Bile salt

The acid and bile tolerance of B. coagulans B37 and B. pumilus B9 strains were assessed as per the methods described previously [20 ,21 ]. Different low pH solutions were prepared to stimulate gastric acidic conditions by adding reagent grade HCl (35.8%) in distilled water drop by drop in a beaker and solutions having pH of 1.0, 2.0, and 3.0 were obtained. Sterile distilled water (pH adjusted to 6.5) was served as control. High bile salt solutions (1.0% and 2.0%) were prepared by dissolving bile salts (Hi-media, India) in distilled water. 1 ml of fresh culture containing approximately 10⁷-10⁸ cfu/ml was added to the different pH solutions (1.0, 2.0, 3.0, and 6.5) as well as bile solutions (1.0% and 2.0%) and mixed thoroughly. 1 ml of each solution was taken from each tube immediately (0 h) and 10-fold serial dilutions were prepared in 0.1% peptone water. Pour plating was done on BC agar media. The inoculated pH solutions were incubated at 37°C for 3 h, and 1 ml of culture was taken hourly from each tube after an interval of 1, 2, and 3 h of incubation followed by plating. The bile solutions containing cultures were incubated at 37°C for 12 h, and 1 ml of bile solution containing culture was taken from each tube after 1, 3, and 12 h of incubation and plated on BCA. All plates were incubated at 37°Cfor 24-72 h and the cfu were counted.

All chemicals used for the analysis of simulated salivary fluids, simulated gastric fluids and simulated intestinal fluids were of analytical grade and were made available from RCI Labscan (Bangkok, Thailand). Salivary α-amylase and pancreatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bile salts, hemin, vitamin K1, cysteine HCl and the appropriate media were purchased from HiMedia (Nashik, India). Standard short chain fatty acids (SCFA), including propionic acid, butyric acid, acetic acid and lactic acid, were of HPLC grade and were obtained from RCI Labscan.

The acid and bile tolerance of YB1701 was determined as described by Duc et al. 2004. [42 (link)] with some modifications. Different pH solutions (2.0, 3.0, and 6.5) were prepared to stimulate gastric acidic conditions by adding reagent grade HCl (35.8%) in LB medium and supplemented with pepsin from porcine gastric mucosa (Sigma-Aldric P7000, Buchs, Switzerland) at 1 mg/mL. High bile salt solutions (1.0% and 2.0%) were prepared by dissolving bile salts (Hi-media, Mumbai, India) in LB medium. One milliliter of fresh culture containing approximately 10⁷–10⁸ CFU/mL was added to the different pH solutions as well as the bile solutions and was mixed thoroughly. Cultures were then incubated at 37 °C with agitation and aliquots were removed after 30, 60 and 90 min for acid tolerance, and after 1 and 3 h for bile salt tolerance. Serial dilution was done in sterile saline (0.9% w/v) and the viable count was enumerated by plating on an LB agar plate. Bacterial cell survival was calculated as follows: NA/NB × 100%, where NA = log₁₀ CFU/mL after incubation and NB = log₁₀ CFU/mL before incubation.

MRS medium with 0.15 and 1.0% (w/v) bile salts was used (HiMedia, Mumbai, India). A total of 20 µL of 0.1 OD₆₀₀ adjusted culture was inoculated into 180 µL of MRS broth with bile salts and oxyresveratrol at specific MIC concentrations in 96-well plates and incubated at 37 °C. Viable counts after exposure to 3 h (log CFU/mL) were determined [20 (link),21 (link)].