Survival and Germination of Bacillus clausii UBBC07 Spores in in vitro Human Gastrointestinal Tract Simulation Model and Evaluation of Clausin Production

Abstract

Bacillus clausii UBBC07 is a commercial spore probiotic known to reduce diarrhea in children and adults. In the present study, survival and germination of UBBC07 spores were investigated under fed and fasted conditions in Simulator of Human Intestinal Microbial Ecosystem. Besides this, lantibiotic production, purification, and characterization were performed. The agar plate analysis showed that spores were 100% tolerant to fed and fasted gastrointestinal tract (GIT) conditions. Simultaneously, flow cytometry revealed that at the end of small intestinal incubation, 120% (fed) and 133% (fasted) spores were in viable germinating state. The transformation of viable germinating spores into viable vegetative cells was observed at 3 h of incubation under fasted GIT conditions. In antimicrobial evaluation, UBBC07 produced low-molecularweight (2107.94 Da) class I lantibiotic clausin. The presence of lanB, lanC, and lanD genes confirms the clausin production. Clausin is stable at proteases (pepsin, proteinase K, and trypsin), temperature (up to 100 • C), and pH (up to 11). Furthermore, the antimicrobial activity toward Gram-positive bacteria including Clostridium difficile is advantageous. In conclusion, B. clausii UBBC07 spore probiotic is capable of surviving and germinating under in vitro upper GIT conditions. The clausin production justifies strain applicability in diarrhea.

A Review of the Effects and Production of Spore-Forming Probiotics for Poultry

“…It was shown in a study using an artificial intestine model that various probiotic Bacillus strains not only proliferated from spores in the intestine (up to 97% of the germination cell), but were also metabolically active under these conditions [54,55]. It has been noted that spore germination occurs at a high rate; for example, in the study by Latorre et al (2014), 90% of spores germinated in the small intestine of chickens within one hour [56].…”

Alkalihalobacillus clausii 088AE as a functional and medical food ingredient: assessment of in vitro protein digestibility and food calorie reduction

“…In a separate study on B . clausii UBBC07, the unchanged numbers of dormant spores (DS) were recorded in the fasting‐state (gastric) SHIME model that was converted into viable germinating spores (VGS) upon entering into the fed‐state intestinal phase (Ahire et al ., 2020). Upon germination, the spores of a probiotic species are converted into the metabolically active cells that can aid in digestion of carbohydrates and proteins by enriching the proteolytic microbial species and reprogramming the gut microbiota structure (Maity et al ., 2021).…”

“…Our finding was corroborated with the report of J€ ager et al (2018) who mentioned on the germination and activation of a spore-forming probiotic B. coagulans GBI-30 in the intestine and aids in the digestion of carbohydrates and proteins. In a separate study on B. clausii UBBC07, the unchanged numbers of dormant spores (DS) were recorded in the fasting-state (gastric) SHIME model that was converted into viable germinating spores (VGS) upon entering into the fed-state intestinal phase (Ahire et al, 2020). Upon germination, the spores of a probiotic species are converted into the metabolically active cells that can aid in digestion of carbohydrates and proteins by enriching the proteolytic microbial species and reprogramming the gut microbiota structure .…”

A tale of caution: prolonged Bacillus clausii bacteraemia after probiotic use in an immunocompetent child

“…The spores germinate into vegetative forms that colonize the small intestine and can persist in faeces for up to 12 days after administration [ 4 ]. In fasting conditions, spore germination can occur rapidly, within 3 h of incubation, producing persistent bacterial growth [ 5 ]. The spores are heat stable and can survive in environments with poor nutrition, dehydration and low pH [ 6 ].…”