Porcine bile extract

The ingredients used to prepare the model systems were dried blackcurrant (Ribes nigrum) pomace (BCP), supplied by the Institute of Natural Materials Technology (Technische Universität Dresden, Germany) and prepared by drying the fresh pomace at 70 °C for 2 h and milling it in a ZM 100 ultracentrifuge mill (Retsch GmbH, Haan, Germany) at 14,000 rpm using a 1 mm sieve [9 (link)] (Reißner et al., 2019). The blackcurrant extract (BCE) was prepared from BCP, olive oil (Consum, Valencia, Spain), native wheat starch (C*Gel, Cargill BV, Amsterdam, Netherlands), and whey protein isolate from milk (Harrison Sport Nutrition S.L, Granada, Spain). All the enzymes (α-amylase from porcine pancreas, pepsin from porcine gastric mucosa, porcine bile extract, lipase from porcine pancreas, and pancreatin from porcine pancreas) used in the in vitro digestion analysis were supplied by Sigma-Aldrich (Barcelona, Spain).

Media to be used for anaerobic cultivation were prepared at least 2 days prior to use. Briefly, TSB was adjusted to pH 7.5, 6.5, 5.5 or 4.5, autoclaved and immediately placed in a Coy Anaerobic Airlock chamber (gas mix 95 % N₂/5 % H₂). Anaerobic conditions were monitored throughout the duration of the study with an oxygen monitor and also with a resazurin control sample.
Isolates from freshly streaked cultures were used to inoculate 5 ml TSB for cultivation at 16 h at 37 °C. Overnight cultures (1 %) were used to inoculate the TSB previously prepared and then were subsequently supplemented with either 0 or 1 % porcine bile extract (Sigma). Cultures were incubated at 37 °C within the anaerobic chamber for 16 h, after which aliquots (0.1 ml) were removed, serially diluted in PBS and plated onto TSA. Plates were incubated anaerobically for 24 h at 37 °C. Per cent survival was determined for each replication based on TSB media only controls. Prism (GraphPad) was used to analyse the differences between the survival of strains in bile under aerobic or anaerobic conditions using an unpaired t-test, with P < 0.05 declared as significant. At least three independent replicates were analysed.

Probiotic Lactobacillus acidophilus (ATCC‐ 4356) was obtained from NIFSAT, University of Agriculture Faisalabad Pakistan. Milk was purchased from local dairy farm. Food additives, media, wall materials (sodium alginate and soy protein isolate), ringer solution, sodium chloride, hydrochloric acid, distilled water, calcium chloride, and porcine bile extract (Sigma‐Aldrich) were purchased from local scientific market.

β-CD was from Roquette Frères (Lestrem, France). Hydroxytyrosol was a generous gift from Pr Visioli (Madrid, Spain). Gallic acid, tyrosol, caffeic acid, p-coumaric acid, pepsin, porcine pancreatin, porcine bile extract, formic acid, water, ethanol, and acetonitrile were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, non-essential amino-acids, penicillin/streptomycin, trypsin-EDTA (500 mg/L and 200 mg/L, respectively), phosphate-buffered saline (PBS), and Hanks’ balanced salt solution (HBSS) were purchased from Life Technologies (Illkirch, France). Fetal bovine serum was from PAA (Vélizy Villacoublay, France). Alperujo was collected from a two-phase centrifuge mill (Moulin Castelas, Baux-de-Provence, France). Foods were purchased from a local Casino supermarket (Marseille, France).

Levosulpiride (LSP) was a generous gift from Bio-Labs Pvt. Ltd. (Islamabad, Pakistan). Precirol^® ATO 5 and Labrasol^® were gifted by Gattefossé (Saint-Priest, France). Tween 80, Span 80, oleic acid, Poloxamer 407, porcine bile extract, pepsin and lipase were purchased from Sigma Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and used without further purification.

The enzymes used during the in vitro digestion process were all purchased from Sigma-Aldrich (St. Louis, MO, USA) and were α-amylase from hog pancreas (≥50 units/mg protein) for the oral phase, pepsin from porcine gastric mucosa (≥400 units/mg protein) for the gastric phase, and pancreatin from porcine and pancreas (8× USP) for the intestinal phase. Furthermore, bile acids were provided during the intestinal phase with the use of porcine bile extract (Sigma-Aldrich, St. Louis, MO, USA).

The analysis of resistance to different bile salts was performed comparing the two human gallbladder bile isolates IPLA60001 and IPLA60002 with the R. gauvreauii type strain DSM-19829. Comparisons were also made with R. bromii strains from the Rowett Institute (L2-63 and 5AMG), the results of which have been described previously (7 ). The analysis was carried out in triplicate by determining the MICs on M2GSC 2% agar plates supplemented with 30% CBRF and different concentrations (ranging from 0% to 12% wt/vol) of CA, TDC GC, TC, GDC, Porcine Bile Extract, Bovine Bile (purchased from Sigma-Aldrich), and Mixed Bile Salts (Oxoid, Waltham, MA). The plates were incubated at 37°C in the anaerobic cabinet for 48 h.