The Study On High Cell Density Culture And Freeze-drying Protection Of Lactobacillus Delbrueckii Subsp. Bulgaricus ND02

Through optimizing the medium and static cultivate condition of Lactobacillus delbrueckii subsp. bulgaricus ND02, investigate its high cell density procedure, as well as the procedure and components of its free-drying protectants. The conclusions below were obtained:(1) Based on MRS medium, screening the factors (such as carbon source, nitrogen source, the ratio of carbon source and nitrogen source, tract elements, buffer salts and growth factors) and optimizing the additive amounts of each components through response surface design by Design Expert7.0software. The optimized culture medium of Lactobacillus delbrueckii subsp. bulgaricus ND02was1.58%glucose,1.05%lactose,1.58%sucrose,0.51%casein peptone,1.53%Nitrogen substancesD,2.55%Nitrogen substances A,0.72%sodium acetate,0.58%K2HPO4,0.43%citrate sodium,0.0358%MgSO4,0.009%MnSO4,0.076%Inorganic salts C,0.1%Tween-80and0.5g/L Growth factor B. The initial pH value of the medium was adjusted to6.5, using6%inoculum, and static cultivated for lOh under38℃. After incubation, the viable count of Lactobacillus delbrueckii subsp. bulgaricus ND02could reach to3.5x109cfu/mL, which increased three times than that of incubated in MRS medium under the same condition (1.05xl09cfu/mL).(2) Based on the optimized medium and static cultivated condition, optimizing the factors (such as constant fermented pH value, types of neutralizer, and stirring speed of impellers) influenced the high cell density cultivation. The optimized high cell density fermentation procedure was fermented at38℃with6%inoculum, the constant pH value was5.0, and the neutralizer was25%ammonia. Under this fermentation condition, the viable count of Lactobacillus delbrueckii subsp. bulgaricus ND02could reach to5.5x10cfu/mL, the fermenting time was6-6.5h. In compared with the static fermentation, the viable count increased57%and the fermenting time decreased almost4hours.(3) Optimizing the anti-freezing temperation, anti-freezing time, ratio and components of protectants of Lactobacillus delbrueckii subsp. bulgaricus ND02, and the best freeze-drying procedure was obtained. The best anti-freezing condition was-40℃for8h, the optimal ratio of bacteria and protectants was1:6. Moreover, the components of protectants were10%skim milk power,8%sugar (T2:T4=1:1),0.1%Vatamin C,1%salt (Y5),1.5%spirits (D), and79.4%distilled water. After the optimization, the viable count of Lactotacillus delbrueckii subsp. bulgaricus ND02in bacterium powder could reach to1.03xl011cfu/g, the survival rate was85.3%. In compared with that of unoptimized freeze-drying condition, the viable count increased66%, and the survival rate increased67%.