Stress survival tests were performed as previously described (Lebeer et al., 2011 (link)). Briefly, simulated gastric juice was prepared using 3.5 g/l glucose, 2.05 g/l NaCl, 0.60 g/l KH2PO4, 0.11 g/l CaCl2, and 0.37 g/l KCl, adjusted to pH 2.0 using 1.0 M HCl, and autoclaved at 121°C for 15 min (Corcoran et al., 2005 (link)). Subsequently, 0.05 g/l porcine bile (Sigma-Aldrich), 0.1 g/l lysozyme (Sigma-Aldrich), and 13.3 mg/l pepsin (Sigma-Aldrich) were added as stock solutions prior to analysis.
For the stress survival assays, 108 CFU/ml (based on estimations via optical density at 600 nm) cells were resuspended in the appropriate volume of either simulated gastric juice or 0.1% vol/vol H2O2. Suspensions were incubated at 37°C for 90 min with constant stirring. The percentage of survivals was calculated by comparing the exact colony forming units (plate counts) before and after addition to simulated gastric juice or 0.1% H2O2. Phosphate-buffered saline (PBS) at pH 7.4 was used as baseline (negative control). Each strain and/or condition was tested threefold, and each experiment was performed at least in triplicate.
For the stress survival assays, 108 CFU/ml (based on estimations via optical density at 600 nm) cells were resuspended in the appropriate volume of either simulated gastric juice or 0.1% vol/vol H2O2. Suspensions were incubated at 37°C for 90 min with constant stirring. The percentage of survivals was calculated by comparing the exact colony forming units (plate counts) before and after addition to simulated gastric juice or 0.1% H2O2. Phosphate-buffered saline (PBS) at pH 7.4 was used as baseline (negative control). Each strain and/or condition was tested threefold, and each experiment was performed at least in triplicate.
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