黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1产生的脂肽类抗生素的分离和鉴别
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摘要
本文旨在对黄曲霉拮抗菌Bacillus amyloliquefaciens B10-6-1的脂肽类抗生素相关基因进行克隆,对其所产生的脂肽类抗生素进行组分分离,对有抑菌活性的脂肽类抗生素进行结构鉴别。以解淀粉芽孢杆菌B10-6-1基因组DNA为模板,采用PCR方法对ItuA、ItuB、ItuC、ItuD、FenA、FenB、FenD、Sfp、bmyA、bmyB、bmyC合成基因进行DNA扩增。将该菌发酵液经过离心、酸沉淀、甲醇抽提等步骤得到脂肽类抗生素的粗提物,采用高效液相色谱法分离,对收集的洗脱峰组分进行体外抑菌试验,对有抑菌活性的组分进行液质(HPLC-ESI-MS)分析。PCR扩增检测结果显示黄曲霉菌拮抗菌B10-6-1能扩增出ItuA、ItuC、ItuD、FenA、FenD、Sfp、bmyA、bmyB、bmyC合成基因,未能扩增出ItuB、FenB合成基因。高效液相色谱法收集到7种组分,体外抑菌试验证明组分5和7有抑菌活性。经液质测定,组分5和组分7分别为脂肽类抗生素C14Bacillocnycin D和C15Bacillocnycin D。本研究探明了黄曲霉菌拮抗菌B10-6-1所产生的天然抑菌活性成分,为该菌用于黄曲霉菌的生物防治奠定了理论基础。
The lipopeptide antibiotic related genes of Bacillus amyloliquefaciens B10-6-1 antagonistic to Aspergillus flavus were cloned, and the components of the antibiotics were separated and structurally identified. Synthesis genes ItuA, ItuB, ItuC, ItuD, FenA, FenB, FenD, Sfp, bmyA, bmyB, bmyC were amplified by PCR amplification using genomic DNA of Bacillus amyloliquefaciens B10-6-1 as template. The crude extract of lipopeptide antibiotics was obtained from the fermentation broth through centrifugation, acid precipitation and methanol extraction. The extract was separated by high performance liquid chromatography(HPLC), and antifungal activity test in vitro of the elution peak components collected was carried out. The components with antimicrobial activity were analyzed by high performance liquid chromatography combined with multi-stage electrospray tandern mass spectrometry(HPLC-ESI-MS). PCR amplification test results showed that B10-6-1 antagonistic to Aspergillus flavus was able to amplify the ItuA, ItuC, ItuD, FenA, FenD, Sfp, bmyA,bmyB, bmyC synthesis genes, but failed to amplify the ItuB, FenB synthesis genes. Seven components were collected by HPLC and antibiotic test in vitro results showed that components 5 and 7 exhibited antimicrobial activity. HPLC-ESI-MS determination results showed that component 5 is lipopeptide antibiotic C14 bacillomycin D and component 7 is lipopeptide antibiotic C15 bacillomycin D. This study laid a theoretical foundation of biological control of Aspergillus flavus by using natural antimicrobial activity of antagonistic bacteria.
The lipopeptide antibiotic related genes of Bacillus amyloliquefaciens B10-6-1 antagonistic to Aspergillus flavus were cloned, and the components of the antibiotics were separated and structurally identified. Synthesis genes ItuA, ItuB, ItuC, ItuD, FenA, FenB, FenD, Sfp, bmyA, bmyB, bmyC were amplified by PCR amplification using genomic DNA of Bacillus amyloliquefaciens B10-6-1 as template. The crude extract of lipopeptide antibiotics was obtained from the fermentation broth through centrifugation, acid precipitation and methanol extraction. The extract was separated by high performance liquid chromatography(HPLC), and antifungal activity test in vitro of the elution peak components collected was carried out. The components with antimicrobial activity were analyzed by high performance liquid chromatography combined with multi-stage electrospray tandern mass spectrometry(HPLC-ESI-MS). PCR amplification test results showed that B10-6-1 antagonistic to Aspergillus flavus was able to amplify the ItuA, ItuC, ItuD, FenA, FenD, Sfp, bmyA,bmyB, bmyC synthesis genes, but failed to amplify the ItuB, FenB synthesis genes. Seven components were collected by HPLC and antibiotic test in vitro results showed that components 5 and 7 exhibited antimicrobial activity. HPLC-ESI-MS determination results showed that component 5 is lipopeptide antibiotic C14 bacillomycin D and component 7 is lipopeptide antibiotic C15 bacillomycin D. This study laid a theoretical foundation of biological control of Aspergillus flavus by using natural antimicrobial activity of antagonistic bacteria.
