驴源短小芽孢杆菌LV2分离鉴定及其生物学特性
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摘要
通过分离培养和形态特征从3头12月龄健康德州驴盲肠内容物样品中分离到芽孢杆菌,采用生理生化和分子生物学方法鉴定分离菌,研究了分离菌的生长曲线、产纤维素酶活性、抗菌活性、药物敏感性及安全性等生物学特性。结果显示,分离到革兰阳性芽孢杆菌1株,鉴定为短小芽孢杆菌,编号为LV2;该菌可降解羧甲基纤维素,产生清晰透明圈,对奶牛乳房炎金黄色葡萄球菌具有较强的抑制作用;在测定的20种临床常用抗生素中仅对林可霉素表现为中度敏感,对其他抗生素均表现为敏感;急性毒性试验和慢性毒性试验结果表明该菌株安全性良好。结果表明,本试验分离到的驴源短小芽孢杆菌具有安全、产纤维素酶、抑制金黄色葡萄球菌等益生活性,在研发驴用微生态制剂或生物发酵粗饲料领域具有潜在的开发价值。
The goal of this work is to isolate Bacillus for studying probiotics of donkey.By means of isolation,culture and morphological characteristics,Bacillus was isolated from caecum content samples of three healthy Dezhou donkeys,with one year old.Isolates was identified by physiological and biochemical characteristics and molecular biology identification.Biological characteristics,such as growth curve,cellulase production,antimicrobial activity,drug susceptibility and safety,was studied by corresponding method.One of the isolates was identified as Gram-positive bacillus,Bacillus pumilus numbering LV2,which could degrade carboxymethyl cellulose and produce clear and transparent circle.Bacillus pumilus LV2 could inhibit Staphylococcus aureus causing cow mastitis.Drug susceptibility results showed that it was only intermediate to lincomycin,and sensitive to other 19 commonly clinically used antibiotics.Acute toxicity test and chronic toxicity test showed that the strain was safe.The Bacillus amyloliquefaciens isolated from this study has the advantages of safety,producing cellulas,inhibiting Staphylococcus aureus,which has potential value in the probiotics and biofermented feed for donkey.
The goal of this work is to isolate Bacillus for studying probiotics of donkey.By means of isolation,culture and morphological characteristics,Bacillus was isolated from caecum content samples of three healthy Dezhou donkeys,with one year old.Isolates was identified by physiological and biochemical characteristics and molecular biology identification.Biological characteristics,such as growth curve,cellulase production,antimicrobial activity,drug susceptibility and safety,was studied by corresponding method.One of the isolates was identified as Gram-positive bacillus,Bacillus pumilus numbering LV2,which could degrade carboxymethyl cellulose and produce clear and transparent circle.Bacillus pumilus LV2 could inhibit Staphylococcus aureus causing cow mastitis.Drug susceptibility results showed that it was only intermediate to lincomycin,and sensitive to other 19 commonly clinically used antibiotics.Acute toxicity test and chronic toxicity test showed that the strain was safe.The Bacillus amyloliquefaciens isolated from this study has the advantages of safety,producing cellulas,inhibiting Staphylococcus aureus,which has potential value in the probiotics and biofermented feed for donkey.
引文
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[14] 汪敬鑫.棉花黄萎病菌拮抗菌的筛选及XSZ5菌株抑菌机制的初步研究[D].安徽合肥:安徽农业大学,2009.
[15] 高坤.短小芽孢杆菌NDY-10的分离、鉴定及抑菌活性和培养条件的研究[D].内蒙古呼和浩特:内蒙古大学,2015.
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[17] 庞浩,陈燕,吴倩倩,等.地衣芽孢杆菌(Bacillus licheniformis)外切葡聚糖酶CelB基因的发掘及功能鉴定[J].生物技术通报,2013(9):157-163.
[18] 张宏梅,黄绍松,李发俊,等.发酵食品中益生菌对抗生素耐药性的研究进展[J].食品与发酵工业,2009(7):119-122.
