Cold stress improves the ability of Lactobacillus plantarum L67 to survive freezing

• Applying cold stress to L. plantarum L67 increases transcription levels of two genes (cspP and cspL) and thirteen proteins.
• Lactobacillus plantarum L67 that have been through cold stress have improved survival rates under freeze-thawing conditions and long-term storage.
• Thus the application of these proteins helps enhance the viability of dairy starter culture.

The stress resistance of bacteria is affected by the physiological status of the bacterial cell and environmental factors such as pH, salts and temperature. In this study, we report on the stress response of Lactobacillus plantarum L67 after four consecutive freeze–thaw cycles. The cold stress response of the cold-shock protein genes (cspC, cspL and cspP) and ATPase activities were then evaluated. The cold stress was adjusted to 5 °C when the bacteria were growing at the mid-exponential phase. A comparative proteomic analysis was performed with two-dimensional gel electrophoresis (2D SDS-PAGE) and a matrix assisted laser desorption/ionization-mass spectrometer. Only 56% of the L. plantarum L67 cells without prior exposure to cold stress survived after four consecutive freeze–thaw cycles. However, 78% of the L. plantarum L67 cells that were treated with cold stress at 5 °C for 6 h survived after freeze–thaw conditions. After applying cold stress to the culture for 6 h, the cells were then stored for 60 days at 5 °C, 25 °C and 35 °C separately. The cold-stressed culture of L. plantarum L67 showed an 8% higher viability than the control culture. After applying cold stress for 6 h, the transcript levels of two genes (cspP and cspL) were up-regulated 1.4 (cspP) and 1.2 (cspL) times compared to the control. However, cspC was not up-regulated. A proteomic analysis showed that the proteins increased after a reduction of the incubation temperature to 5 °C. The importance of the expression of 13 other relevant proteins was also determined through the study. The exposure of L. plantarum cells to low temperatures aids their ability to survive through subsequent freeze–thaw processes and lyophilization.