Optimal combination of multiple cryoprotectants and freezing-thawing conditions for high lactobacilli survival rate during freezing and frozen storage

The optimal cryoprotectants combination, freezing rate and thawing temperature were investigated for preserving Lactobacillus plantarum and Lactobacillus casei during freezing and frozen storage. It is indicated that, among all cryoprotectants applied singly, 150 g/L and 100 g/L sucrose provided the best protective effect for L. plantarum and L. casei, respectively (survival rate = 74.4% and 84.8%, respectively). By using orthogonal experimental design for cryoprotectant mixtures, the mixture of trehalose, sucrose, glycerol and skimmed milk at final concentration of 12.5, 12.5, 37.5 and 25.0 g/L, respectively, appeared to enhance significantly the survival rate of L. plantarum (92.8%) and L. casei (91.2%). Among all freezing and thawing conditions examined, the survival rate of L. plantarum was the highest at a freezing rate of −10 °C/min and thawing temperature of 0 °C; and that of L. casei, at a freezing rate of −1 °C/min and thawing temperature of 0 °C. The cryoprotectant effect exerted by keeping the integrity of cell membrane and therefore maintaining the lactobacilli survival.