溶藻弧菌冷冻保护剂配方与冻干工艺研究

Research on the Formula and Lyophilization Process of Vibrio alginolyticus Cryoprotectant

【目的】研究溶藻弧菌(Vibrio alginolyticus)真空冷冻干燥保护剂配方与优化冻干工艺条件,并探究冷冻干燥对菌株毒力及药物敏感性的影响,为菌种在冷冻干燥保藏上的应用提供理论依据。【方法】采用真空冷冻干燥技术,以溶藻弧菌HY9901为研究对象,通过单因素试验及正交试验,探究L-谷氨酸钠、海藻糖、脱脂乳、葡萄糖、甘露醇、甘氨酸6种单一保护剂对菌株活性的影响,筛选最佳冷冻保护剂复配配方,并研究菌液浓度、预冻时间、预冻温度及复水介质4个因素对菌株冻干工艺的影响,优化溶藻弧菌冻干工艺条件。同时,通过储藏试验、半数致死量(LD_(50))试验和药敏试验,探究冻干菌株的活性变化规律及其生物特性。【结果】6种单一保护剂对溶藻弧菌冻干的保护效果存在显著差异,其效果从强到弱依次为:L-谷氨酸钠、海藻糖、脱脂乳、葡萄糖、甘露醇、甘氨酸;菌株最佳冷冻干燥保护剂复配配方为海藻糖50 g/L+L-谷氨酸钠50 g/L+脱脂乳50 g/L,其存活率为62.12%,与单一保护剂的保护效果差异显著;冻干工艺最适条件:菌液浓度1×10~8~10~9 CFU/mL、预冻时间3 h、预冻温度-80℃、复水介质为PBS或50 g/L脱脂乳,此条件下菌株存活率可达到最大值(63.72%)。最佳冻干条件下,菌株储藏60 d期间,其活性无显著差异;菌株冻干前后的LD_(50)分别为1.22×10~8、1.20×10~8 CFU/mL,LD_(50)无显著差异。菌株在冻干前后的药敏抑菌圈直径无显著差异,对氟苯尼考、氯霉素、环丙沙星等药物均表现敏感;对新霉素、卡那霉素均表现中度敏感;对丁胺卡那、哌拉西林等药物均表现耐药。【结论】溶藻弧菌HY9901的冷冻干燥复配配方为海藻糖50 g/L+L-谷氨酸钠50 g/L+脱脂乳50 g/L,冻干工艺条件为菌液浓度1×10~8~10~9 CFU/mL、预冻时间3 h、预冻温度-80℃、复水介质为PBS或50 g/L脱脂乳,该配方及工艺合理可行,具有良好的储藏性及生物稳定性。

【Objective】The study aims to screen the formula of vacuum freeze-drying protective agent for Vibrio alginolyticus,optimize the freeze-drying process conditions,and explore the influence of freeze-drying on the virulence and drug sensitivity of the strains,with a view to providing a theoretical basis for the application of freeze-drying in the preservation of the strains.【Method】With Vibrio alginolyticus HY9901 as the research object,the vacuum freeze-drying technology was used to freeze-dry the strains.Through single factor test and orthogonal test,the effects of six single cryoprotectants (L-glutamate sodium,trehalose,skim milk,glucose,mannitol and glycine) on the activity of the strains were investigated,and the optimal formula of cryoprotectants was selected.The influences of four factors,namely,concentration of bacterial solution,pre-freezing time,pre-freezing temperature and rehydration medium,on the freeze-drying process of strains were studied,and the freeze-drying process conditions for Vibrio alginolyticus were optimized.At the same time,storage test,LD_(50) test and antimicrobial susceptibility test were used to investigate the changes in the activity of lyophilized strains and their of biological characteristics.【Result】There were significant differences in the protective effects of 6 cryoprotectants on lyophilized Vibrio alginolyticus,and the effects from strongest to weakest showed as:L-glutamate sodium,trehalose,skim milk,glucose,mannitol and glycine.The optimum freeze-drying compound formula of cryoprotectants was trehalose 50 g/L+ L-glutamate sodium 50 g/L+ skim milk 50 g/L,with a survival rate of 62.12%,which was significantly different from that of single cryoprotectant.The optimal conditions of freeze-drying process were as follows:bacterial solution concentration 1×10~8-10~9 CFU/mL,pre-freezing time 3 h,pre-freezing temperature-80 ℃,rehydration medium PBS or 50 g/L skim milk,under which the activity of the strains could reach the maximum of 63.72%.There was no significant difference in the activity of the strains during 60 days of storage under optimal freeze-drying conditions.The LD_(50) values of the strains before and after freeze-drying were 1.22×10~8 CFU/mL and 1.20×10~8 CFU/mL,respectively,with no significant difference in LD_(50).There was no significant difference in the diameter of the antimicrobial inhibition zone before and after freeze-drying,and the strains were sensitive to florfenicol,chloramphenicol,ciprofloxacin and other drugs.They were moderately sensitive to neomycin and kanamycin,and resistant to drugs like amimcana and piperacillin.【Conclusion】The freeze-drying formula for Vibrio alginolyticus HY9901 is trehalose 50 g/L+ L-glutamate sodium 50 g/L+ skim milk 50 g/L,and the freeze-drying process conditions are:bacterial solution concentration 1×10~8-10~9 CFU/mL,pre-freezing time 3 h,pre-freezing temperature-80 ℃,rehydration medium PBS or 50 g/L skim milk.The fomulaand process for this strain are reasonable and feasible,with good storage and stability of biological characteristics.