Taurodeoxycholic acid

Gallic acid, rat intestinal acetone powder, porcine pancreatic α-amylase, 4-methylumbelliferone, glucose oxidase kits and 3,5-dinitrosalicylic acid p-nitrophenylbutylrate (p-NPB), oleic acid, phosphatidylcholine, glycodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, porcine cholesterol esterase, porcine pancreatic lipase were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cholesterol test kits were purchased from HUMAN GmbH Co. (Wiesbaden, Germany). Total bile acid kit was purchased from Bio-Quant Co. (San Diego, CA, USA). All other chemical reagents used in this study were of analytical grade.

Cecal contents (5 controls and 8 from L. paracasei treated mice) from trial 3 were used to quantify primary and secondary bile acids. Bile acids analysis was performed at Bioaster (Lyon, France). Samples were prepared from 50 to 60 mg of −80 °C frozen cecal content using aqueous methanol extraction process. Bile acids extracts were analysed by LC-MS/MS on a triple quadrupole Thermo Quantum Ultra (SN: TQU00665) combined to a Dionex Ultimate 3000 HPLC system (SN: 8074045 & 8087183). Samples were separated on a C18 column (2,7 μM, 150 × 2,1 mm) from Ascentis Express using a methanol/water gradient containing 5 mM ammonium acetate and 0,012% formic acid. All bile acid standards: LithoCholic acid, ChenoDeoxyCholic acid, DeoxyCholic acid, UrsoDeoxyCholic acid, Cholic acid, GlycoDeoxyCholic acid, GlycoursoDeoxyCholic acid, GlyChenoDeoxyCholic acid, Glychocolic acid, TauroLithoCholic acid, TauroDeoxyCholic acid, TauroCholic acid, were bought from Sigma Aldrich. Results of bile salt quantification were expressed in peak area, corrected for sample weight.

BAM8-22 was purchased from Tocris (Bristol, UK) and dissolved in 1× phosphate buffered saline (PBS). Cholic acid (CA), tauro-Cholic acid (TCA), glycol-Cholic acid (GCA), chenodeoxyCholic acid (CDCA), tauro-chenodeoxyCholic acid (TCDCA), glycol-chenodeoxyCholic acid (GCDCA), deoxyCholic acid (DCA), tauro-deoxyCholic acid (TDCA), lithoCholic acid (LCA), and tauro-lithoCholic acid (TLCA) were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Protease inhibitor cocktail (PIC) was purchased from Bio Vision (Milpitas, CA 95035 USA).

To determine Bifidobacterium survival in bile, isolates were first grown in RCM and then subcultured using a 1:50 dilution into MRS ± 0.3% unfractionated bovine bile salt (Sigma-Aldrich), as described by [57 (link)]. After 48 h of stationary growth in an anaerobic chamber at 37 °C OD_600 nm using the Benchmark Plus microplate spectrophotometer (Bio-Rad) for both conditions. For the MRS plate the mean blank OD_600 nmvalue was 0.1585 and for the anaerobic plate it was 0.1825. Data shown are mean values from three experimental repeats.To assess bile salt hydrolyase activity, overnight cultures were spotted (3 mL) onto MRS plates supplemented with l-cysteine and 0.5% w/v of either taurocholic acid, taurodeoxycholic acid, and sodium glycodeoxycholate bile salt (Sigma-Aldrich). Bile salt precipitation was assessed after a maximum of 96 h of anaerobic incubation at 37 °C. For both assays, uninoculated MRS media were used as a control.

Bacterial cells from overnight (18 h) cultures were harvested (10,000 x g, 5 min, 4 °C), washed twice with PBS buffer (pH 7.2), before inoculating in PBS solution (pH 8.0), containing 0.3 %, 0.5 %, 1 % and 2 % (w/v) bile salt (Oxgall, Difco). Resistance was assessed in triplicates in terms of viable colony counts and enumerated after incubation at 37 °C for 4 h.
For the determination of bile salt hydrolysis (BSH), fresh bacterial cultures were streaked in triplicates on MRS agar containing 0.5 % (w/v) taurodeoxycholic acid (Sigma). The hydrolysis effect was indicated by different colony morphology from the control MRS plates, after 48 h of anaerobic incubation at 37 °C.