摘要
【目的】准确高效分离红树林等海洋环境中产脂肽芽孢杆菌。【方法】将前期从红树林环境筛出具有抗菌活性的12株芽孢菌株作为研究菌株,采用溶血活性及排油作用对其中的可能产脂肽菌株进行初步筛选,并基于脂肽合成酶基因PCR扩增法对脂肽合成酶基因携带菌复筛,采用飞行时间质谱(TOF-MS)对所产脂肽进行鉴定。【结果】初步发现6株芽孢杆菌具有溶血活性,其中HY-5、HY-4和HN-9这3株具有排油能力,油层透明圈的直径分别为45、32和3 mm。PCR扩增检测到HY-5含有iturin、surfactin和bacillomycin D合成酶基因,HY-4含有iturin、surfactin合成酶基因,HN-9含有iturin合成酶基因。TOF-MS检测到HY-5和HY-4的发酵液含surfactin、iturin和bacillomycin 3种脂肽,HN-9的发酵液只含iturin。【结论】基于PCR与TOF-MS为核心的级联技术,建立一种海洋源脂肽产生菌快速筛选以及脂肽谱快速鉴定的方法,解决海洋源芽孢杆菌在常规培养基难合成脂肽造成的漏筛情况。
【Objective】To accurately and efficiently isolate lipopeptide-producing bacillus spp.from mangrove and other marine environments.【Methods】The 12 bacillus with antibacterial activity that were identified from the mangrove area in our previously study were used as research strains.Blood plate and oil drainage method were performed out to evaluate the hemolytic activity and surface activity of the fermentation,following PCR was then used to detect the four lipopeptide synthase genes.Finally,TOF-MS method was used to detect the lipopeptide components.【Results】6 bacillus with hemolytic activity were identified,and three of them have the ability to drain oil.The diameter of transparent zone of HY-4,HY-9 and HN-9 were 45,32 and 3 mm,respectively.PCR results showed that HY-5 contained iturin,surfactin and bacillomycin D synthase genes,HY-4 contained iturin and surfactin synthetase genes,and HN-9 contained iturin synthase genes.Surfactin,iturin and bacillomycin were detected by TOF-MS both in the fermentation of HY-5 and HY-4,while only iturin was detected in the fermentation of HN-9.【Conclusion】Based on the combination of PCR and TOF-MS,a cascade method was established to identify and screen marine bacillus and their lipopeptide profile rapidly.The method can solve the problem of false negative screening caused by leakage of lipopeptides produced by marine bacillus in conventional medium.
作者
邓旗
花梅芳
杨秀文
孙力军
蒲月华
王雅玲
廖建萌
刘颖
房志家
DENG Qi;HUA Mei-fang;YANG Xiu-wen;SUN Li-jun;PU Yue-hua;WANG Ya-ling;LIAO Jian-meng;LIU Ying;FANG Zhi-jia(College of Food Science and Technology,Guangdong Ocean University//Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety//Guangdong Province Engineering Laboratory for Marine Biological Products//Guangdong Provincial Engineering Technology Research Center of Seafood//Key Laboratory of Advanced Processing of Aquatic Product of Guangdong Higher Education Institution,Zhanjiang 524088,China;Collaborative Innovation Center of Seafood Deep Processing,Dalian Polytechnic University,Dalian 116034,China;Guangdong Institute of Special Equipment Inspection and Research Zhanjiang Branch,Zhanjiang 524088,China;Zhanjiang Institute of Food and Drug Control,Zhanjiang 524022,China)
出处
《广东海洋大学学报》
CAS
北大核心
2021年第1期72-80,共9页
Journal of Guangdong Ocean University
基金
广东海洋大学科研启动经费资助项目(R19050)
广东省现代农业产业共性关键技术研发创新团队建设项目-水产品质量安全与环境协调关键技术团队(2019KJ151)
广东省大学生创新项目(S202010566021)。
关键词
海洋
脂肽
PCR扩增
芽孢杆菌
质谱
marine
lipopeptide
PCR amplification
bacillus
mass spectrum