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J. Hefei Univ. Tech. (Nat. Sci.)

合肥工业大学学报(自然科学版)

JOURNAL OF HEFEI UNIVERSITY OF TECHNOLOGY (NATURAL SCIENCE)

Competent Authorities: Ministry of Education of the People's Republic of China

Sponsor by: Hefei University of Technology

ISSN 1000-1646, CN 34-1001/N

Isolation of antimicrobial substances from cell-free supernatant of Bacillus velezensis A4 and study of its antibacterial effect

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贝莱斯芽孢杆菌 A4 发酵液抗菌物质的分离及抑菌作用研究

Journal Information

J. Hefei Univ. Tech. (Nat. Sci.), Dec.2025, Vol.48 No.12: 1646-1651, 1704

DOI: 10.3969/j.issn.1003-5060.2025.12.010

Authors

ZHU Hongyan, MIAO Min, SONG Wu, WU Zirui, XU Yu

(School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China)

Abstract and Keywords

Abstract: The present study aimed to isolate crude protein, crude lipopeptides, and crude polysaccharides from the cell-free supernatant of Bacillus velezensis A4 through ammonium sulfate precipitation, acid precipitation, and alcoholic extraction. The antimicrobial activities of the crude extracts were investigated through plate confrontation and kiwifruit inoculation experiments. The antibacterial active substances in components were analyzed by liquid chromatograph-mass spectrometer (LC-MS) and SDS-PAGE. The results indicated that crude protein and lipopeptides possessed substantial inhibitory effects on four distinct types of kiwifruit soft rot pathogens. In vitro inhibition assays showed that crude protein had the most potent inhibitory ability against $ Epicoccus\ nigrum $, while crude lipopeptides demonstrated the most significant inhibitory effect on Alternaria alternata. Furthermore, crude protein and lipopeptides effectively suppressed the expansion of kiwifruit lesions caused by pathogens. The LC-MS analysis revealed that the active constituents present in crude lipopeptides consisted of surfactants, iturin, and fengycin, and the results obtained from SDS-PAGE demonstrated that the substances that exhibited antibacterial activities in the crude protein were two specific protein molecules that weighed 20 kDa and 40 kDa. This study provides theoretical basis and technical support for the use of Bacillus velezensis in preventing and controlling plant pathogenic fungi.

Keywords: Bacillus velezensis; cell-free supernatant; antimicrobial substances; isolation

作者信息

祝红艳，苗敏，宋武，吴子睿，徐雨

（合肥工业大学食品与生物工程学院，安徽合肥230601）

摘要和关键词

摘要: 文章分别利用硫酸铵分级沉淀、酸沉法、醇提法从贝莱斯芽孢杆菌（Bacillus velezensis）A4的无细胞上清液中分离粗蛋白、粗脂肽、粗糖，通过平板对峙和猕猴桃接菌实验探究粗提物质的抑菌活性，并利用液相色谱质谱联用仪（liquid chromatograph-mass spectrometer，LC-MS）和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析组分中的抗菌活性物质。结果表明粗蛋白和粗脂肽对4种猕猴桃软腐病原菌具有显著抑制作用；体外平板抑制实验显示，粗蛋白对黑附球菌（Epicoccus nigrum）的抑制能力最强，粗脂肽对链格孢菌（Alternaria alternata）的抑制效果最显著，且粗蛋白和粗脂肽有效地抑制了病原菌引起的猕猴桃病斑扩展；LC-MS分析显示，粗脂肽的活性成分包括表面活性素、伊枯草菌素、芬荠素；SDS-PAGE结果表明，粗蛋白中发挥抗菌活性的物质是分子量为20、40 kDa的2个蛋白分子。研究结果为生防菌贝莱斯芽孢杆菌在防治植物病原真菌等方面提供了一定的理论依据和技术支持。

关键词: 贝莱斯芽孢杆菌；无细胞上清液；抗菌物质；分离

Funding

国家自然科学基金资助项目（31970345）

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