天然低共熔溶剂对冷冻干燥期间嗜热链球菌活力的影响

[目的]以天然低共熔溶剂（NADES）作为低温保护剂，研究其在冷冻干燥期间对嗜热链球菌活力的影响。[方法]通过透射电子显微镜观察嗜热链球菌冷冻期间的损伤机制，并利用平板计数法测定嗜热链球菌的存活率。采用控制变量法分析嗜热链球菌细胞的最优裂解条件，测定嗜热链球菌中乳酸脱氢酶（LDH）和β-半乳糖苷酶活性，同时通过低场核磁共振仪测定NADES中的水分活度。[结果]嗜热链球菌冷冻后细胞中的细胞膜部分消失，细胞内的细胞质聚集成团，细胞间的链状结构消失，细胞表面蛋白完整。当体系中含有50% NADES时，嗜热链球菌的存活率明显高于体系中含有80% NADES的。在冷冻干燥后，只有氯化胆碱与山梨糖醇形成的NADES显著提高了嗜热链球菌冷冻干燥后的存活率（88.28%）。最优的裂解条件为：在1.2 mg？mL-1溶菌酶和0.5%（体积分数）TritonX-100的溶液中37℃裂解30 min。NADES对胞内LDH和β-半乳糖苷酶没有显著的保护作用。嗜热链球菌在含有氯化胆碱类的NADES中冷冻干燥的存活率较高。[结论]在冷冻期间细胞内冰晶对细胞膜和细胞质的损伤是主要的损伤机制，使用氯化胆碱和山梨糖醇形成的天然低共熔溶剂能显著提高嗜热链球菌冷冻干燥后的活力，嗜热链球菌的存活率与NADES中的水分活度有关。
[Objectives]In this paper,the important effects of natural deep eutectic solvents(NADES) as chemical protectants on Streptococcus thermophilus during freeze-drying were studied.[Methods]The damage mechanism was explored by transmission electron microscopy. The survival rate of S.thermophilus was determined by plate counting method. The control variables were used to explore the method of cell lysis. And lactate dehydrogenase(LDH) and β-galactosidase activities in S.thermophilus were determined. In addition,the experiment of water activity measurement was carried out by low-field nuclear magnetic resonance.[Results]The damage mechanism of S.thermophilus after freezing was studied,showing that the cell membrane disappeared,the intracellular cytoplasm agglomerated,the chain structure disappeared,and the cell surface protein was complete. The effects of different concentrations of NADES on S.thermophilus during pre-freezing period were different. It was found that the survival rate of S.thermophilus was significantly higher than that of 80% NADES,when 50% NADES was contained in the solvents. After freeze-drying,only NADES formed by choline chloride and D-sorbitol significantly increased the survival rate of S.thermophilus with a survival rate of 88.28%. The results showed that the best condition for cell lysis was the solution of 1.2 mg？mL-1 lysozyme and 0.5% TritonX-100 for 30 min at 37℃. NADES was found to have no significant protective effects on intracellular LDH and β-galactosidase. The survival rate of S.thermophilus was higher in NADES based on choline chloride after freeze-drying.[Conclusions]In the freezing period,intracellular ice crystals damage to the cell membrane and cytoplasm was the main damage mechanism. NADES formed by choline chloride and D-sorbitol significantly increased S.thermophilus viability after freeze-drying. The survival rate of S.thermophilus related to the changes of water activities in NADES