Study On The Physiology Mechanism Of Cell Damage Caused By Freeze-drying And Protection Strategies Of Lactobacillus

Lactic acid bacteria （LAB） play an important role in food production, and as starter cultures and probiotic product for the food fermentation industry. Freeze-drying is commonly used to preserve LAB, but it could cause cell damage and loss of viability. Over the years, attempts to improve the vitality of lactic acid bacteria during freeze-drying have been the focus of freeze drying study. Based on the related research of our group, the physiological changes of Lactobacillus and its potential mechanisms have been examined during freeze-drying process. Meanwhile some protection strategies were suggested to reduce the damage of LAB and protection mechanisms were further discussed in the paper.Taking Lactobacillus reuteri CICC6226, Lactobacillus plantarum ST-Ⅲand Lactobacillus bulgaricus as study objects, the survival rates and viability have been investigated in the presence of cryoprotectants after freeze-drying. Activities of sugar metabolic enzymes and changes of cell membrane have been determined immediately following the freeze-drying. Furthermore, the main factor leading to the inactivation of Lactobacillus and the phisiological damage mechanism of Lactobacillus were analyzed. The results showed that the cryoprotectants can significantly improve the survival rate and vitality of Lactobacillus, but the effects of cryoprotectants were differencent to some extent. The best cryoprotectant for Lactobacillus plantarum ST-Ⅲwas 10% trehalose, and that for Lactobacillus reuteri CICC6226 and Lactobacillus bulgaricus was10 % skim milk. Due to the decrease of metabolism of lactic acid bacteria during freeze-drying, the key metabolic enzyme was analysised. The results showed that lactate dehydrogenase was affected significantly by the freeze-drying process, but hexokinase and pyruvate kinase were not. This result implied that inactivation of lactate dehydrogenase was one of the key factors of damage caused by freeze-drying. Meanwhile, the results showed that the maintenance of the membrane integrity and fluidity was decreased; the membrane permeability was changed; the intracellular Ca²⁺ and protein leaked out, and membrane protein function （ATPase） was damaged during freeze-drying. These results have provided direct biochemical and metabolic evidence of injured cell during freeze-drying.Taking Lactobacillus reuteri CICC6226 as the main study objects, stress protection strategy was used, and the survival of lactic acid bacteria, vitality, key enzymes, membrane permeability, fluidity, integrity and fatty acid ratio was tested. The results showed that the survival rate of Lactobacillus reuteri CICC6226 was increased by stress treatment. The survival rate was increased by a 30min heat shock at 45°C, or by a 3h cold shock at 4°C. The survival rates were 91.90%, 93.23%, increased by 6.13% and 7.46% respectively. Meanwhile, the loss of the lactate dehydrogenase and ATPase activity was reduced; the extracellularβ-galactosidase activity was less by a 30min heat shock at 45°C, or by a 3h cold shock at 4°C. This indicated the better maintenance of cell permeability, which has been confirmed by detecting the integrity of the cell membrane. It was found that ion concentration ratio has changed dramatically after freeze-drying, but there was no correlation between the change of ion ratios and the survival rate of Lactobacillus. These results indicated that change of ion ratios could only reflect the damage of bacterial cell membrane and change of the cell membranes permeability, rather than cell death. The fluorescence polarization and microviscosity was the smallest by a 30min heat shock at 45°C. These results suggested that the membrane fluidity of Lactobacillus was the best. It was found that unsaturated fatty acid ratio was increased by a 30min heat shock at 45°C, or by a 3h cold shock at 4°C. This result suggested that the resistance of bacteria to lyophilization was improved, due to induced unsaturated fatty acid ratio by stress treatments.Taking Lactobacillus reuteri CICC6226 and Lactobacillus plantarum ST-Ⅲas the main objects of the study, pre-incubation protection strategy was used, and the survival of lactic acid bacteria, vitality, key enzymes, membrane permeability, fluidity, integrity was tested. The mathematical model of pre-incubation for trehalose concentration, temperature and time corresponding to the survival was established, which was based on three elements quadratic current revolving design. The optimum pre-incubation conditions of Lactobacillus reuteri CICC6226 were established: 11.45% trehalose, pre-incubation temperature of 38°C, pre-incubation time of 31.5min, and Lactobacillus reuteri survival was 95.34±4.35%. The optimum pre-incubation conditions of Lactobacillus plantarum ST-Ⅲwere established: 11.85% trehalose, pre-incubation temperature of 37.5°C, pre-incubation time of 29min and the survival was 94.64±3.26%. The study showed that the vitality of bacteria was improved, and ATPase LDH enzyme activity was restored; the cell membrane fluidity was maintained by pre-incubation during freeze-drying.The cell membrane integrity, intracellular pH and Ca²⁺ were determined by flow cytometry. The results showed that the membrane integrity was improved, and the change of intracellular pH and Ca²⁺ was less by pre-incubation during freeze-drying. These results suggested that the cell membrane integrity and permeability was maintained by pre-incubation during freeze-drying.The effect of different temperature on the activity of lactic acid bacteria was studied , and the survival of lactic acid bacteria, vitality, LDH and ATPase was tested during storage. The results showed that after 90 days of storage and at 4°C, the survival rate of Lactobacillus reuteri CICC6226 and Lactobacillus plantarum ST-Ⅲwas maintained at 82.56%, 82.38%, respectively. In the storage process, it was found that inactivation of lactate dehydrogenase and ATPase was correlated positively with death of bacteria cells, which suggested that inactivation of key enzymes caused the death of bacteria cells.