Porcine bile

Once mid-log phase was reached, cells were immediately challenged with simulated gallbladder bile. The in vitro bile fluid was prepared as previously described (Versantvoort et al., 2005 (link)). Briefly, 0.5 ml of 2 × 6% porcine bile (Sigma, United States), pH 8.2 ± 0.2 (30 ml of 175.3 g/l NaCl, 68.3 ml of 84.7 g/l NaHCO₃, 4.2 ml of 89.6 g/l KCl, 150 μl of 37% g/g HCl, 10 ml of 25 g/l urea, 10 ml of 22.2 g/l CaCl₂.2H2O, and 1.8 g BSA to 500 ml distilled water) was added to 0.5 ml of mid-log phase culture. The challenged cultures were incubated at 37°C with shaking for 10 and 20 min, respectively. Survival ability was determined at 10 and 20 min after stress (t = 10 and t = 20). A 100 μl aliquot of the pre-treated control, untreated control (culture with additional 20 min incubation) along with t = 10 and t = 20 cultures were ten-fold serially diluted in PBS (pH 7.4); 10 μl of each dilution was plated onto BHI agar plates for subsequent enumeration. Experiments were conducted in triplicate on separate days.

All bacterial strains used in this study are listed in Table 3. Bifidobacterial strains were routinely cultured in reinforced clostridial medium pH 6.8 (RCM, Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) or reinforced clostridial agar (RCA, Oxoid Ltd.). RNAseq experiments were carried out using cultures that had been grown in filtered RCM (fRCM). All bifidobacterial strains were grown anaerobically in a modular atmosphere controlled system (Davidson and Hardy, Belfast, Ireland). Where required, media was supplemented with tetracycline (Tet, 10 µg ml⁻¹) or porcine bile, 0.5% (w/v) or 1% (w/v) (Sigma- Aldrich, Steinheim, Germany). Individual bile salts were purchased from Sigma-Aldrich.Table 3

Strains and plasmids used in this work.

Bacterial strain/plasmid	Features	References
Bifidobacterium breve
UCC2003		48 (link)
UCC2003::Bbr_430	Insertional mutant in Bbr_430 gene of the EPS cluster	51 (link)
UCC2003-luxS	Insertion mutant in luxS – (Bbr_0541)	43 (link)
JCM 7017
JCM 7019
NCTC 11815
Bifidobacterium longum subsp. longum
NCIMB 8809
CCUG 30698
Bifidobacterium longum subsp. infantis
ATTC 15697
Bifidobacterium dentium
DSM 20436
Bifidobacterium adolescentis
DSM 20083
Bifidobacterium pseudolongum
DSM 20438

S. epidermidis DSM 20042, DSM 28764, 9^c and F530B were grown in triplicate in 20 ml of BHI supplemented with 0.3% v/v porcine bile (Sigma, United Kingdom). One milliliter of each culture was collected at 24 and 48 h and kept at −20°C for further analysis. Solid phase extraction (SPE) clean-up was performed using Waters Oasis Prime HLB 1 30 mg SPE cartridges in a SPE vacuum system and washed with 1 ml of 5% methanol. Elution was performed with 500 μl 100% methanol using the same procedure and 25 μl of internal standards for each BA (Steraloids, United States) were added. The final volume was transferred to low volume autosampler tubes for liquid chromatography-mass spectrometry (LC-MS) analysis.

Strains (grown to OD₆₀₀ = 0.5) were assessed for their ability to survive 0.2% porcine bile (Sigma, St. Louis, MO) in 0.85% NaCl for 1 hr at room temperature. Pond water survival assays were done as described previously (Kamp et al., 2013 (link)) at 30°C for 48 hr. Competition experiments were performed between the strain of interest (lacZ⁺) and the appropriate control strain (ΔlacZ), and outputs were plated on LB agar plates containing 100 µg/ml Sm and 40 µg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal).

Whole wheat flours were subjected to simulated gastrointestinal digestion [15 (link)] as follows: (i) 1 g of flour was incubated (2 min; 37 °C under constant gentle mixing) with 1 mL simulated saliva containing porcine amylase (Sigma-Aldrich, St. Louis, MI, USA; 75 U/mL of digesta); (ii) 2 mL simulated gastric juice containing porcine pepsin (Sigma-Aldrich, St. Louis, MI, USA; 2000 U/mL of digesta) was added and incubated (2 h; 37 °C under constant gentle mixing) after pH adjustment to 3; (iii) 4 mL duodenal juice containing porcine pancreatin (SigmaAldrich, St. Louis, MI, USA; 100 U trypsin activity/mL of digesta) and porcine bile (Sigma-Aldrich, St. Louis, MI, USA; 10 mmol/L in the total volume) was added and incubated (2 h; 37 °C under constant gentle mixing) after pH adjustment to 7. Afterward, to inactivate the enzymes, the sample was boiled for 10 min at 95 °C. After centrifugation (3220 g, 4 °C, 45 min), the supernatant (295 μL) was added to 5 μL of internal standard solution (TQQPQQPF(d5)PQQPQQPF(d5)PQ; 1.6 mM); samples were then subjected to reverse phase liquid chromatography coupled to mass spectrometry (RP-UPLC/ESI-MS) to quantify peptides associated with CD [20 (link)]. All the samples were digested in duplicate.

A commercially available, food grade Plantago psyllium husk, sodium alginate (pure (>99%) food grade, viscosity 15–25 cp), porcine bile, xanthan gum (XG, viscosity; 1785 cps), citrus pectin with a high degree of esterification (53%), sodium acetate (≥99%), sodium citrate (99.5), sodium chloride (≥99%), potassium phosphate (≥98%), acetate buffer (pH 5.2 ± 0.1 (25 °C)), α-Amylase from porcine pancreas, potassium chloride (≥99%), pancreatin from porcine pancreas (8 × USP), pepsin (powder, ≥250 units/mg solid), magnesium chloride (anhydrous, ≥98%), bovine bile (dried, unfractionated, NA.85), HCl (ACS reagent, 37%), ferric chloride (ACS reagent, 37%), ethanol (95%), calcium chloride (99%), ammonium carbonate (ACS reagent, ≥30.0% NH3 basis), were procured from Sigma Aldrich (Johannesburg, South Africa). All other reagents used were of analytical grade purity.

Bacterial growth was assessed under various stress culture conditions (Khaleghi et al., 2010 (link); Grosu-Tudor et al., 2016 (link)). Overnight L. acidophilus cultures were used to inoculate 96-well microplates (Corning Costar, Corning, NY) containing 200 μl of MRS broth or MRS broth supplemented with 2.5% (w/v) NaCl (Fisher Scientific, Hampton, NH, United States), 0.2% (w/v) porcine bile (Sigma) or 0.5% (w/v) oxgall (Difco). Plates were sealed with clear adhesive film then incubated at 37°C in a Fluostar Optima microplate reader (BMG Labtech, Cary, NC, United States). The culture turbidity was recorded at OD₆₀₀ every hour for 30 h. To validate results obtained with the plate reader, strains grown in MRS and MRS containing 2.5% NaCl were also enumerated on MRS solid media. Overnight bacterial cultures were used to inoculate broth treatments. Aliquots were taken at 0, 4, 6, 8, 10, and 24 h and plated on MRS agar to obtain viable cell counts.