Bacillus amyloliquefaciens MBRC1的鉴定与抗菌活性研究
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摘要
从台州市剑门港海区的海底淤泥中采集样品分离得到4株具抗菌活性的微生物,其中一株具有较强的抗菌活性。对该菌作了形态学、生理生化、分子生物学、(G+C)mol%含量等研究,确定该菌株属于芽孢杆菌属,命名为Bacillus amyloliquefaciens MBRC1。采用BLAST Clustal W(V1.83)及Mega(V 5.1)等软件对其进行了系统发育树的分析,确立了该菌株在系统发育上的地位。采用不同的培养基对该菌株进行发酵培养,利用牛津杯法对该菌胞外的抑菌活性物质的抑菌效果进行了研究,结果显示:Bacillus amyloliquefaciens MBRC1胞外具有较强的抑菌活性物质,且对金黄色葡萄球菌(S. aureus)、枯草杆菌(B. subtilis)和大肠杆菌(E. coli)均有较好的抑菌效果。
Four bacterial strains with antibacteria activity are isolated from Jianmen harbour seafloor mud in Taizhou of Zhejiang Province,China. Thus,further studying on one of the stronger antibacteria activity strain is undertaken. This strain belongs to the genus Bacillus amyloliquefaciens,named as Bacillus amyloliquefaciens MBRC1 through molecular biology,(G+C) mol% content,colony and morphological identification. The separation position also has been identified by the analysis on phylogenetic tree with such softwares:BLAST Clustal W(V 1.83) and Mega(V 5.1).Fermented Bacillus amyloliquefaciens MBRC1 with different media and then investigates the effect of antimicrobial in extra using the method of oxford cup. The results show that Bacillus amyloliquefaciens MBRC1 could generate better antimicrobial substances and has good antimicrobial effect on S. Aureus、B. Subtilis and E. coli.
Four bacterial strains with antibacteria activity are isolated from Jianmen harbour seafloor mud in Taizhou of Zhejiang Province,China. Thus,further studying on one of the stronger antibacteria activity strain is undertaken. This strain belongs to the genus Bacillus amyloliquefaciens,named as Bacillus amyloliquefaciens MBRC1 through molecular biology,(G+C) mol% content,colony and morphological identification. The separation position also has been identified by the analysis on phylogenetic tree with such softwares:BLAST Clustal W(V 1.83) and Mega(V 5.1).Fermented Bacillus amyloliquefaciens MBRC1 with different media and then investigates the effect of antimicrobial in extra using the method of oxford cup. The results show that Bacillus amyloliquefaciens MBRC1 could generate better antimicrobial substances and has good antimicrobial effect on S. Aureus、B. Subtilis and E. coli.
引文
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[2]ZHANG Min,LIU Qian. Study on present situation and development trends of fruit&vegetable preservation in the world[J].Journal of Food Science and Biotechnology,2014,33(8):785-792.(in Chinese)
[3]ZHANG Baojun,ZHANG Jiarong,HAN Jucai,et al. Purification and characterization of an antifungal protein from endophytic Bacillus amyloliquefaciens LP-5[J]. Acta Phytophylacica Sinica,2010,37(2):143-147.(in Chinese)
[4]LIU Qiao,QUAN Chunshan,FAN Shengdi. Extraction of antimicrobial substances from fermentation of Bacillus amyloliquefaciens Q-12 by ultra filtration[J]. Food Science and Technology,2006,32(7):70-72.(in Chinese)
[5]XIN Shanshan,QI Gaofu,ZHU Fayin,et al. Research on optimization of fermentation medium of Bacillus amyloliquefaciens and its control effect to colletotrichum gloeosporioides[J]. Journal of Huazhong Agricultural University,2011,30(4):411-415.(in Chinese)
[6]CALDEIRA A T,ARTEIRO J M S,COELHO A V,et a1. Combined use of LC-ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI 1051[J]. Process Bidchemistry,2011,46(9):1738-1746.
