Survival Kinetics of Strains in Simulated Gastric Juice

The survival kinetics of the strains in simulated gastric juice (SGJ) were measured during 2 h of incubation, as previously described by [29 (link)]. The SGF was prepared with KCL 6.9 mM, HCl 15.6 mM, KH₂PO₄ 0.9 mM, NaHCO₃ 25 mM, NaCl 47.2 mM, MgCl₂ 0.1 mM, (NH₄)₂CO₃ 0.5 mM (all from Sigma-Aldrich, Saint Quentin Fallavier, France) and sterilized through filtration. The pH was then adjusted to 3 using HCl 1 M, and CaCl₂ and porcine pepsin (Sigma-Aldrich, Saint Quentin Fallavier, France) were added to achieve a final concentration of 0.075 mM and 2.000 U/mL, respectively, in the final digestion mixture. The bacterial suspension was centrifugated (2000 rpm, 10 min), washed twice with PBS, and standardized at 10⁹ CFU/mL. Then, 950 µL of SGF (with pepsin and at pH 3) was inoculated with 50 µL of the bacterial suspension to reach a final concentration of 5 × 10⁷ CFU/mL and incubated at 37 °C for 2 h with sampling at 0 and 120 min. Bacterial viability was measured by the numeration of a serial dilution plated on BBA plates after 48 h incubation in anaerobiosis. The death rate was calculated by dividing the number of CFU/mL at 120 min by the CFU/mL measured at time zero, as the 14 studied strains have been previously shown to survive under aerobic conditions without any indication of mortality for 6 h [26 (link)].