Research On Cryoprotectant Selection And Protective Mechanism For Lactobacillus Casei

Vacuum freeze-drying is widely used to produce commercial lactic acid bacterial starter,but this technique brings about undesirable side effects,such as denaturation of sensitive proteins and decreased viability of many cell types.Adding appropriate cryoprotectants is effective in the case of LAB,so as to increase their survival rates during dehydration,however,the protective effect depends on cryoprotectants and bacteria.In China, the research on protective mechanism was less than picking an efficient protective agent.This dissertation optimized cryoprotectants for Lactobacillus casei,evaluated their protective effects by comparing the changes of survival rate,recovery capability and acidification activity,concluded protective mechanism through detecting inactivation of intercellular enzymes,integrity and oxidation of membrane.Based on survival rate,the formulation of cryoprotectants for lyophilized Lactobacillus casei was optimized by means of response surface methodology in this study.Results indicated the optimal amounts in100mL of cryoprotectants formula were skimmed milk15.8g,lactose5.73g and monosodium glutamate0.42g.The optimal cell survival rate was observed to be92.4%using the optimal compound cryoprotectant.In the absence of protectant,survival rate was merely5.25%,tOD0.2and tpH5.5were separately90min and20h.Results were improved when adding cryoprotectant,the different protective effects were as follows:optimized> skimmed milk> lactose> monosodium glutamate.92.4%for survival rate,20min for tODo.2and90min for tpH5.5were the best results observed by adding the optimized protectant.By using fluorescent probes PI,5(6)-CFDA and C11-BODIPY581/591,combined with multiparametric flow cytometry,membrane integrity,intracellular esterase activity,cellular vitality,and membrane oxidation were quantified before and after freeze-drying.Results indicated that cryoprotectants can reduce "mechanical damage" and "solution effect" as the activity of intracellular esterase,(3-galactosidase and ATPase were all improved.Lactose and monosodium glutamate may act as antioxidant,though,oxidantion of lipid on membrane was not the main reason for reducing cell viability during freeze-drying.