Screening of Bacillus Strains with Glutenolytic Activity and Probiotic Potential: Balancing Efficacy and Biosafety

The development of alternative strategies for gluten detoxification represents an important approach for the management of celiac disease and other gluten-related disorders. This study aimed to identify gluten-degrading Bacillus strains with probiotic potential by integrating enzymatic screening with comprehensive biosafety and functional characterization. A total of 45 isolates obtained from diverse ecological niches in Uzbekistan were evaluated for glutenolytic activity using an agar diffusion assay. Fifteen strains demonstrated detectable gluten hydrolysis (16–32 mm). The highest activity was observed in Bacillus cereus isolates (up to 32 mm), whereas Bacillus amyloliquefaciens and Bacillus licheniformis exhibited moderate activity (16–24 mm). However, biosafety profiling revealed pronounced hemolytic, lecithinase, and DNase activities in most B. cereus strains, along with reduced antibiotic susceptibility, which precludes their probiotic application. In contrast, B. amyloliquefaciens and B. licheniformis strains exhibited γ-hemolysis, lacked lecithinase and DNase activities, showed high susceptibility to clinically relevant antibiotics, and demonstrated satisfactory tolerance to simulated gastrointestinal conditions. None of the strains demonstrated detectable antimicrobial activity under standard in vitro conditions. Culture supernatants exhibited pronounced antioxidant activity (73.38–90.34% DPPH radical scavenging), indicating the production of extracellular bioactive metabolites. The results demonstrate functional divergence within the Bacillus genus between maximum glutenolytic capacity and probiotic safety. Species belonging to the Bacillus group, including Bacillus amyloliquefaciens, B. subtilis, and B. licheniformis, are widely recognized as safe for food and probiotic applications (GRAS and/or QPS when appropriately characterized at the strain level). Accordingly, while B. cereus strains represent promising sources of industrial gluten-degrading enzymes. B. amyloliquefaciens 6/4/2, together with selected B. subtilis and B. licheniformis strains, demonstrated the most favorable balance between glutenolytic activity and biosafety, making them the most promising candidates for further probiotic development.