Cross-protection studies.
Wild-type L. plantarum WCFS1 was grown in MRS in the absence or presence of ethanol 8% (vol/vol) until the OD600 was 1.0 at 20°C. Cells were washed in PBS before exposure to various stresses. For all stress tolerance assays, serial dilutions of the samples were prepared immediately after stress exposure and these serial dilutions were immediately plated on MRS agar. Plates were incubated at 30°C for 2 days for CFU enumeration according to the technique described by Sieuwerts et al. (46). Oxidative stress tolerance was determined upon suspending the L. plantarum cells in PBS containing 40 mM hydrogen peroxide, a concentration which is lethal to L. plantarum WCFS1 (52). Cells were collected every 5 min for 60 min for CFU enumeration. To quantify L. plantarum survival at low pH, cells grown in MRS or MRS with 8% (vol/vol) ethanol were suspended in PBS with an adjusted pH of 2.4 (acidified by 5 M HCl) and subsequently sampled at 5-min intervals, followed by assessment of the amounts of viable cells as described above.