拮抗黄曲霉大花杓兰内生细菌的筛选及抑菌研究  

Screening of endophytic bacteria from Cypripedium macranthum and antifungal activity against Aspergillus flavus

作　　者：付亚娟[1,2,3] 许笑晴 范佳宁 樊双虎 韩卉 侯晓强 FU Yajuan;XU Xiaoqing;FAN Jianing;FAN Shuanghu;Han Hui;HOU Xiaoqiang(College of Life Science,Langfang Normal University,Langfang 065000,China;Technical Innovation Center for Utilization of Edible and Medicinal Fungi in Hebei Province,Langfang 065000,China;Key Laboratory of Biological Sample Analysis and Pesticide Residue Detection,Langfang 065000,China)

机构地区：[1]廊坊师范学院生命科学学院,河北廊坊065000 [2]河北省食药用菌资源高值利用技术创新中心,河北廊坊065000 [3]廊坊市生物样品分析及农残检测重点实验室,河北廊坊065000

出　　处：《食品与发酵工业》2024年第18期154-160,共7页Food and Fermentation Industries

基　　金：河北省高等学校科学研究重点项目(ZD2020194);河北省自然科学基金面上项目(H2020408003)。

摘　　要：为获得对黄曲霉菌丝生长具有良好抑菌活性的细菌资源,该研究以31株大花杓兰内生细菌为对象,通过平板对峙法和菌丝生长速率法筛选拮抗黄曲霉的内生细菌;菌丝生长速率法测定无菌发酵液对黄曲霉抑菌活性的稳定性;PCR扩增芽孢杆菌脂肽合成酶基因和多聚酮合酶基因;酸沉醇提制备芽孢杆菌脂肽粗提物,牛津杯法评估其对黄曲霉的抑菌活性。结果显示,3株大花杓兰内生芽孢杆菌(CmR-1n、CmR-3a、CmR-4b)对黄曲霉有较好抑菌活性;CmR-1n对黄曲霉的抑菌效果最好,平板拮抗率为57.88%,而CmR-1n无菌发酵液对黄曲霉的抑菌率达63.31%;芽孢杆菌CmR-1n无菌发酵液具有较好的耐热性、耐酸碱性、抗紫外线性和贮藏稳定性;CmR-1n基因组含有表面活性素基因Srf、伊枯草菌素基因Itu和丰原素基因(fenB和fenD),且体外抑菌实验证实CmR-1n脂肽粗提物甲醇液对黄曲霉菌丝生长的抑制率达62.58%。推测芽孢杆菌CmR-1n合成的脂肽可能是抑制黄曲霉的主要活性物质,使其在黄曲霉防治领域具有一定的应用潜力。To obtain the endophytic bacteria of Cypripedium macranthum with efficient antagonistic effect on Aspergillus flavus,31 endophytic bacteria were screened against A.flavus through plate confrontation test and mycelia growth rate method.A series of methodologies were carried out as follows:The stability of antifungal substances test was measured by using mycelium growth rate method;the lipopeptide related and polyketide synthase genes from the genomic DNA of strain CmR-1n were amplified by PCR;the antifungal activity of the crude lipopeptide extracted from CmR-1n fermentation broth against A.flavus was evaluated by using cup and saucer method.The results showed that 3 endophytic bacteria strains(CmR-1n,CmR-3a and CmR-4b)possessed better antagonistic effects on A.flavus.Briefly,Bacillus sp.CmR-1n had the best antagonistic effect on A.flavus with the inhibition rate of 57.88%,while the inhibition rate of CmR-1n sterile fermentation broth against A.flavus reached 63.31%.Antifungal substances in CmR-1n fermentation broth had significant heat,acid,alkali,and UV resistance and storage stability.Besides,the CmR-1n genome contained gene fragments of iturin(Itu),surfactin(Srf),fengycin(fenB and fenD).Moreover,the crude lipopeptides obtained from CmR-1n fermentation broth in vitro inhibited A.flavus mycelium growth with the inhibition rate up to 62.58%.Therefore,it is speculated that lipopeptides from Bacillus sp.CmR-1n may be active substance against A.flavus.Bacillus sp.CmR-1n has certain application potential in biological prevention and control of A.flavus.

关 键 词：大花杓兰 内生芽孢杆菌 黄曲霉 脂肽粗提物 抑菌活性 

分 类 号：TS201.3[轻工技术与工程—食品科学]