引文
Anne LM,Thomas EC,Sadik T,2004.Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D.FEMSMicrobiology Letter,234:43-49
Cazorla FM,Romero D,Perez-Garcia A,Lugtenberg BJJ,de Vincente A,Bloemberg G,2007.Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocotrol activity.Journal of Applied Microbiology,103:1950-1959
Chen H,Wang L,Yuan CL,Zheng ZM,Yu ZL,2008.Separation and identification of lipopeptide compounds from Bacillus subtilis by high performance liquid chromatography-electrospray mass spectrometry.Chromatographic Column,2008(3):343-347(in Chinese)
Cvetni?Z,Pepeljnjak S,2007.Interaction between certain moulds and aflatoxin B1 producer Aspergillus flavus NRRL 3251.Archives of Industrial Hygiene and Toxicology,58(4):429-434
Deabes M,El-Soud N,El-Kassem L,2011.In vitro inhibition of growth and aflatoxin B1 production of Aspergillus flavus strain(ATCC 16872)by various medicinal plant essential oils.Macedonian Journal of Medical Sciences,4(4):345-350
Delcambe L,Devignat R,1957.L’iturine,nouvel antibiotique d’origine congolaise.Académie Royale des Sciences Coloniales,6:1-77
Denardi LB,Dalla-Lana BH,Kunz de Jesus FP,Severo CB,Santurio JM,Zanette RA,Alves SH,2017.In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil.Brazilian Journal of Infectious Diseases,22(1):e0188520
Deng JL,2009.Studies on the inhibitory effect of Bacillus amyloliquefaciens YN-1 on the active substances of plant pathogenic fungi.Master Thesis,Huazhong Agricultural University,Wuhan.1-65(in Chinese)
Gong AD,2015.Isolation and antagonistic mechanism of biocontrol bacteria against Fusarium and Aspergillus flavus.PhD Dissertation,Huazhong Agricultural University,Wuhan.1-157(in Chinese)
Gong QW,2012.Isolation and purification of antibacterial lipid peptide from Bacillus sp.and study on the inhibitory effect of bacillomycin D against Aspergillus flavus.Master Thesis,Agricultural University of Nanjing,Nanjing.1-133(in Chinese)
Hou HM,Jin Y,Jin MF,Yu XJ,Zhang W,2006.Structure function and biosynthesis of cyclic lipopeptidic biosurfactant.Microbiology China,33(5):122-128(in Chinese)
Leclere V,Bechet M,Adam A,Guez JS,Wathelet B,Ongena M,Thonart P,Gancel F,Chollet-Imbert M,Jacques P,2005.Mycosubtilin overproduction by Bacillus subtilis BBG100enhances the organism’s antagonistic and biocontrol activities.Applied and Environmental Microbiology,71(8):4577-4584
Li HY,Li SN,Wang SX,Wang Q,Li W,Zhu BC,2017.Analysis of lipopeptide antimicrobial constituents of Bacillus probiotics by high performance liquid chromatographyelectrospray tandem mass spectrometry.Journal of Animal Nutrition,29(4):1191-1197(in Chinese)
Liu LX,Gao L,Bie XM,Lu ZX,Zhang C,LüFX,2014.Reconstruction of Bacillus subtilis 168 to produce surfactin.Journal of Nanjing Agricultural University,37(6):97-102(in Chinese)
Moyne AL,Shelby R,Cleveland TE,Tuzun S,2001.Bacillomycin D:an iturin with antifungal activity against Aspergillus flavus.Journal of Applied Microbiology,90:622-629
Ongena M,Jacques P,2008.Bacillus lipoeptides:versatile weapons for plant disease biocontrol.Trends in Microbiology,6(3):115-125
