拮抗黄曲霉海洋放线菌的筛选及发酵条件优化
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摘要
该研究从黄海海域沉积物中筛选一株能高效抑制黄曲霉(Aspergillus flavus)的海洋放线菌,通过形态观察、生理生化试验及分子生物学技术对其进行鉴定,并以抑菌圈直径为响应值,通过Plackett-Burman试验、最陡爬坡试验及响应面试验对其产抑菌活性物质的发酵条件进行优化。结果表明,筛选获得一株对黄曲霉抑制活性较高的菌株B11,并鉴定其为锈赤蜡黄链霉菌(Streptomyces rubiginosohelvolus);菌株B11产抑菌活性物质的主要影响因素为发酵温度、发酵时间及接种量,最优发酵条件为:发酵温度29℃、发酵时间11d、接种量5%。在此最优发酵条件下,抑菌圈直径达到(2.9±0.15)cm,抑菌圈直径比优化前(1.4±0.15)cm增大51.72%。
A strain of marine Actinomycetes that could effectively inhibit Aspergillus flavus was screened from the sediments of the Yellow Sea, and identified by morphological observation, physiological and biochemical tests and molecular biology techniques. Using the inhibitory zone diameter as the response value, the fermentation conditions for strain producing inhibiting active substances were optimized by Plackett-Burman tests, steepest ascent design and response surface methodology. The results showed that the strain B11 with high inhibiting activity against A. flavus was screened,and identified as Streptomyces rubiginosohelvolus. The main factors affecting the inhibiting substances production of the strain B11 were fermentation temperature, time and inoculum. The optimum fermentation conditions were fermentation temperature 29 ℃, time 11 d, inoculum 5%. Under the optimum fermentation conditions, the inhibitory circle diameter reached(2.9±0.15) cm, which was 51.72% higher than that before optimization(1.4±0.15) cm.
A strain of marine Actinomycetes that could effectively inhibit Aspergillus flavus was screened from the sediments of the Yellow Sea, and identified by morphological observation, physiological and biochemical tests and molecular biology techniques. Using the inhibitory zone diameter as the response value, the fermentation conditions for strain producing inhibiting active substances were optimized by Plackett-Burman tests, steepest ascent design and response surface methodology. The results showed that the strain B11 with high inhibiting activity against A. flavus was screened,and identified as Streptomyces rubiginosohelvolus. The main factors affecting the inhibiting substances production of the strain B11 were fermentation temperature, time and inoculum. The optimum fermentation conditions were fermentation temperature 29 ℃, time 11 d, inoculum 5%. Under the optimum fermentation conditions, the inhibitory circle diameter reached(2.9±0.15) cm, which was 51.72% higher than that before optimization(1.4±0.15) cm.
引文
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[17]梁光杰,车程川,巩志金,杨革.一株海洋链霉菌发酵条件的优化及其抑菌活性物质的研究[J].中国酿造,2018,37(12):101-105.
[18]李培京.扫描电镜生物样品制备与观察[J].现代科学仪器,2008(3):124-125.
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[22]李志华.基于PB试验和响应面分析法对谷氨酸棒杆菌CN1021发酵培养基优化[J].中国酿造,2014,33(2):23-27.
[23]熊志强,徐平,涂国全,等.利用响应面法优化红谷霉素发酵培养基[J].微生物学通报,2006,33(4):5-9.
[24]宋萍,戚小灵,谢宁昌,等.响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基[J].中国生物工程杂志,2010,30(8):100-105.
[2]VAN BURIK J A,COLVEN R,SPAEH D H.Cutaneous aspergillosis[J].J Clin Microbiol,1998,36:3115-3121.
[3]ZHANG Q Q,LI L,ZHU M,et al.Primary cutaneous aspergil-losis due to Aspergillus flavus:a case report[J].Chinese Med J,2005,18(3):255-257.
[4]邢小黑.放线菌次生代谢的分子调控研究进展[J].生物技术,1999,9(5):47-48.
[5]朱昌雄,蒋细良,孙动园,等.新农用抗生素-中生菌素[J].精细与专用化学品,2002(16):14-17.
[6]赵蕾,宋家华,杨合同,等.一种农用拮抗链霉菌的初步研究[J].中国生物防治,1998,14(1):18-20.
[7]施跃峰,桑金隆,竺利红,等.新微生物农药抑霉菌素的研究[J].核农学报,2004,18(1):68-71.
[8]SCHUMACHER R W,TALMAGE S C,MILLER S A,et al.Isolation and structure determination of an antimicrobial ester from a marine sedimentderived bacterium[J].J Nat Prod,2003,66(9):1291-1293.
[9]KLICH M A.Soil fungi of some low-altitude desert cotton fields and ability of their extracts to inhibit Aspergillus flavus[J].Mycopathologia,2003,142(2):97-100.
[10]ONO M,SAKUDA S,SUZUKI A.Aflastatin A,a novel inhibitor of aflatoxin production by aflatoxigenic fungi[J].Antibiotics,1997,50(2):111-117.
[11]刘姝,陆兆新,吕凤霞,等.一株海洋放线菌的分类鉴定及抗菌活性研究[J].南京农业大学学报,2007,30(4):124-129.
[12]HAYAKAWA M,NONOMURA H.Humic acid-vitamin agar,a new medium for the selective isolation of soil actinomycetes[J].J Ferment Technol,1987,65(5):501-509.
[13]YUAN M,YU Y,LI R,et al.Phylogenetic diversity and biological activity of actinobacteria isolated from the Chukchi Shelf marine sediments in the Arctic Ocean[J].Mar Drug,2014,12(3):1281-1297.
[14]WILLIAMS S T,GOODFELLOW M,ALDERSON G.Genus Streptomyces Waksman and Henrici 1943,339AL[J].Berg Man Syst Bacteriol,1989,4:2452-2492.
[15]ATALAN E,MANFIO GP,WARD AC,et al.Biosystematic studies on novel streptomycetes from soil[J].Anton Leeuw Int J G,2000,77(4):337-353.
[16]刘丽.耐盐短杆菌Y-1-4产脂肽类生物表面活性剂的研究[D].青岛:青岛科技大学,2016.
[17]梁光杰,车程川,巩志金,杨革.一株海洋链霉菌发酵条件的优化及其抑菌活性物质的研究[J].中国酿造,2018,37(12):101-105.
[18]李培京.扫描电镜生物样品制备与观察[J].现代科学仪器,2008(3):124-125.
[19]R E布坎南,N E吉布斯.伯杰氏细菌手册[M].北京:科学出版社,1994:241-287.
[20]中国科学院微生物研究所放线菌分类组.链霉菌鉴定手册[M].北京:科学出版社,1975:658-685.
[21]HANSEN P,BLAKESLEY R.Simple archiving of bacterial and plasmid DNAs for future use[J].Focus,1998,20(3):72-74.
[22]李志华.基于PB试验和响应面分析法对谷氨酸棒杆菌CN1021发酵培养基优化[J].中国酿造,2014,33(2):23-27.
[23]熊志强,徐平,涂国全,等.利用响应面法优化红谷霉素发酵培养基[J].微生物学通报,2006,33(4):5-9.
[24]宋萍,戚小灵,谢宁昌,等.响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基[J].中国生物工程杂志,2010,30(8):100-105.