In vitro probiotic properties and anti-Vibrio activity of Bacillus subtilis B13 and its ethyl acetate extract

This study is carried out to examine the in vitro probiotic properties of Bacillus subtilis B13 (= VTCC 910231), isolated from brackish water in the Cau Hai lagoon, Vietnam. The strain was confirmed to be safe, showing no hemolytic activity and exhibiting strong biofilm formation. It demonstrated notable environmental adaptability, growing across a wide pH range (5-9), showing moderate tolerance at 7% NaCl. B13 produced multiple extracellular enzymes, with the highest activities observed for lipase, protease, and cellulase, and additionally metabolized 25 of the 49 carbohydrates tested (51.0%). Antibiotic susceptibility profiling revealed broad sensitivity, with resistance detected to only 4 of 15 antibiotics, and it was highly sensitive to fluoroquinolones and phenicols. The ethyl acetate extract of its cell-free culture supernatant displayed noticeable inhibitory activity against five Vibrio species, which exhibited inhibition diameters varying from 13.3 ± 0.6 to 21.7 ± 0.6 mm. Moreover, the extract exhibited significant anti-inflammatory effects, achieving concentration-dependent nitric oxide inhibition (IC₅₀ = 28.6 µg/mL) while maintaining high macrophage viability (94.9-97.1%). Chemical analysis by GC-MS and LC-ESI-QTOF/MS identified 34 metabolites, of which 26 were tentatively characterized, including organic acids, fatty acid derivatives, volatile hydrocarbons, and a diverse range of cyclic dipeptides, many of which have previously been reported with antimicrobial and anti-inflammatory activities. Notably, the detection of pre-aurantiamine and leupyrrin A1, together with the broad diversity of cyclic dipeptides, represents a rare feature among B. subtilis strains. These findings emphasise the potential of B13 as a probiotic in aquaculture practices.