Peypoux F,Bonmatin JM,Wallach J,1999.Recent trends in the biochemistry of surfactin.Applied Microbiology and Biotechnology,51(5):553-563
Qiao JQ,Liu YZ,Zhang RS,Liu YF,2018.Optimization of shaking flask fermentation process for lipopeptideproducing antibiotics of Bacillus subtilis T-500.Journal of Nanjing Agricultural University,41(3):447-455(in Chinese)
Qu XX,Liu HX,Gao L,Lu ZX,LüFX,Zhang C,Zhao HZ,Bie XM,2016.Rapid detection of lipid peptide regulatory gene from Bacillus sp.Journal of Nanjing Agricultural University,39(5):858-864(in Chinese)
Rao SQ,Chen SY,Gao L,Yin YQ,Yang ZQ,Fang WM,2015.Screening,Identification and antimicrobial characteristics of an antagonistic bacterium F1 from Aspergillus flavus.Modern Food Technology,31(12):99-105(in Chinese)
Shi CR,Zhang CS,Du YM,Yu LN,Bi J,Xu TT,Wang MQ,Feng CC,Zhang JC,Zhao SC,Sun J,2017.Effects of different plant inhibitors on the growth and toxicity of Aspergillus flavus.Journal of Quality Inspection of Food Safety,8(8):2892-2897(in Chinese)
Sui M,2018.Effects of probiotics mixed culture on the growth and toxicity of Aspergillus flavus.China Fodder,2018(11):45-48(in Chinese)
Sun Q,2016.Effects of cinnamaldehyde on growth and toxicity of Aspergillus flavus.PhD Dissertation,Chinese Academy of Agricultural Sciences,Beijing.1-112(in Chinese)
Wang DP,Meng H,Guan XL,Luo XG,2010.Identification of Bacillus amyloliquefaciens BI_2 and its inhibitory effect on Aspergillus flavus.Journal of Tianjin University of Science and Technology,25(6):5-9(in Chinese)
Wang L,Ran YP,LüYY,Shang HH,Hu YS,2017.Advances in biocontrol of aflatoxin B1.Journal of Quality Inspection of Food Safety,8(5):1527-1532(in Chinese)
Xu MQ,Cai GL,Zhu DW,Wang LL,Lu J,2015.Isolation and identification of aflatoxin B1 detoxified strain and its application in peanut meal.Chinese Oils and Fats,40(3):20-24(in Chinese)
Yu GY,Sinclair JB,Hartman GL,Bertagnolli BL,2002.Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani.Soil Biology and Biochemistry,34:955-963
陈华,王丽,袁成凌,郑之明,余增亮,2008.高效液相色谱-电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物.色谱,2008(3):343-347
邓建良,2009.解淀粉芽孢杆菌YN-1抑制植物病原真菌活性物质研究.华中农业大学硕士论文,武汉.1-65
宫安东,2015.镰刀菌和黄曲霉菌生防菌的分离及拮抗机理研究.华中农业大学博士论文,武汉.1-157
龚庆伟,2012.芽孢杆菌抗菌脂肽的分离纯化及Bacillomycin D抑制黄曲霉作用的研究.南京农业大学硕士论文,南京.1-133
侯红漫,靳艳,金美芳,虞星炬,张卫,2006.环脂肽类生物表面活性剂结构、功能及生物合成.微生物学通报,33(5):122-128
李红亚,李术娜,王树香,王全,李文,朱宝成,2017.益生芽孢杆菌脂肽类抑菌成分的高效液相色谱-电喷雾串联质谱分析.动物营养学报,29(4):1191-1197
刘丽霞,高玲,别小妹,陆兆新,张充,吕凤霞,2014.Bacillus subtilis 168菌株产surfactin改造.南京农业大学学报,37(6):97-102
乔俊卿,刘邮洲,张荣胜,刘永锋,2018.枯草芽胞杆菌T-500产脂肽类抗生素的摇瓶发酵工艺优化.南京农业大学学报,41(3):447-455
曲晓旭,刘洪霞,高玲,陆兆新,吕凤霞,张充,赵海珍,别小妹,2016.芽孢杆菌产抗菌脂肽调控基因快速检测.南京农业大学学报,39(5):858-864
饶胜其,陈素雅,高璐,尹永祺,杨振泉,方维明,2015.一株黄曲霉拮抗细菌F1的筛选、鉴定及其抑菌特性.现代食品科技,31(12):99-105
石程仁,张初署,杜咏梅,于丽娜,毕洁,许婷婷,王明清,冯楚楚,张建成,赵善仓,孙杰,2017.不同植物抑制剂对黄曲霉菌生长和产毒的影响.食品安全质量检测学报,8(8):2892-2897
隋明,2018.益生菌混合培养物对黄曲霉菌生长以及产毒效果的影响.中国饲料,(11):45-48
孙琦,2016.肉桂醛对黄曲霉菌生长和产毒的影响机制研究.中国农业科学院博士论文,北京.1-112
王德培,孟慧,管叙龙,罗学刚,2010.解淀粉芽孢杆菌BI_2的鉴定及其对黄曲霉的抑制作用.天津科技大学学报,25(6):5-9
王乐,冉艳朋,吕扬勇,商海红,胡元森,2017.生物法防治黄曲霉毒素B1研究进展.食品安全质量检测学报,8(5):1527-1532
徐铭乾,蔡国林,朱德伟,王璐璐,陆健,2015.黄曲霉毒素B1脱毒菌株的分离鉴定及在花生粕中的应用.中国油脂,40(3):20-24
