Abstract

Bile salt hydrolase (BSH)-producing probiotics can assimilate cholesterol from the body through de novo synthesis. The BSH enzyme was found in 23 of 513 isolates accessed from various sources. Five of the 23 BSH-positive strains have been selected for further study, based on their BSH activity, compared to two positive controls, Lactobacillus acidophilus and Enterococcus lactis. The Grams nature of the strains was determined and further examined for hemolytic activity, gelatinase, and catalase assay as per Indian Council for Medical Research—Department of Biotechnology recommendations. Two Enterococcus faecalis (CGz3 and CGz4) strains with γ-hemolytic, negative catalase, and gelatinase activity are selected for probiotic characterization, evaluating the organisms surface hydrophobicity, autoaggregation tests, tolerance to lysozyme, gastric acidity, bile salt and gastric juices (pepsin and pancreatin). The strain which withstands the harsh gastrointestinal conditions was considered for further experiments. To establish a standardized method to quantify the BSH activity of the potential probiotic isolate, substrate utilization was performed by screening sodium glycocholate (GCA) and taurocholic acid (TCA) at different concentrations. The optimal BSH activity was observed at the 16th hour and 0.1% (v/v) GCA. Based on the standardized protocol, factorial optimization of process parameters, such as pH, inoculum percentage, temperature, and revolutions per minute (RPM) was carried out for increased BSH activity. The optimal BSH activity was observed at pH 5.5 and 1% inoculum (v/v). The highest BSH activity was obtained at 40°C and 200 RPM. Among the other BSH-positive strains, E. faecalis CGz3 shows the best probiotic potential. The strain would be further studied for its ability to alleviate symptoms associated with non-alcoholic fatty liver disease (NAFLD), using a cell line-based study and associated gene regulation. In conclusion, E. faecalis CGz3 would have the potential to be used as a dietary supplement to treat metabolic disorders, such as hypercholesterolemia and NAFLD/metabolic-associated fatty liver disease.

Key words: Bile Salt Hydrolase, cholesterol assimilation, probiotics, process parameter optimisation and thin-layer chromatography (TLC).