芽孢杆菌B13的分离鉴定及其抑菌作用研究

doi:10.16431/j.cnki.1671-7236.2019.02.029

摘要/Abstract

摘要：

试验旨在发掘有效的抗性菌以替代部分抗生素，并研究其抑菌作用，以大肠杆菌K88、鼠伤寒沙门氏菌和金黄色葡萄球菌为指示菌，采用凝胶打孔法从猪肠道内容物中筛选出一株对3种病原菌均具有抑菌活性的菌株，利用形态特征观察、革兰氏染色、16S rDNA序列同源性比对鉴定菌株，并对其抑菌条件和生长条件进行优化。结果显示，试验筛选到1株对大肠杆菌K88、鼠伤寒沙门氏菌和金黄色葡萄球菌均具有较强抑制作用的菌株，经鉴定确定其为芽孢杆菌（Bacillus sp.），并命名为芽孢杆菌B13。此分离菌为革兰氏阳性菌，单生或成对生长。生长试验结果表明，该分离菌在pH为6.0，温度为34℃，以蔗糖为碳源、蛋白胨为氮源时，生长速度最快；在pH为7.0，温度为35℃，以麦芽糖为碳源、蛋白胨为氮源时，抗鼠伤寒沙门氏菌、黄色葡萄球菌和大肠杆菌K88活性最高。本试验通过对芽孢杆菌B13的特性进行研究发现，此菌株具有较好的抑菌效果，在抑菌方面具有良好的开发应用前景。

关键词: 芽孢杆菌; 分离; 鉴定; 条件优化

Abstract:

The aim of the experiment was to find a new antimicrobial bacterium,then analyze its main metabolic antimicrobial characterization.A strain was isolated from pig intestinal contents with resistance to E.coli K88,Staphylococcus aureus and Salmonella Typhimurium by gel punching.Its taxonomic status determined by colonial morphology,Gram staining and 16S rDNA sequence analysis.Then the fermentation medium and conditions for its growth and antimicrobial activity were optimized.The results showed that the isolated strain was identified as Bacillus sp.and showed an excellent antimicrobial activity against E.coli K88,Staphylococcus aureus and Salmonella Typhimurium,and then was named as Bacillus sp.B13.It was Gram-positive,single-birth or in pairs of growth.Auxanographic analysis showed that the optimal growth condition of Bacillus sp.B13 was that the pH was 6.0,temperature was 34℃,sucrose as carbon source and peptone as nitrogen source.Results of antimicrobial activity against E.coli K88,Staphylococcus aureus and Salmonella Typhimurium showed the optimal condition was that pH was 7.0,temperature was 35℃,maltose as carbon source and peptone as nitrogen source.In conclusion,Bacillus sp.B13 had good bacteriostatic activities and had great development potential applied prospects in the production.

Key words: Bacillus sp.; isolation; identification; optimization

中图分类号:

S852.61

刘韶娜, 张斌, 相德才, 赵智勇, 赵彦光. 芽孢杆菌B13的分离鉴定及其抑菌作用研究[J]. 《中国畜牧兽医》, 2019, 46(2): 573-581.

LIU Shaona, ZHANG Bin, XIANG Decai, ZHAO Zhiyong, ZHAO Yanguang. Isolatiog,Identification and Study of a Bacillus sp.B13 with High Antibiotic Character[J]. , 2019, 46(2): 573-581.

