Assessment of Phenotypical Changes

In order to assess improved thermal capacity, cell viability at 55–75°C was assessed. Cells in the mid-exponential phase were prepared to OD₆₀₀ at 1.3, corresponding to approximately 1 × 10⁹ CFU/ml. A hundred microliters of cells were injected into 900 μl of pre-heated saline by heat block. After heat was applied for 1 min, cells were plated on MRS agar to quantify the viable cell count. Thermal resistance was presented as the survival ratio. To check other phenotypical changes induced by heat stress, acid and salinity tolerance were measured. Inoculated cells were harvested by centrifugation at 12,000 rpm for 10 min at 4°C. After the supernatant was removed, the pellet was washed two times with 1X phosphate buffer (1X PBS). It was resuspended into 1 ml of PBS buffer adjusted to acidified solutions (up to pH 2.0 and 7.0) or salted solutions (7.5 and 15.0%). Cells were exposed to the acidic solution for 2.5 h and the high salt solution for 3.5 h at 37°C. The viable cell count was measured in order to assess cell survival, and it was calculated by the same method as thermal resistance.