Evaluation of cholesterol-related functional traits of Bacillus infantis 63-11 under in vitro simulated small intestinal conditions and genomic analysis

Suwapat Saejie; Varapha Kongpensook; Nattida Chotechuang; Cheunjit Prakitchaiwattana

Authors

Suwapat Saejie Department of Food Technology, Faculty of Science, Chulalongkorn University, Phayathai Road, Wang Mai, Pathum Wan, Bangkok 10330, Thailand
Varapha Kongpensook Department of Food Technology, Faculty of Science, Chulalongkorn University, Phayathai Road, Wang Mai, Pathum Wan, Bangkok 10330, Thailand
Nattida Chotechuang Department of Food Technology, Faculty of Science, Chulalongkorn University, Phayathai Road, Wang Mai, Pathum Wan, Bangkok 10330, Thailand; Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), Paseo Miramon 182 C, 20014 Donostia-San Sebastian, Spain
Cheunjit Prakitchaiwattana Department of Food Technology, Faculty of Science, Chulalongkorn University, Phayathai Road, Wang Mai, Pathum Wan, Bangkok 10330, Thailand

Keywords:

Bacillus infantis, bile salt hydrolase, cholesterol reduction, whole-genome sequencing

Abstract

Bacillus infantis 63-11 is a microorganism isolated from traditional Thai fermented fish and shows potential for probiotic development. This study evaluated cholesterol-related functional traits of strain 63-11 together with genome-based taxonomic characterization and bile salt hydrolase-associated activity. Species identification was confirmed by 16S rRNA gene sequence analysis, which showed sequence similarity with reference strains of B. infantis ranging from 98.13–99.35%. Whole-genome analysis also supported the classification, with an average nucleotide identity (ANIb) of 96.34% and a digital DNA–DNA hybridization (dDDH) value of 86.0%, both above the commonly accepted threshold for species identification. Phylogenomic analysis further showed that strain 63-11 clusters within the B. infantis group. Genome annotation identified putative BSH homologs. Phenotypic testing using a taurodeoxycholic acid (TDCA) halo assay produced precipitation halos around the colonies, with an average diameter of 1.88 ± 0.48 mm, indicating the enzyme bile salt hydrolase (BSH) activity. In vitro analysis also showed that cholesterol levels decreased by 78.21% after 48 h of cultivation, suggesting a possible relationship with bile salt deconjugation. Under simulated small intestinal conditions containing 0.3% bile salts, strain 63-11 remained detectable during the experiment in both vegetative cells and spores, indicating adaptation to bile stress. Metabolite analysis using ¹H NMR at 48 h detected bile-related compounds. From this study, the persistence of strain 63-11 under simulated small intestinal conditions, together with its cholesterol reduction, suggests that BSH-related processes may contribute to the observed phenotype. However, further studies, especially in vivo experiments, are needed to confirm these findings and clarify their biological significance.

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