参考文献

[1] TADASU I,THOMAS B,VICTORIA R,et al.Gut microbiome and antibiotics[J].Archieves of Medical Research,2017,48(8):727-734.
[2] ZHANG Y J,LIU Q,ZHANG W M,et al.Gastrointestinal microbial diversity and short-chain fatty acid production in pigs fed different fibrous diets with or without cell wall-degrading enzyme supplementa-tion[J].Livestock Science,2018,207:105-116.
[3] HYEUN B K,RICHARD E I.The pig gut microbial diversity:Understanding the pig gut microbial ecology through the next generation high throughput sequencing[J].Veterinary Microbiology,2015,177(3-4):242-251.
[4] 柳饶波,凌泽春.猪胃肠道微生物菌群的研究现状浅谈[J].山东农业科学,2011,10:90-94. LIU R B,LING Z C.Study on microflora of pig gastrointestinal tract[J].Shandong Agricultural Sciences,2011,10:90-94.(in Chinese)
[5] 冀海龙,王红英,周英男,等.瘤胃源益生菌对小鼠生长和生理特性的影响[J].中国畜牧兽医,2018,45(9):2463-2470. JI H L,WANG H Y,ZHOU Y N,et al.Effects of rumen-derived probiotics on the growth and physiological characteristics of mice[J].China Animal Husbandry & Veterinary Medicine,2018,45(9):2463-2470.(in Chinese)
[6] 苗玉和,刘岩,闫艳丽,等.猪源乳酸杆菌的分离鉴定及生物学特性分析[J].中国兽药杂志,2011,45(8):12-14. MIAO Y H,LIU Y,YAN Y L,et al.Isolation,identification and bionomics analysis of Lactobacillus from piglets intestine[J].Chinese Journal of Veterinary Drug,2011,45(8):12-14.(in Chinese)
[7] 梁艳琼,黄兴,吴伟怀,等.解淀粉芽孢杆菌TWC2发酵条件的优化[J].中国糖科,2017,39(6):17-22. LIANG Y Q,HUANG X,WU W H,et al.Optimizing fermentation condition for Bacillus amyloliquefaciens TWC2[J].Sugar Crops of China,2017,39(6):17-22.(in Chinese)
[8] 罗璋,陈昌福,白晓慧,等.枯草芽孢杆菌BHI344培养条件的优化[J].中国饲料,2007,10:41-43. LUO Z,CHEN C F,BAI X H,et al.Optimizing culture condition for Bacillus subtilis BHI344[J].China Feed,2007,10:41-43.(in Chinese)
[9] 杜瑾,张爱君,郝建安,等.芽孢杆菌dhs-330产脂肽-糖脂混合型表面活性剂的抑菌性能[J].生物技术通讯,2017,28(6):818-822. DU J,ZHANG A J,HAO J A,et al.Antimicrobial activity of a blend-biosurfactant of lipopeptide and glycolipid produced by Bacillus sp.dhs-330[J].Letters in Biotechnology,2017,28(6):818-822.(in Chinese)
[10] 朱大恒,刘丽,喻锐,等.芽孢杆菌LWM1的分离鉴定及其对蜡样芽孢菌的抑制作用[J].中国酿造,2017,36(7):27-31. ZHU D H,LIU L,YU R,et al.Isolation and identification of Bacillus LWM1 and its inhibition effect on Bacillus cereus[J].China Brewing,2017,36(7):27-31.(in Chinese)
[11] ONIEL K,KARLY W,ROWIDA M,et al.Avis.Bacillus and Pseudomonas spp.provide antifungal activity against gray mold and Alternaria rot on blueberry fruit[J].Biological Control,2018,126:136-141.
[12] 张雪,谈诗瑶,钱斯日古楞,等.一株解淀粉芽孢杆菌的特性及抑菌活性研究[J].中国畜牧兽医,2018,45(5):1251-1257. ZHANG X,TAN S Y,QIAN S,et al.Study on the characteristics and antibacterial activity of a Bacillus amyloliquefaciens[J].China Animal Husbandry & Veterinary Medicine,2018,45(5):1251-1257.(in Chinese)
[13] 权春善,王军华,徐洪涛,等.一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究[J].微生物学通报,2006,46(1):7-12. QUAN C S,WANG J H,XU H T,et al.Identification and characterization of a Bacillus amyloliquefaciens with high antifungal activity[J].Acta Microbiologica Sinica,2006,46(1):7-12.(in Chinese)
[14] XU J H,WANG H J,ZHU Z Z,et al.Isolation and characterization of Bacillus amyloliquefaciens ZDS-1:Exploring the degradation of Zearalenone by Bacillus spp.[J].Food Control,2016,68:244-250.
[15] 张雪花,李琳玲,程华,等.解淀粉芽孢杆菌CMN1308抗真菌特性研究[J].北方园艺,2016,2:117-121. ZHANG X H,LI L L,CHENG H,et al.Characterizing a novel strain of Bacillus amyloliquefaciens CMN1308 for potential biological control in pathogenic bacterium[J].Northern Horticulture,2016,2:117-121.(in Chinese)
[16] 谭才邓,朱美娟,杜淑霞,等.抑菌试验中抑菌圈法的比较研究[J].食品工业,2016,11:122-125. TAN C D,ZHU M J,DU S X,et al.Study on the inhibition zone method in antimicrobial test[J].The Food Industry,2016,11:122-125.(in Chinese)
