解淀粉芽孢杆菌素Amylocyclicin W5的纯化及其抑菌机理

doi:10.7506/spkx1002-6630-20200407-082

食品科学 ›› 2021, Vol. 42 ›› Issue (7): 29-34.doi: 10.7506/spkx1002-6630-20200407-082

解淀粉芽孢杆菌素Amylocyclicin W5的纯化及其抑菌机理

王伟，李津津，迟海

（1.中国水产科学研究院东海水产研究所，上海 200090；2.上海理工大学医疗器械与食品学院，上海 200093）

出版日期:2021-04-15 发布日期:2021-05-17
基金资助:
国家自然科学基金青年科学基金项目（31801663）；上海市自然科学基金项目（18ZR1449900）；中央级公益性科研院所基本科研业务费专项资金项目（2016M02）

Purification and Antimicrobial Mechanism of Amylocyclicin W5 Produced by Bacillus amyloliquefaciens DH8030

WANG Wei, LI Jinjin, CHI Hai

(1. East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China; 2. School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China)

Online:2021-04-15 Published:2021-05-17

摘要/Abstract

摘要： 为研究解淀粉芽孢杆菌素Amylocyclicin W5的抑菌机理，本实验通过硫酸铵沉淀、离子交换以及?KTA蛋白纯化系统对Amylocyclicin W5进行分离纯化，并对Amylocyclicin W5进行三羟甲基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；此外，以蜡样芽孢杆菌（Bacillus cereus LMGT2805）为指示菌对Amylocyclicin W5进行抑菌实验，同时通过扫描电子显微镜、透射电子显微镜观察细胞形态变化以及细胞内部结构变化。结果表明，最适宜沉淀细菌素的硫酸铵饱和度为70%，0.5 mol/L NaCl可以明显洗脱吸附在HiPrep SP XL 16/10柱上的Amylocyclicin W5，将洗脱液通过?KTA蛋白纯化系统得到2 个对应的收集峰（F1和F2）；对不同收集峰对应的洗脱液进行抑菌实验，结果发现只有F2对应的组分液对B. cereus LMGT2805有抑菌效果。三羟甲基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示Amylocyclicin W5组分F2的分子质量约为12.3 kDa。高浓度（4 倍最低半抑菌浓度）条件下的Amylocyclicin W5可以完全抑制B. cereus LMGT2805，其主要抑菌机制是破坏其细胞壁，使其形成孔洞，导致其内容物外泄，细菌（营养或芽孢细菌）无法正常进行代谢，从而造成细菌的死亡。实验结论为新型广谱细菌素的开发利用及其在食品安全领域的应用研究提供了数据支持。

关键词: 解淀粉芽孢杆菌；Amylocyclicin W5；分离纯化；抑菌机理

Abstract: In this study, amylocyclicin W5, a bacteriocin produced by Bacillus amyloliquefaciens DH8030, was purified by sequential use of ammonium sulfate precipitation, ion exchange chromatography and ?KTA purification system, and it was analyzed by Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Bacillus cereus LMGT2805 was employed as an indicator strain to evaluate the antibacterial effect of amylocyclicin W5. The changes in cell morphology and the intracellular structure induced by the bacteriocin were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that the optimal degree of saturation of ammonium sulfate precipitation was 70%. In addition, 0.5 mol/L NaCl could significantly elute adsorbed bacteriocin from HiPrep SP XL 16/10 column. The eluate was fractionated by using ?KTA purification system into two fractions (F1 and F2). From bacteriostatic experiments, it was found that F2 but not F1 had a bacteriostatic effect against Bacillus cereus LMGT2805. The molecular mass of F2 was determined to be about 12.3 kDa. Amylocyclicin W5 at high concentration (four-fold higher than the half-minimal inhibitory concentration (MIC50) could completely inhibit B. cereus LMGT2805, and its antimicrobial mechanism was by destroying the cell wall to form holes and thus causing the leakage of intracellular contents, abnormal metabolism and finally cell death. These results provide data supporting the development of amylocyclicin W5 and its application in the field of food safety.

Key words: Bacillus amyloliquefaciens; amylocyclicin W5; isolation and purification; antimicrobial mechanism

中图分类号:

TS201.3

王伟，李津津，迟海. 解淀粉芽孢杆菌素Amylocyclicin W5的纯化及其抑菌机理[J]. 食品科学, 2021, 42(7): 29-34.

WANG Wei, LI Jinjin, CHI Hai. Purification and Antimicrobial Mechanism of Amylocyclicin W5 Produced by Bacillus amyloliquefaciens DH8030[J]. FOOD SCIENCE, 2021, 42(7): 29-34.

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解淀粉芽孢杆菌素Amylocyclicin W5的纯化及其抑菌机理

Purification and Antimicrobial Mechanism of Amylocyclicin W5 Produced by Bacillus amyloliquefaciens DH8030

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