一种脱氧雪腐镰刀菌烯醇制备工艺的研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0325

摘要/Abstract

摘要：

脱氧雪腐镰刀菌烯醇（DON）是镰刀菌属产生的一种真菌毒素,具有细胞毒性,可导致胚胎发育畸形,严重威胁人畜健康。为了获得大量的高纯度DON用于毒理学及脱毒研究,本研究开发了一种操作简单、经济高效的DON分离纯化方法：抽提：采用84%的乙腈水溶液从禾谷镰刀菌PH-1的大米培养物中提取DON获得DON粗毒素;净化：使用石油醚和饱和食盐水净化DON粗毒素;精制：使用乙酸乙酯：石油醚（1∶1,V/V）洗涤净化后的粗毒素,再利用乙酸乙酯和饱和食盐水之间的极性差异,通过液液分配的方式萃取DON,进一步去除杂质;纯化：使用制备型高效液相色谱纯化DON,获得DON纯品。该方法可以从每千克PH-1大米培养物中获得61.4 mg DON纯品,其优势是经过精制的DON纯度已经达到80%以上,可以满足常规研究的需求,如需通过制备液相色谱进一步纯化,可显著降低色谱柱损耗,降低经济成本。

关键词: 脱氧雪腐镰刀菌烯醇, 分离纯化, 禾谷镰刀菌

Abstract:

Deoxynivalenol（DON）is a mycotoxin produced by Fusarium. It is cytotoxic and can cause embryo deformity,which seriously threatens human and animal health. To provide enough high-purity DON for scientific research in toxicology and detoxification,we developed a simple,economical and effective process for the separation and purification of DON. 1. Extraction：84% acetonitrile aqueous solution was used to extract DON from the rice culture of Fusarium graminearum PH-1 to obtain DON crude extract. 2. Cleaning：Petroleum ether and saturated saline aqueous solution were used to remove the impurity from the DON crude extract. 3. Refining：The product obtained in step 2 was washed by ethyl acetate：petroleum ether (1∶1 of V/V). Then more impurity was removed by liquid-liquid distribution based on the polarity difference between ethyl acetate and saturated saline aqueous solution. 4. Purification：Helped by preparative high performance liquid chromatography DON in high purity was obtained. With this method 61.36 mg highly purified DON was obtained from one kilogram PH-1 culture. Its main feature is that the purity of the refined DON has reached 80%,which could be directly applied in conventional scientific research. If further purification needed via preparative liquid chromatography,the column loss can be remarkably reduced,therefore the economic cost decreases.

Key words: deoxynivalenol, separation and purification, Fusarium graminearum

段佳琪, 李琲琲, 任杰, 邱德文, 李广悦. 一种脱氧雪腐镰刀菌烯醇制备工艺的研究[J]. 生物技术通报, 2020, 36(10): 256-262.

DUAN Jia-qi, LI Bei-bei, REN Jie, QIU De-wen, LI Guang-yue. Research on a Preparation Process of Deoxynivalenol[J]. Biotechnology Bulletin, 2020, 36(10): 256-262.

图/表 7

图1 脱氧雪腐镰刀菌烯醇的分子结构

图2 接种禾谷镰刀菌PH-1的大米培养基在发酵0 d、7 d、14 d、21 d、28 d、35 d后的状态 A-F：0 d、7 d、14 d、21 d、28 d、35 d

图3 禾谷镰刀菌PH-1的产毒曲线

图4 不同溶剂提取液色谱图 A：50 mg/L DON标准品;B：乙腈-水（84：16,V/V）;C：甲醇-水（9：1,V/V）;D：甲醇-水（8：2,V/V）;E：甲醇-水（7：3,V/V）

表1 不同提取剂提取效果对比

指标	乙腈-水（84:16,V/V）	甲醇-水（9:1,V/V）	甲醇-水（8:2,V/V）	甲醇-水（7:3,V/V）
提取率/%	100	22.7	96.9	102
DON/粗毒素/%	2.9	0.5	2.2	1.9

图5 不同分离阶段色谱图 A：粗毒素;B：净化后的粗毒素;C：EA/PE萃取后的水相;D：EA萃取后的饱和NaCl溶液;E：精制后的DON

图6 纯化产物纯度分析 A：纯化产物色谱图;B：纯化产物光谱图;C：纯化产物1H-NMR谱图

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