广谱拮抗菌株的筛选诱变及抗菌物质分离鉴定

doi:10.7506/spkx1002-6630-20191112-161

食品科学 ›› 2021, Vol. 42 ›› Issue (2): 143-150.doi: 10.7506/spkx1002-6630-20191112-161

广谱拮抗菌株的筛选诱变及抗菌物质分离鉴定

高兆建，王秋芬，丁飞鸿，许祥，赵宜峰，焦魏，陈腾

（1.徐州工程学院食品（生物）工程学院，江苏徐州 221018；2.海滨林场，河北秦皇岛 066100；3.长江桂柳食品睢宁有限公司，江苏徐州 221000；4.邳州市金大地肥料有限公司，江苏徐州 221300）

出版日期:2021-01-18 发布日期:2021-01-27
基金资助:
江苏省苏北科技计划项目（XZ-SZ201819；BC2013417；BN2015021）；徐州市科技计划项目（KC17083）

Screening and Mutagenesis of Broad-Spectrum Antagonistic Bacillus licheniformis and Purification and Identification of Antimicrobial Substances Produced by Its Mutant

GAO Zhaojian, WANG Qiufen, DING Feihong, XU Xiang, ZHAO Yifeng, JIAO Wei, CHEN Teng

(1. College of Food (Biological) Engineering, Xuzhou Institute of Technology, Xuzhou 221018, China; 2. Haibin Forest Farm, Qinhuangdao 066100, China; 3. Yangtze River Guiliu Food Suining Co. Ltd., Xuzhou 221000, China; 4. Pizhou Golden Earth Fertilizer Co. Ltd., Xuzhou 221300, China)

Online:2021-01-18 Published:2021-01-27

摘要/Abstract

摘要： 分离和鉴定产生广谱抗菌活性物质的菌株，并对抗菌物质分离鉴定。通过形态学、生理生化特征和16S rDNA序列分析鉴定菌株。采用紫外线-硫酸二乙酯化学复合诱变的方法，获得抗菌能力强、遗传稳定的高产菌株。分别用甲醇提取、凝胶过滤色谱（Sephadex G-10）和半制备高效液相色谱分离纯化抗菌物质，基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry，MALDI-TOF-MS）技术对抗菌物质鉴定。本研究从土壤中分离出67 株芽孢杆菌。其中8 株表现出较强的抗菌作用，菌株XF32表现出显著广谱抑制活性，并鉴定为地衣芽孢杆菌（Bacillus licheniformis）。菌株XF32对金黄色葡萄球菌、枯草芽孢杆菌、蜡样芽孢杆菌、大肠杆菌、沙门氏菌、志贺氏菌、铜绿假单胞菌7 种病原菌具有抗菌活性，对酿酒酵母、黑曲霉、深绿木霉和尖孢镰刀菌有抗真菌活性。经诱变筛选得到突变菌株XF32-22，对金黄色葡萄球菌的抑菌活性比原始菌株显著提高，分离纯化抗菌物质，得到单一的抗菌成分。MALDI-TOF-MS分析表明抗菌物质为Fengycin系脂肽化合物。采用复合诱变技术，可以有效地提高地衣芽孢杆菌脂肽的产量。成功分离得到对多种病原细菌具有较强抑制作用的Fengycin类脂肽，同时该菌株在拮抗植物病原真菌方面也有明显效果。XF32-22菌株在食品保鲜和农业生物防治方面具有很好的应用前景。

关键词: 地衣芽孢杆菌；诱变；抗菌；纯化；脂肽

Abstract: The aims of this study were (1) to isolate and characterize bacterial strains capable of producing broad-spectrum antimicrobial substances and (2) to separate and identify these antimicrobial compounds. Strain identification was performed based on morphology, physiological and biochemical characteristics and 16S rDNA sequence analysis. To obtain genetically stable strains able to produce a high yield of antimicrobial compounds, we carried out mutagenesis by ultraviolet irradiation combined with diethyl sulfate treatment. Then, the antimicrobial substances were isolated and purified sequentially using methyl alcohol extraction, gel filtration chromatography (Sephadex G-10) and semi preparative high performance liquid chromatography (HPLC), and were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). In this study, a total of 67 Bacillus strains were isolated from soils, eight of which showed strong antimicrobial effects, including strains XF32, which showed significant broad-spectrum antimicrobial and was identified as B. licheniformis. It displayed an antibacterial activity against seven pathogenic bacterial species, including Staphylococcus aureus, B. subtilis, B. cereus, Escherichia coli, Salmonella sp., Shigella sp., and Pseudomonas aeruginosa. The strain also exhibited antifungal activity against Saccharomyces cerevisiae, Aspergillus niger, Trichoderma atroviride and fusarium oxysporum. Finally, mutant XF32-22 was obtained, whose antibacterial activity against S. aureus was increased significantly as compared to that of the original strain (XF32). Then, one antimicrobial substance was isolated and purified from the fermentation broth of XF32-22, and was identified as fengycin, a lipopeptide. The mutant produced significantly more fengycin than did its parental strain. Fengycin could strongly inhibit multiple bacterial pathogens as well as plant pathogenic fungi. Mutant XF32-22 holds great promise for application in food preservation and agricultural biological control.

Key words: Bacillus licheniformis; mutagenesis; antifungal activity; purification; lipopeptides

中图分类号:

Q819

高兆建，王秋芬，丁飞鸿，许祥，赵宜峰，焦魏，陈腾. 广谱拮抗菌株的筛选诱变及抗菌物质分离鉴定[J]. 食品科学, 2021, 42(2): 143-150.

GAO Zhaojian, WANG Qiufen, DING Feihong, XU Xiang, ZHAO Yifeng, JIAO Wei, CHEN Teng. Screening and Mutagenesis of Broad-Spectrum Antagonistic Bacillus licheniformis and Purification and Identification of Antimicrobial Substances Produced by Its Mutant[J]. FOOD SCIENCE, 2021, 42(2): 143-150.

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广谱拮抗菌株的筛选诱变及抗菌物质分离鉴定

Screening and Mutagenesis of Broad-Spectrum Antagonistic Bacillus licheniformis and Purification and Identification of Antimicrobial Substances Produced by Its Mutant

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