[17] 李静泉,高坤,许继飞,等.土壤芽孢杆菌NDD-1及其拮抗菌短小芽孢杆菌NDY-10的分离、鉴定和抑菌特性[J].安徽农业科学,2017,45(15):91-95. LI J Q,GAO K,XU J F,et al.Identification characteristics and antibacterial activity of soil Bacillus NDD-1 and its antibacterial bacterium Bacillus pumilus NDY-10[J].Journal of Anhui Agricultural Sciences,2017,45(15):91-95.(in Chinese)
[18] 段绪果,周素雅,沈镇炎,等.一株产生淀粉酶杆菌Bacillus sp.GEL-09的筛选、鉴定及发酵条件优化[J].微生物学通报,2018,45(6):1180-1189. DUAN X G,ZHOU S Y,SHEN Z Y,et al.Identification characterization and fermentation of a Bacillus amyloliquefaciens GEL-09[J].Microbiology,2018,45(6):1180-1189.(in Chinese)
[19] 严涛,朱建国,姜甜,等.一株凝结芽孢杆菌的分离筛选及产孢条件优化[J].微生物学通报,2018,45(2):238-249. YAN T,ZHU J G,JIANG T,et al.Optimization of fermentation medium and conditions for antimicrobial substances production by Bacillus amyloliquefaciens PC2[J].Microbiology,2018,45(2):238-249.(in Chinese)
[20] 张涛,李雪艳,杨红梅,等.新疆棉花黄萎病菌拮抗细菌的分离、筛选与鉴定[J].微生物学通报,2018:1-13. ZHANG T,LI X Y,YANG H M,et al.Isolation,screening and identification of antagonistic bacteria against Verticillium dahliae Kleb.in Xinjiang[J].Microbiology,2018:1-13.(in Chinese)
[21] 何红,沈兆昌,邱思鑫,等.内生拮抗枯草芽孢杆菌BS-2菌株的发酵条件[J].中国生物防治,2004,20(1):38-41. HE H,SHEN Z C,QIU S X,et al.Fermentation conditions of endophytic antagonistic strain BS-2 of Bacillus subtilis[J].Chinese Journal of Biological Control,2004,20(1):38-41.(in Chinese)
[22] 黄宇,孙宝盛,孙井梅,等.枯草芽孢杆菌发酵条件的优化[J].河南科学,2007,25(1):70-72. HUANG Y,SUN B S,SUN J M,et al.Optimizing fermentation condition for Bacillus subtilis[J].Henan Science,2007,25(1):70-72.(in Chinese)
[23] 宋卡魏,王星云,张荣意.培养条件对枯草芽孢杆菌B68芽孢产量的影响[J].中国生物防治,2007,23(3):255-259. SONG K W,WANG X Y,ZHANG R Y,et al.Culture conditions affecting spore production of Bacillus subtilis B68[J].Chinese Journal of Biological Control,2007,23(3):255-259.(in Chinese)
[24] 王健华.枯草芽孢杆菌DPG-01发酵条件的优化[J].安徽农业科学,2007,35(26):8101-8102. WANG J H.Optimization of fermentation conditions for Bacillus subtilis DPG-01[J].Journal of Anhui Agricultural Sciences,2007,35(26):8101-8102.(in Chinese)
[25] 张荣胜,梁雪杰,刘永锋,等.解淀粉芽胞杆菌Lx-11生物发酵工艺优化[J].中国生物防治学报,2013,29(2):254-262. ZHANG R S,LIANG X J,LIU Y F,et al.Optimization of fermentation process of Bacillus amyloliquefaciens Lx-11[J].Chinese Journal of Biological Contral,2013,29(2):254-262.
[26] 张淑梅,王玉霞,孟力强,等.内生解淀粉芽孢杆菌TF28液体发酵条件研究[J].东北农业大学学报,2013,44(11):19-24. ZHANG S M,WANG Y X,MENG L Q,et al.Study on liquid fermentation conditions of Bacillus amyloliquefaciens TF28[J].Journal of Northeast Agricultural University,2013,44(11):19-24.(in Chinese)
[27] 孙力军,陆兆新,别小妹,等.培养基对解淀粉芽孢杆菌ES-2菌株产抗菌脂肽的影响[J].中国农业科学,2008,41(10):3389-3398. SUN L J,LU Z X,BIE X M,et al.Influence of medium on antimicrobial lipopeptide production by Bacillus amyloliquefaciens ES-2[J].Scientia Agricultura Sinica,2008,41(10):3389-3398.(in Chinese)
[28] 申光辉,郑丽君,张志清,等.解淀粉芽胞杆菌PC2产抑菌物质培养基及发酵条件优化[J].微生物学通报,2017,44(6):1358-1369. SHEN G H,ZHENG L J,ZHANG Z Q,et al.Optimization of fermentation medium and conditions for antimicrobial substances production by Bacillus amyloliquefaciens PC2[J].Microbiology,2017,44(6):1358-1369.(in Chinese)
[29] 卢彩鸽,董红平,张殿朋,等.解淀粉芽胞杆菌MH71摇瓶发酵培养基及发酵条件优化[J].中国生物防治学报,2015,31(3):369-377. LU C G,DONG H P,ZHANG D P,et al.Optimization the flask-shaking fermentation medium and conditions of Bacillus amylolyticus MH71[J].Chinese Journal of Biological Control,2015,31(3):369-377.(in Chinese)
[30] 周映华,李秋云,增艳,等.益生菌蜡状芽孢杆菌发酵条件及培养基的优化[J].饲料研究,2007,7:56-58. ZHOU Y H,LI Q Y,ZENG Y,et al.Optimization of fermentation conditions and culture condition for Bacillus cereus[J].Feed Research,2007,7:56-58.(in Chinese)