去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

Optimization of cryopreservation and lyophilization for demethylvancomycin producing strain—an assessment of 10-year preservation

摘要:

【目的】针对去甲基万古霉素产生菌不耐保藏的问题，改进菌种保藏方法，对超低温液氮保藏、?80 °C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性，评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和?80 °C低温冷冻保藏，采用脱脂牛奶作基础保护剂进行冷干保藏，针对超低温液氮保藏进行降温速率考察，研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对 3种保藏方法的冻存影响，对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为：?80 °C低温冷冻保藏>超低温液氮保藏>冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方：超低温液氮保藏为甘油8.0%，海藻糖3.5%；?80 °C低温冷冻保藏为甘油6.0%，PVP5.0%；冷干保藏为脱脂牛奶，6.0%海藻糖。采用优化保藏条件，液氮保藏10年存活率稳定在70.6%，菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下，尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。

Abstract:

[Objective] Due to the difficulties in preserving demethylvancomycin producing strain, we optimized the preservation conditions, and assessed the 10-year stability of different preservation methods with liquid nitrogen preservation, ?80 °C cryopreservation and lyophilization outcomes. [Methods] Using glycerol and skim milk as the basic protective agent for cryopreservation and lyophilization, the effect of cooling rate for liquid nitrogen cryopreservation and impermeability protective agents such as trehalose and polyvinylpyrrolidone (PVP) to these three preservation methods were studied. Ten years tracking assessment on optimal preservation method was performed. [Results] Among the three preservation methods, ?80 °C cryopreservation showed the highest survival rate for demethylvancomycin producing strain, followed by liquid nitrogen cryopreservation and lyophilization. The optimum cooling rate for liquid nitrogen cryopreservation was rapid freezing, and the optimum protective agent was as follows: 8.0% glycerol with 3.5% trehalose for liquid nitrogen cryopreservation, 6.0% glycerol with 5.0% PVP for ?80 °C cryopreservation, skimmed milk with 6.0% trehalose for lyophilization. The 10-year survival rate of liquid nitrogen preservation reached 70.6% through optimum preservation condition, productivity of demethylvancomycin remained 92.9% of the original level. [Conclusion] Under optimum preservation condition, liquid nitrogen cryopreservation is suitable for long-term preservation.