肺炎链球菌4型菌株冷冻真空干燥工艺保护剂配方的筛选

Screening of protective agent formula for freezing and vacuum drying process of Streptococcus pneumoniae type 4 strain

目的通过正交实验筛选肺炎链球菌4型菌株冷冻真空干燥工艺保护剂配方,并对该配方下菌种的活性、遗传稳定性以及生长特性进行分析,找到符合长期、稳定保存菌种要求的冻干保护剂配方。方法以肺炎链球菌4型菌株为试验菌种,以菌种冻干前后存活率为评价指标,通过采用三因素正交试验确定冻干保护剂配方,冻干后对该配方下制备的菌种进行活性、遗传稳定性以及生长特性的分析。结果通过正交实验确定最佳冻干保护剂配方为:5%脱脂奶粉+10%海藻糖+10%谷氨酸钠;冻干前菌液浓度为2.35×108 cfu/mL,冻干后菌种保存不同时间(冻干后,存放90、180、360 d)取样检测活性分别为:81.2%,80.4%,80.1%和79.8%,同源性分析为100%,生长特性无差异;该配方对6A、18C、19F型肺炎链球菌在冷冻真空干燥工艺下同样具有较好的保护性,三批样品0 d取样存活率均在78%以上。结论冻干保护剂配方优化后使得菌种存活率以及稳定性都保持较高水平,并且该配方工艺可用于肺炎链球菌不同血清型菌株冷冻真空干燥保存,为肺炎链球菌多糖疫苗的生产提供稳定可靠的菌株。

Objective To screen the formula of the protective agent for the freezing and vacuum drying process of Streptococcus pneumoniae type 4 strain through orthogonal experiments,and to analyze the activity,genetic stability and growth characteristics of the strains under the formula,and find the long-term and stable freeze-dried protective agent formulation.Methods Streptococcus pneumoniae type 4 strain was selected as the test strain.The survival rate of the strain before and after freeze-drying was used as an evaluation index.The freeze-dried protective agent formula was determined by using a three-factor orthogonal test.After freeze-drying,the activity,genetic stability and growth characteristics of the bacteria under the formula were analyzed.Results Through orthogonal experiment,the best freeze-drying protective agent formula was determined as:5%skimmed milk powder+10%trehalose+10%sodium glutamate.The concentration of the bacterial solution before lyophilization was 2.35×108 cfu/mL.After lyophilization,the strains were stored for different times(after lyophilization,storage 90,180,360 days)to test their activity,genetic stability,and growth characteristics.The activities were 81.2%,80.4%,80.1%and 79.8%.The homology analysis was 100%,and there was no difference in growth characteristics.This formulation compatible with types of 6A,18C and 19F Streptococcus pneumoniae also have good protection under the freeze-vacuum drying process,and the survival rate of the three batches of samples at day 0 was all above 78%.Conclusion After the optimization of the freeze-dried protective agent formulation,the survival rate and stability of the strains maintain a high level,and the formulation process can be used for freeze-vacuum drying and storage of different serotypes of Streptococcus pneumoniae,providing stable and reliable strains for the production of Streptococcus pneumoniae polysaccharide vaccines.