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

doi:10.7506/spkx1002-6630-20191112-161

Abstract

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

CLC Number:

Q819

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

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