色谱

色谱 ›› 2016, Vol. 34 ›› Issue (11): 1091-1096.DOI: 10.3724/SP.J.1123.2016.06044

• 研究论文 • 上一篇    下一篇

青枯雷尔氏菌特征菌株高效离子交换色谱快速分离条件的优化

郑雪芳, 刘波, 朱育菁, 陈德局   

  1. 福建省农业科学院农业生物资源研究所, 福建 福州 350003
  • 收稿日期:2016-06-27 出版日期:2016-11-08 发布日期:2016-11-03
  • 通讯作者: 刘波
  • 基金资助:

    国家公益性行业（农业）科研专项基金（201303015）；福建省自然科学基金（2015J01103）；福建省公益类科研院所专项基金（2014R1018-8）.

Optimization of rapid separation conditions of Ralstonia solanacearum using high performance ion-exchange chromatography

ZHENG Xuefang, LIU Bo, ZHU Yujing, CHEN Deju   

  1. Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
  • Received:2016-06-27 Online:2016-11-08 Published:2016-11-03
  • Supported by:

    Chinese Special Fund for Agro-Scientific Research in the Public Interest (No. 201303015); Fujian Provincial Natural Science Foundation of China (No. 2015J01103); Fujian Provincial Special Fund for Non-profit Institutions (No. 2014R1018-8).

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摘要：

建立了高效离子交换色谱和紫外检测系统快速分离青枯雷尔氏菌的细菌色谱方法。通过比较青枯雷尔氏菌悬浮在哌嗪-HCl缓冲体系和双蒸水后的菌体数变化及细胞形态变化，分析该缓冲液对青枯雷尔氏菌生长活性及细胞表面特性的影响。结果表明，青枯雷尔氏菌悬浮在平衡缓冲液、洗脱缓冲液和双蒸水中的菌体数量无明显差异，分别为6.467×109、6.267×109和6.233×109 cfu/mL。透射电镜观察发现，3种溶液处理后，青枯雷尔氏菌均保持完整的细胞结构。研究了缓冲液pH值、流速及菌体细胞浓度对青枯雷尔氏菌色谱分离效果的影响，确定青枯雷尔氏菌的最佳色谱分离条件为：缓冲液pH值为8.0，流速为2 mL/min，菌体浓度大于1.0×108 cfu/mL且小于1.0×1010 cfu/mL。该分离条件缩短了分离时间，提高了分离效率，为快速分离青枯雷尔氏菌提供了一种有效的手段，同时也为细菌等微生物的分离提供了新途径。

关键词: 分离, 高效离子交换色谱, 青枯雷尔氏菌, 细胞表面特性

Abstract:

Rapid separation of Ralstonia solanacearum with intact cell using high performance ion-exchange chromatography equipped with a UV detector was described. The bacterial growth abilities and cell morphologies in different suspension buffers were compared. The results showed that the bacterial numbers in the balance buffer (0.02 mol/L piperazine-hydrochloric acid), elution buffer (0.02 mol/L piperazine-hydrochloric acid+1 mol/L NaCl) and double distilled water were 6.467×109, 6.267×109 and 6.233×109 cfu/mL, respectively, exhibiting no significant difference. Moreover, the cell morphologies were observed under a transmission electron microscope which showed that the bacterial cells could remain intact. Effects of buffer pH, flow velocity and bacterial cell concentration on the separation efficiency were analyzed. The results showed that the optimum chromatographic separation parameters were as follows: the buffer pH was 8, the flow velocity was 2 mL/min and the bacterial cell concentration was from 1.0×108 cfu/mL to 1.0×1010 cfu/mL. Under the chromatographic separation conditions, the separation time was shortened and the separation efficiency was improved. Therefore, the chromatographic method developed here may be a promising tool for the rapid separation of Ralstonia solanacearum and other bacteria.

Key words: Ralstonia solanacearum, cell surface properties, high performance ion-exchange chromatography, separation

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