一种定量保存金黄色葡萄球菌真空冷冻干燥技术方法的设计  

Design of a technical method of vacuum freeze-drying for quantitative preservation of Staphylococcus aureus

作　　者：崔雯 赵良 时玉雯[1] 陈潇 刘辉[1] CUI Wen;ZHAO Liang;SHI Yu-wen;CHEN Xiao;LIU Hui(Bacterial Disease Laboratory,Jinan City Center for Disease Control and Prevention,Shandong 250021,China;Department of Anesthesiology,Jinan Children's Hospital)

机构地区：[1]济南市疾病预防控制中心细菌性疾病检验所,山东250021 [2]济南市儿童医院麻醉科

出　　处：《预防医学论坛》2024年第8期614-619,共6页Preventive Medicine Tribune

基　　金：山东省济南市卫生健康委员会公共卫生计划专项(2021-公-03)。

摘　　要：目的 探究一种利用真空冷冻干燥技术定量保存标准菌株金黄色葡萄球菌的新方法。方法 在真空冷冻干燥的条件下,使用微滤孔膜制备金黄色葡萄球菌冻干菌片,分别从生长曲线、单一冻干保护剂的筛选、保护剂的优化复配、真空冷冻预冻温度和冷冻温度、保存条件以及有效期分析对冻干菌片的菌种存活率的影响。结果 生长曲线结果显示,金黄色葡萄球菌4 h后进入生长对数期,16 h后进入生长稳定期。单一保护剂分别从糖类、蛋白质类和抗氧化剂中筛选,结果显示脱脂牛奶、海藻糖和抗坏血酸的细菌存活率最高(F=6.7,P<0.05,Adj R^(2)=0.785 5),利用响应曲面法优化冻干保护剂的配方为脱脂牛奶(3%)、海藻糖(10%)以及抗坏血酸(0.75%)。在该保护剂的作用下,冻干菌片的细菌存活率可达90%以上,与预测值较为相似,证明响应曲面法优化保护剂配方有效。冻干菌片在预冻温度-20℃、冷冻温度-40℃的冷冻条件下,细菌存活率最高(F=147.0,R^(2)=0.982 9,P<0.001)。在-40℃的冰箱保存6个月后金黄色葡萄球菌的存活率可达93.3%,保存2年后细菌存活率仍可达80%以上(F=79.42,R^(2)=0.969 5,P<0.001)。结论 利用真空冷冻干燥技术制备菌片用于定量保存金黄色葡萄球菌,用复配的保护剂制备的金黄色葡萄球菌冻干菌片可达到有效的定量保存效果。Objective To investigate a new approach for quantitative preservation of staphylococcus aureus standard strains utilizing vacuum freeze-drying.Methods Staphylococcus aureus lyophilized pellets were prepared using microfiltration pore membranes under vacuum freeze-drying conditions,and the effects of various factors,including growth curves,screening of single lyophilized protectants,optimal compounding of protectants,vacuum freezing pre-freezing and freezing temperatures,preservation conditions,and expiration dates,on the viability of the lyophilized pellets were investigated.Results The growth curve data revealed a logarithmic phase after 4 hours and a stabilizing phase after 16 hours.Single protective agents were selected among sugars,proteins,and antioxidants,and the findings revealed that skim milk,trehalose,and ascorbic acid had the highest rates of bacterial survival(F=6.7,P<0.05,adj R^(2)=0.7855).The lyophilized protectant formulation was improved utilizing the response surface approach using skimmed milk(3%),trehalose(10%),and ascorbic acid(0.75%).The bacterial survival of the lyophilized bacterial tablets could reach more than 90%in the presence of this protective agent,which is more comparable to the projected value.The results demonstrated the effectiveness of of the response surface approach in improving the protectant composition.Bacterial pellets were freeze-dried at-40℃ after being pre-frozen at-20℃(F=147.0,R^(2)=0.9829,P<0.001).After 6 months of storage in a refrigerator at-40℃,the survival rate of bacteria can reach 93.3%,and it can still reach more than 80%after two years(F=79.42,R^(2)=0.9695,P<0.001).Conclusion The results demonstrated that lyophilized pellets of Staphylococcus aureus made using the compounded protective agent could accomplish effective quantitative preservation by determining the survival rate of the lyophilized pellets.

关 键 词：金黄色葡萄球菌 定量保存 真空冷冻干燥 冻干保护剂 细菌存活率 

分 类 号：R378.11[医药卫生—病原生物学] R155.5[医药卫生—基础医学]