S9888

During the experiment, simulated gastric juices of the following composition (g L⁻¹) were used: 3.50 dextrose (361105; Roqquette, Lestrem, France); 2.05 sodium chloride (S9888; Sigma-Aldrich, Munich, Germany); 0.60 potassium dihydrogen phosphate (A1043; PanReac Applichem, Barcelona, Spain); 0.11 calcium chloride (131232; PanReac Applichem, Barcelona, Spain); and 0.37 potassium chloride (191494; PanReac Applichem, Barcelona, Spain), pH 2.0. The solution was autoclaved at 115 °C for 20 min. The following components were sterilized by filtration (0.2 μm, Millipore, St. Louis, MO, USA) and added to the solution after cooling: 0.05 g L⁻¹ pork bile (48305; Sigma, St. Louis, MO, USA); 0.10 g L⁻¹ lysozyme (CAS Number 9066-59-5; Caglificio Clerici S.p.A., Cadorago, Italy), and 13.30 mg L⁻¹ pepsin (12762; JSC “Plant of Endocrine Enzymes”, Moscow, Russia) [23 (link)].

During the experiment, a medium [26 (link)] of the following composition was used: 30 mL of sodium chloride (S9888; Sigma-Aldrich, Munich, Germany), 175.3 g L⁻¹ solution; 68.3 mL of sodium hydrogen carbonate (131638; PanReac Applichem, Barcelona, Spain), 84.7 g L⁻¹ solution; 4.2 mL of potassium chloride (191494; PanReac Applichem, Barcelona, Spain), 89.6 g L⁻¹ solution; 200 mL of hydrochloric acid (170266; LenReaktiv, Moscow, Russia), 37% w/v solution; 10 mL of urea (U5378; Sigma-Aldrich, Munich, Germany), 25 g L⁻¹ solution; 10 mL of calcium chloride dihydrate (121214; PanReac Applichem, Barcelona, Spain), 22.2 g L⁻¹ solution; albumin (CAS Number 9048-46-8; “Sonac Loenen BV,” Netherlands), 1.8 g L⁻¹; and pork bile (48305; Sigma, St. Louis, MO, USA), 6.0 g L⁻¹, pH 8.0.

LDL (1.019 < d < 1.063), HDL₂ (1.063 < d < 1.125), and HDL₃ (1.125 < d < 1.225) were isolated from the sera of young and healthy human males (mean age, 22 ± 2 years, n = 20), who voluntarily donated blood after fasting overnight via sequential ultracentrifugation; the density was adjusted appropriately by adding NaCl (Sigma # S9888)and NaBr (Sigma # 310506), as detailed elsewhere [39 (link)], and procedures were carried out in accordance with standard protocols [40 (link)]. The samples were centrifuged for 24 h at 10 °C at 100,000× g using a Himac CP-100NXα (Hitachi, Tokyo, Japan) at the LipoLab of Yeungnam University. After centrifugation, each lipoprotein sample was dialyzed extensively against Tris-buffered saline (TBS; 10 mM Tris-HCl, 140 mM NaCl, and 5 mM EDTA (pH 8.0)) for 24 h to remove NaBr.

Senescence was determined by measuring the senescence-associated β-galactosidase (SA-β-Gal) activity. Briefly, cells were fixed for 3–5 min at room temperature in 3% formaldehyde and incubated with fresh SA-β-Gal staining at 37°C for 3 h. The staining solution included 1 mg/mL of 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (B4252, Sigma-Aldrich), 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide (P3289, Sigma), 5 mM potassium ferricyanide (P4066, Sigma), 150 mM NaCl (S9888, Sigma), and 2 mM MgCl₂ (M9272, Sigma). The percentage of senescent cells was calculated by the number of β-galactosidase positive cells (blue) out of a total number of cells per field. Images were acquired with an IX51 Olympus inverted fluorescent microscope.

The cultures were centrifuged (Eppendorf, Hamburg, Germany Centrifuge 5417C, 7000 rpm, 15 min) after 24 h of growth (daily culture) and washed once in a 1:1 v/v ratio with a Ringer solution of the following composition (g L⁻¹): 8.69 sodium chloride (S9888; Sigma-Aldrich, Munich, Germany); 0.30 potassium chloride (191494; PanReac Applichem, Barcelona, Spain); 0.48 calcium chloride (131232; PanReac Applichem, Barcelona, Spain), pH 6.4 [21 (link)]. The precipitate was resuspended in simulated gastric juices (see below) in a 1:1.33 ratio [22 ] and incubated at 37 °C. Sampling was carried out for 20, 40, 60, and 90 min of the experiment for bifidobacteria and for 30, 60, and 90 min for lactobacilli and the viable cells was counted. After 90 min, the samples were centrifuged, the supernatant was decanted, and a medium simulating the conditions of the duodenum (see below) was added in a 1:3 v/v ratio [22 ]. If the strain was found to completely lose viability in gastric juices, the simulated intestinal conditions test was repeated with fresh daily culture. Samples were taken at 60, 120, and 180 min.
The effect of the bile medium on the viability of bifidobacteria and lactobacilli was also studied [22 ]. The precipitates were obtained as described above and resuspended in a bile medium (1:1). The incubation was carried out at 37 °C with sampling at 60, 120, and 180 min.

All strains were cultivated on trypticase soy agar and broth (236920 and 211825, BD, France) at 37 °C, except Acinetobacter baumannii, which was grown at 22 °C, as previously described [7 (link)]. The bacteria were grown on the plate and then a single colony was transferred to a second plate. The inoculum was prepared from isolates of the second agar plate in TPS solution (Tryptone 0.1% (211705, BD, Le Pont-de-Claix, France) and sodium chloride 0.85% (S9888, Sigma Aldrich, Saint-Quentin-Fallavier, France)), and was then diluted in adequate broth at 5 × 10⁵–1 × 10⁶ CFU/mL.