Abstract Description

Bacillus paranthracis has recently gained attention for its metabolic flexibility and potential as a probiotic candidate. A key feature underlying probiotic functionality is the ability to utilize complex and non-digestible carbohydrates through carbohydrate-active enzymes (CAZymes). These enzymes enable the breakdown of dietary polysaccharides into simpler metabolites that can influence host health and microbial interactions. However, little is known about the substrate specificity and enzymatic activity of B. paranthracis. This study aimed to characterize carbohydrate utilization at both the genomic and biochemical levels to assess its potential probiotic functions. Genome annotation of B. paranthracis was conducted to identify CAZyme families associated with carbohydrate metabolism. To validate in silico predictions, in vitro biochemical assays were performed. The 3,5-dinitrosalicylic acid (DNS) assay was used to quantify reducing sugars released from selected substrates, including starch, cellulose, chitin, and glycogen. Cultures were incubated, supernatants harvested, and enzymatic activity measured at two-hour intervals up to 12 hours. Absorbance at 540 nm was recorded, and results were expressed as mean values of triplicate experiments. Genomic analysis revealed diverse CAZymes associated with glycoside hydrolase, glycosyltransferase, and carbohydrate-binding module families, suggesting broad carbohydrate metabolism potential. In vitro assays confirmed enzymatic activity against all tested substrates. Starch and cellulose showed the highest reducing sugar release, while glycogen and chitin demonstrated moderate but consistent activity. Activity generally increased with time, peaking between 8 and 12 hours of incubation. Negative controls showed negligible absorbance changes, confirming substrate-specific enzymatic hydrolysis. This study demonstrates that B. paranthracis possesses both genomic potential and measurable enzymatic activity for the breakdown of complex carbohydrates. These findings highlight its ability to metabolize dietary polysaccharides, supporting its relevance as a potential probiotic candidate. By combining genome-based predictions with biochemical validation, this work provides novel insights into the substrate specificity of B. paranthracis and lays the groundwork for future studies on its functional role in gut health and host–microbe interactions.