枯草芽孢杆菌YN145的发酵条件优化及活性物质分析  

Optimization of fermentation conditions and analysis of active substances of Bacillus subtilis YN145

作　　者：毛思业 周瑚 郑昕威 梁海迪 周佳 郑晶晶 刘二明 任佐华 MAO Siye;ZHOU Hu;ZHENG Xinwei;LIANG Haidi;ZHOU Jia;ZHENG Jingjing;LIU Erming;REN Zuohua(College of Plant Protection,Hunan Provincial Key Laboratory of Plant Pest and Disease Biology and Prevention and Control,Hunan Agricultural University,Changsha 410128,China;Hunan Yueyang City Inspection and Testing Center,Yueyang 414000,Hunan,China)

机构地区：[1]湖南农业大学植物保护学院植物病虫害生物学与防控湖南省重点实验室,长沙410128 [2]湖南省岳阳市检验检测中心,湖南岳阳414000

出　　处：《农药》2025年第2期106-114,141,共10页Agrochemicals

基　　金：国家自然科学基金青年基金(32102286);湖南省重点领域研发计划(2023NK2024)。

摘　　要：[目的]Bacillus subtilis YN145是一株对稻瘟病菌具有显著的拮抗作用的拮抗菌株。为推动该菌株在生产实际中的运用,挖掘其潜在的抑菌活性物质和最适的发酵条件。[方法]以菌株发酵液OD600值和菌株对稻瘟病菌的抑菌率为评价指标,将单因素试验法、正交试验和响应面试验相结合,探寻该菌株的最优发酵条件;并基于antiSMASH注释结果,采用气相色谱-质谱联用技术(HPLC-MS)对菌株YN145的活性物质进行分析。[结果]菌株YN145最佳发酵培养基配方为黄豆粉10 g/L、蔗糖10 g/L、胰蛋白胨3 g/L、CaCl24 g/L、CaCO31 g/L,最佳发酵条件为发酵温度28℃、转速220 r/min、培养时间48 h、接种量3%、瓶装量40 mL/250mL,在此条件下菌株单位体积中的菌体浓度提升,对稻瘟病菌的抑菌率提升12.5%。antiSMASH注释结果显示,菌株YN145基因组含有10个次级代谢产物基因簇,包含5种与抑菌作用相关基因簇,分别是surfactin、bacillibactin、fengycin、bacillaene和bacilysin。通过HPLC-MS定性分析发现菌株含10个表面活性素(surfactin)同系物、2个丰原素(fengycin)同系物、2个环酯类抗生素(plipastatin)同系物以及嗜铁素(bacillibactin)和多烯类抗生素(bacillaene)等16个同抑菌作用相关的化合物。分析结果大致对应antiSMASH注释结果,确定菌株YN145能产生多种与抑菌代谢相关的化合物,推测该菌株能通过这些化合物协同作用来抑制稻瘟病菌的生长,为深入了解生防菌YN145的抑菌活性物质提供了参考,为帮助菌株进一步走向生产实践提供理论依据。[Aims]Bacillus subtilis YN145 is a strain of bacterium with significant antagonistic effects against rice blast fungus.To promote the utilization of this strain in production practice,its potential fungistatic active substances and optimal fermentation conditions were explored.[Methods]Using the OD600value of the fermentation broth and the inhibition rate of the strain against Magnaporthe oryzae as the evaluation indexes,the optimal fermentation conditions of the strain were explored by combining the one-way test method,the orthogonal test and the response surface test;and based on the results of the annotation of antiSMASH,the active substances of strain YN145 were analyzed by HPLC-MS.[Results]The optimal fermentation medium formulation for strain YN145 was 10 g/L soybean flour,10 g/L sucrose,3 g/L tryptone,4 g/L CaCl2and 1 g/L CaCO3.The optimal fermentation conditions were:fermentation temperature 28℃,rotational speed 220 r/min,incubation time 48 h,inoculum volume 3%,and bottle volume 40 mL/250mL,in which the strain′s concentration of organisms in unit volume was improved.Under these conditions,the concentration per unit volume of the strain was increased,and the inhibition rate of M.oryzae was increased by 12.5%.AntiSMASH annotation results showed that the genome of strain YN145contained 10 secondary metabolite gene clusters,including 5 clusters of genes related to the inhibition of fungistatic activity,which were surfactin,bacillibactin,fengycin,bacillary and bacitracin.Qualitative analysis by HPLC-MS revealed that the strain contained 10 surfactin homologs,2 fengycin homologs,2 plipastatin homologs,and 16 fungistatic compounds including bacillibactin and bacillaene.The analysis results generally corresponded to the results of the antiSMASH annotation,and it was determined that strain YN145 could produce a variety of compounds related to fungistatic metabolism,and it was hypothesized that the strain could synergistically inhibit the growth of M.oryzae through these compounds,which provided a reference for

关 键 词：Bacillus subtilis 发酵条件优化 黑色素 次级代谢产物分析 抑菌活性物质 

分 类 号：S482.2[农业科学—农药学]