溶藻弧菌冷冻保护剂配方与冻干工艺研究

邵新飞; 阙莉文; 黄郁葱; 蔡 佳; 黄 瑜; 王 蓓; 简纪常

文章摘要

邵新飞，阙莉文，黄郁葱，蔡佳，黄瑜，王蓓，简纪常.溶藻弧菌冷冻保护剂配方与冻干工艺研究[J].广东农业科学,2024,51(6):117-127

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Research on the Formula and Lyophilization Process of Vibrio alginolyticus Cryoprotectant

DOI：10.16768/j.issn.1004-874X.2024.06.012

中文关键词: 溶藻弧菌冷冻干燥冷冻保护剂冻干工艺毒力药敏试验

英文关键词: Vibrio alginolyticus freeze-drying cryoprotectant freeze-drying process virulence antimicrobial susceptibility test

基金项目:国家自然科学基金（32073006，32002426，U20A2065）

作者	单位
邵新飞，阙莉文，黄郁葱，蔡佳，黄瑜，王蓓，简纪常	（广东海洋大学水产学院 / 广东省水产动物病害防控与健康养殖重点实验室 / 广东高等学校水产经济动物病害控制重点实验室，广东湛江 524088）

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中文摘要:

【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（LD50）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 1×108 ~109 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 LD50 分别为 1.22×108 、1.20×108 CFU/mL，LD50 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 1×108 ~109 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。

英文摘要:

Abstract: 【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 freezedrying process conditions for Vibrio alginolyticus were optimized. At the same time, storage test, LD50 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 1×108 -109 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 LD50 values of the strains before and after freeze-drying were 1.22×108 CFU/mL and 1.20×108 CFU/mL, respectively, with no significant difference in LD50. 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 1×108 - 109 CFU/mL, pre-freezing time 3 h, pre-freezing temperature -80 ℃ , rehydration medium PBS or 50 g/L skim milk. The fomula and process for this strain are reasonable and feasible, with good storage and stability of biological characteristics.

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