Cazorla FM,Romero D,Perez-Garcia A,Lugtenberg BJJ,de Vincente A,Bloemberg G,2007.Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocotrol activity.Journal of Applied Microbiology,103:1950-1959
Chen H,Wang L,Yuan CL,Zheng ZM,Yu ZL,2008.Separation and identification of lipopeptide compounds from Bacillus subtilis by high performance liquid chromatography-electrospray mass spectrometry.Chromatographic Column,2008(3):343-347(in Chinese)
Cvetni?Z,Pepeljnjak S,2007.Interaction between certain moulds and aflatoxin B1 producer Aspergillus flavus NRRL 3251.Archives of Industrial Hygiene and Toxicology,58(4):429-434
Deabes M,El-Soud N,El-Kassem L,2011.In vitro inhibition of growth and aflatoxin B1 production of Aspergillus flavus strain(ATCC 16872)by various medicinal plant essential oils.Macedonian Journal of Medical Sciences,4(4):345-350
Delcambe L,Devignat R,1957.L’iturine,nouvel antibiotique d’origine congolaise.Académie Royale des Sciences Coloniales,6:1-77
Denardi LB,Dalla-Lana BH,Kunz de Jesus FP,Severo CB,Santurio JM,Zanette RA,Alves SH,2017.In vitro antifungal susceptibility of clinical and environmental isolates of Aspergillus fumigatus and Aspergillus flavus in Brazil.Brazilian Journal of Infectious Diseases,22(1):e0188520
Deng JL,2009.Studies on the inhibitory effect of Bacillus amyloliquefaciens YN-1 on the active substances of plant pathogenic fungi.Master Thesis,Huazhong Agricultural University,Wuhan.1-65(in Chinese)
Gong AD,2015.Isolation and antagonistic mechanism of biocontrol bacteria against Fusarium and Aspergillus flavus.PhD Dissertation,Huazhong Agricultural University,Wuhan.1-157(in Chinese)
Gong QW,2012.Isolation and purification of antibacterial lipid peptide from Bacillus sp.and study on the inhibitory effect of bacillomycin D against Aspergillus flavus.Master Thesis,Agricultural University of Nanjing,Nanjing.1-133(in Chinese)
Hou HM,Jin Y,Jin MF,Yu XJ,Zhang W,2006.Structure function and biosynthesis of cyclic lipopeptidic biosurfactant.Microbiology China,33(5):122-128(in Chinese)
Leclere V,Bechet M,Adam A,Guez JS,Wathelet B,Ongena M,Thonart P,Gancel F,Chollet-Imbert M,Jacques P,2005.Mycosubtilin overproduction by Bacillus subtilis BBG100enhances the organism’s antagonistic and biocontrol activities.Applied and Environmental Microbiology,71(8):4577-4584
Li HY,Li SN,Wang SX,Wang Q,Li W,Zhu BC,2017.Analysis of lipopeptide antimicrobial constituents of Bacillus probiotics by high performance liquid chromatographyelectrospray tandem mass spectrometry.Journal of Animal Nutrition,29(4):1191-1197(in Chinese)
Liu LX,Gao L,Bie XM,Lu ZX,Zhang C,LüFX,2014.Reconstruction of Bacillus subtilis 168 to produce surfactin.Journal of Nanjing Agricultural University,37(6):97-102(in Chinese)
Moyne AL,Shelby R,Cleveland TE,Tuzun S,2001.Bacillomycin D:an iturin with antifungal activity against Aspergillus flavus.Journal of Applied Microbiology,90:622-629
Ongena M,Jacques P,2008.Bacillus lipoeptides:versatile weapons for plant disease biocontrol.Trends in Microbiology,6(3):115-125
