nheB</i>, <i>bceT</i>, and <i>cytK</i>). Cytotoxicity assays using HepG-2 cells indicated non-toxicity, with supernatants potentially enhancing cell viability. While exhibiting lower biofilm formation than <i>B. clausii</i>, <i>A. wakoensis</i> displayed promising traits. These findings suggest <i>A. wakoensis</i> PTCC 1596 is a potential probiotic candidate warranting further investigation, particularly given its alkaliphilic nature.