[7]YU G Y,SINCLAIR J B,HARTMAN G L,et a1. Production of iturin A by Bacillus amyloliquefaciens suppressing rhizoctonia solani[J]. Soil Biology and Biochemistry,2002,34(7):955-963.
[8]RAO Y K,LIN H Y,WU W S,et a1. Evaluation of HPLC and MEKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens[J]. International Journal of Applied Science and Engineering,2008,6(2):85-96.
[9]CHEN X H,KOUMOUTSIA A,SCHOLZA R,el a1. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens[J]. Journal of Biotechnology,2009,140(1/2):27-37.
[10]CHEN X H,SCHOLZ R,BORRISS M,et a1. Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease[J]. Journal of Biotechnology,2009,140(1/2):38-44.
[11]SUN Yanxia,ZHAN Runqing,LIU Baoyou,et al. Control effect of Geotrichum candidum and Bacillus amyloliquefaciens on cucumber fusarium wilt[J]. Shandong Agricultural Sciences,2010(12):55-57.(in Chinese)
[12]CHEN Shiyun,YANG Baoyu,GAO Meiying,et al. Inhibition of sclerotia formation of sclerotinia sclerotiorum by Bacillus amyloliquefaciens[J]. Chinese Journal of Applied&Environmental Biology,2005,1l(3):373-376.(in Chinese)
[13]SHI Wei,XIE Weitian,ZHANG Yan,et al. Preliminary research on effect of fermentation broth of Bacillus amyloliquifaciens on fresh-keeping of chilled meat[J]. China Brewing,2011,235(10):51-53.(in Chinese).
[14]KMPFER P,YOUNG C C,SRIDHAR K,et al. Transfer of[Flexibacter]sancti,[Flexibacter]filiformis,[Flexibacter]japonensis and[Cytophaga]arvensicola to the genus Chitinophaga and description of Chitinophaga skermanii sp. nov[J]. Int J Syst Evol Micr,2006,56(9):2223-2228.
[15]R.E.布坎南,N.E.吉本斯.伯杰细菌鉴定手册[M].北京:科学出版社,1984.
[16]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994.
[17]MARMUR J. A procedure for the isolation of deoxyribonucleic acid from microorganisms[J]. J Mol Bio,1961,3(2):208-218.
[18]MESBAH M,PREMACHANDRAN U,WHITMAN W B. Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography[J]. Int J Syst Evol Micr,1989,39(2):159-167.
[19]BROSIUS J,DULL T J,SLEETER D D,et al. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli1[J]. Journal of Molecular Biology,1981,148(2):107-127.
[20]XI Fengyuan,XUE Changyan,HAO Zhikui. Research on antibacterial activity and identification of Nocardia asteroidesⅪ[J].Chinese Journal of Antibiotics,2014,39(5):394-398.(in Chinese)
[21]WEON H,KIM B,YOO S,et al. Andreprevotia chitinilytica gen. nov.,sp. nov.,isolated from forest soil from Halla Mountain,Jeju Island,Korea[J]. Int J Syst Evol Micr,2007,57(7):1572-1575.
[22]SAITOU N,NEI M. The neighbor-joining method:a new method for reconstructing phylogenetic trees[J]. Mol Biol Evol,1987,4(4):406-425.
[23]KIMURA M. A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[J]. J Mol Evol,1980,16:111-120.
[24]岑沛霖,蔡谨.工业微生物学[M].北京:化学工业出版社,2000.
[25]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[26]DOBRETSOV S,DAHMS H U,QIAN P Y. Antibacterial and antidiatom activity of Hong Kong sponges[J]. Aquat Microb Ecol,2005,38:191-201.
[27]HENTSCHEL U,SCHMID M,WAGNER M,et al. Isolation and phylogenetic analysis of bacteria with antimicrobial activeties from Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola[J]. FEMS Microbiol Ecol,2001,35:305-312.