Peypoux F,Bonmatin JM,Wallach J,1999.Recent trends in the biochemistry of surfactin.Applied Microbiology and Biotechnology,51(5):553-563
Qiao JQ,Liu YZ,Zhang RS,Liu YF,2018.Optimization of shaking flask fermentation process for lipopeptideproducing antibiotics of Bacillus subtilis T-500.Journal of Nanjing Agricultural University,41(3):447-455(in Chinese)
Qu XX,Liu HX,Gao L,Lu ZX,LüFX,Zhang C,Zhao HZ,Bie XM,2016.Rapid detection of lipid peptide regulatory gene from Bacillus sp.Journal of Nanjing Agricultural University,39(5):858-864(in Chinese)
Rao SQ,Chen SY,Gao L,Yin YQ,Yang ZQ,Fang WM,2015.Screening,Identification and antimicrobial characteristics of an antagonistic bacterium F1 from Aspergillus flavus.Modern Food Technology,31(12):99-105(in Chinese)
Shi CR,Zhang CS,Du YM,Yu LN,Bi J,Xu TT,Wang MQ,Feng CC,Zhang JC,Zhao SC,Sun J,2017.Effects of different plant inhibitors on the growth and toxicity of Aspergillus flavus.Journal of Quality Inspection of Food Safety,8(8):2892-2897(in Chinese)
Sui M,2018.Effects of probiotics mixed culture on the growth and toxicity of Aspergillus flavus.China Fodder,2018(11):45-48(in Chinese)
Sun Q,2016.Effects of cinnamaldehyde on growth and toxicity of Aspergillus flavus.PhD Dissertation,Chinese Academy of Agricultural Sciences,Beijing.1-112(in Chinese)
Wang DP,Meng H,Guan XL,Luo XG,2010.Identification of Bacillus amyloliquefaciens BI_2 and its inhibitory effect on Aspergillus flavus.Journal of Tianjin University of Science and Technology,25(6):5-9(in Chinese)
Wang L,Ran YP,LüYY,Shang HH,Hu YS,2017.Advances in biocontrol of aflatoxin B1.Journal of Quality Inspection of Food Safety,8(5):1527-1532(in Chinese)
Xu MQ,Cai GL,Zhu DW,Wang LL,Lu J,2015.Isolation and identification of aflatoxin B1 detoxified strain and its application in peanut meal.Chinese Oils and Fats,40(3):20-24(in Chinese)
Yu GY,Sinclair JB,Hartman GL,Bertagnolli BL,2002.Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solani.Soil Biology and Biochemistry,34:955-963
陈华,王丽,袁成凌,郑之明,余增亮,2008.高效液相色谱-电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物.色谱,2008(3):343-347
邓建良,2009.解淀粉芽孢杆菌YN-1抑制植物病原真菌活性物质研究.华中农业大学硕士论文,武汉.1-65
宫安东,2015.镰刀菌和黄曲霉菌生防菌的分离及拮抗机理研究.华中农业大学博士论文,武汉.1-157
龚庆伟,2012.芽孢杆菌抗菌脂肽的分离纯化及Bacillomycin D抑制黄曲霉作用的研究.南京农业大学硕士论文,南京.1-133
侯红漫,靳艳,金美芳,虞星炬,张卫,2006.环脂肽类生物表面活性剂结构、功能及生物合成.微生物学通报,33(5):122-128
李红亚,李术娜,王树香,王全,李文,朱宝成,2017.益生芽孢杆菌脂肽类抑菌成分的高效液相色谱-电喷雾串联质谱分析.动物营养学报,29(4):1191-1197
刘丽霞,高玲,别小妹,陆兆新,张充,吕凤霞,2014.Bacillus subtilis 168菌株产surfactin改造.南京农业大学学报,37(6):97-102
乔俊卿,刘邮洲,张荣胜,刘永锋,2018.枯草芽胞杆菌T-500产脂肽类抗生素的摇瓶发酵工艺优化.南京农业大学学报,41(3):447-455
曲晓旭,刘洪霞,高玲,陆兆新,吕凤霞,张充,赵海珍,别小妹,2016.芽孢杆菌产抗菌脂肽调控基因快速检测.南京农业大学学报,39(5):858-864
饶胜其,陈素雅,高璐,尹永祺,杨振泉,方维明,2015.一株黄曲霉拮抗细菌F1的筛选、鉴定及其抑菌特性.现代食品科技,31(12):99-105
石程仁,张初署,杜咏梅,于丽娜,毕洁,许婷婷,王明清,冯楚楚,张建成,赵善仓,孙杰,2017.不同植物抑制剂对黄曲霉菌生长和产毒的影响.食品安全质量检测学报,8(8):2892-2897
隋明,2018.益生菌混合培养物对黄曲霉菌生长以及产毒效果的影响.中国饲料,(11):45-48
孙琦,2016.肉桂醛对黄曲霉菌生长和产毒的影响机制研究.中国农业科学院博士论文,北京.1-112
王德培,孟慧,管叙龙,罗学刚,2010.解淀粉芽孢杆菌BI_2的鉴定及其对黄曲霉的抑制作用.天津科技大学学报,25(6):5-9
王乐,冉艳朋,吕扬勇,商海红,胡元森,2017.生物法防治黄曲霉毒素B1研究进展.食品安全质量检测学报,8(5):1527-1532
徐铭乾,蔡国林,朱德伟,王璐璐,陆健,2015.黄曲霉毒素B1脱毒菌株的分离鉴定及在花生粕中的应用.中国油脂,40(3):20-24