[19] 韦露,陈偿,龙云映,等.1株短小芽孢杆菌B1的筛选鉴定及其抗菌特性研究[J].水产科学,2015,34(3):161-168.
[2] 赵金香,赵义龙,姚冰冰,等.厌氧培养箱法对新疆驴盲肠中纤维素分解菌的初步分离与鉴定[J].黑龙江畜牧兽医,2014(10):61-63.
[3] 赵红艳,孙泽威.马属动物肠道微生物的研究进展[J].经济动物学报,2014,18(1):59-62.
[4] 曹广添,占秀安,张玲玲,等.解淀粉芽孢杆菌对肉鸡生长性能、小肠发育和血清生化指标及免疫功能的影响[J].中国畜牧杂志,2016,52(3):31-35.
[5] 朱博.解淀粉芽孢杆菌对仔猪生长性能、抗氧化能力、免疫功能和肠道菌群的影响[D].江苏南京:南京农业大学,2016.
[6] 郑敏.芒果炭疽病挥发性抑菌物质产生菌的筛选及拮抗机理研究[D].山东泰安:山东农业大学,2013.
[7] CHOWDHURY S P,UHL J,GROSCH R,et al.Cyclic lipopeptides of Bacillus amyloliquefaciens subsp.plantarum colonizing the lettuce rhizosphere enhance plant defense responses toward the bottom rot pathogen Rhizoctonia solani[J].Mol Plant Microbe Interact,2015,28(9):984.
[8] KANG S M,RADHAKRISHNAN R,LEE I J.Bacillus amyloliquefaciens subsp.plantarum GR53,a potent biocontrol agent resists Rhizoctonia disease on Chinese cabbage through hormonal and antioxidants regulation[J].World J Microbiol Biotechnol,2015,31(10):1517-1527.
[9] KADAIKUNNAN S,REJINIEMON T S,KHALED J M,et al.In vitro antibacterial,antifungal,antioxidant and functional properties of Bacillus amyloliquefaciens[J].Ann Clin Microbiol Antimicrob,2015,14(1):9.
[10] GUAN Z B,SONG C M,ZHANG N,et al.Overexpression,characterization,and dye-decolorizing ability of a thermostable,pH-stable,and organic solvent-tolerant laccase from Bacillus pumilus W3[J].J Mol Catal B Enzym,2014,101(1/2):1-6.
[11] 王岩,陈楠楠,侯美如,等.解淀粉芽孢杆菌SSY1株的安全性试验[J].中国畜牧兽医,2017,44(3):928-934.
[12] 布坎南.伯杰细菌鉴定手册[M].北京:科学出版社,1984.
[13] 曹凤明,沈德龙,李俊,等.应用多重 PCR 鉴定微生物肥料常用芽孢杆菌[J].微生物学报,2008,48(5):651-656.
[14] 汪敬鑫.棉花黄萎病菌拮抗菌的筛选及XSZ5菌株抑菌机制的初步研究[D].安徽合肥:安徽农业大学,2009.
[15] 高坤.短小芽孢杆菌NDY-10的分离、鉴定及抑菌活性和培养条件的研究[D].内蒙古呼和浩特:内蒙古大学,2015.
[16] 冯志珍.解淀粉芽孢杆菌FB-16的筛选及其对作物疫霉菌的抑制作用[D].陕西杨凌:西北农林科技大学,2012.
[17] 庞浩,陈燕,吴倩倩,等.地衣芽孢杆菌(Bacillus licheniformis)外切葡聚糖酶CelB基因的发掘及功能鉴定[J].生物技术通报,2013(9):157-163.
[18] 张宏梅,黄绍松,李发俊,等.发酵食品中益生菌对抗生素耐药性的研究进展[J].食品与发酵工业,2009(7):119-122.
[19] 韦露,陈偿,龙云映,等.1株短小芽孢杆菌B1的筛选鉴定及其抗菌特性研究[J].水产科学,2015,34(3):161-168.