Isolation and Probiotic Characterization of Bacillus stercoris VR19 from the polyherbal Ayurvedic fermented formulation Madhookasavam

Abstract Probiotics are live microorganisms that provide health benefits to the host when administered in adequate amounts. In this study, bacterial isolates from Madhookasavam, a polyherbal Ayurvedic fermented formulation, were screened to identify potential probiotic candidates. Preliminary safety assessment included Gram staining, hemolysis testing, and antibiotic susceptibility profiling. Among the isolates, VR19 was the only strain to exhibit γ-hemolysis, indicating a non-hemolytic phenotype, and was susceptible to several clinically relevant antibiotics, including Chloramphenicol and azithromycin. The isolate VR19 demonstrated tolerance to acidic conditions and simulated gastrointestinal stresses, including pepsin (0.1–1%), bile salts (0.1–3%), and NaCl (1–10%). Functional characterization revealed cellulase activity of up to 1.95 U/mL, corresponding to the release of 0.959 mg/mL reducing sugars. The isolate also exhibited concentration-dependent antioxidant activity, reaching a maximum DPPH radical-scavenging activity of 72.9%. Probiotic performance was further evaluated using Caenorhabditis elegans as an in vivo model. VR19 showed effective intestinal colonization and persistence, as demonstrated by bacterial enumeration and Gram staining of colonies recovered from the nematode intestine. Molecular identification based on, 16S rRNA gene sequencing, and phylogenetic analysis identified VR19 as Bacillus stercoris . Collectively, these findings show that Bacillus stercoris VR19 possesses promising probiotic properties, including a non-hemolytic phenotype, gastrointestinal stress tolerance, cellulolytic and antioxidant activities, and effective intestinal colonization. The strain therefore warrants further safety and efficacy evaluation for potential therapeutic and functional-food applications.