动物粪便中纤维素分解菌的分离和鉴定
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摘要
纤维素分解菌可用于动物微生态添加剂和除臭剂。此试验采用羧甲基纤维素平板法及刚果红染色法,从鹅的粪便中初步分离筛选出7株能够降解纤维素的细菌,结合摇瓶发酵法进行复筛,最终选出3株羧甲基纤维素酶活性高的菌株,并对其进行形态学和16SrRNA鉴定。结果表明:3株纤维素分解菌的菌落形态不一,但其表面均微粗糙,革兰氏染色均呈阳性,短杆状或细杆状,有芽孢;结合16SrRNA鉴定结果,最终将分离到3个菌株鉴定为芽孢杆菌属的地衣芽孢杆菌、蜡样芽孢杆菌和沙福芽孢杆菌,分别命名为Bacillus licheniformis G-1、Bacillus cereus G-4和Bacillus safensis G-10。
Cellulolytic bacteria can be used in animal micro-ecological agent and the deodorant.In this experiment,seven strains of cellulolytic bacteria from the goose's feces with the carboxymethyl cellulose medium and Congo red staining were isolated,and three strains of bacteria with high carboxymethyl cellulase activity were screened finally through re-screening with the method of the shake flask fermentation.At the same time,they were identified through the method of the morphological and 16 SrRNA genes.The results show that the three strains of cellulolytic bacteria isolated have different colony characteristics,but their surfaces all slightly rough,Gram stain displaying positive with short or thin rod,and with spores.Based on the results of 16 SrRNA gene molecular identification,the three strains are identified as Bacillus licheniformis,Bacillus cereus and Bacillus safensis,which are separately named as Bacillus licheniformis G-1,Bacillus cereus G-4 and Bacillus safensis G-10.
Cellulolytic bacteria can be used in animal micro-ecological agent and the deodorant.In this experiment,seven strains of cellulolytic bacteria from the goose's feces with the carboxymethyl cellulose medium and Congo red staining were isolated,and three strains of bacteria with high carboxymethyl cellulase activity were screened finally through re-screening with the method of the shake flask fermentation.At the same time,they were identified through the method of the morphological and 16 SrRNA genes.The results show that the three strains of cellulolytic bacteria isolated have different colony characteristics,but their surfaces all slightly rough,Gram stain displaying positive with short or thin rod,and with spores.Based on the results of 16 SrRNA gene molecular identification,the three strains are identified as Bacillus licheniformis,Bacillus cereus and Bacillus safensis,which are separately named as Bacillus licheniformis G-1,Bacillus cereus G-4 and Bacillus safensis G-10.
引文
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[22]徐爱蕾,黄毅,王为.16SrRNA基因序列分析技术在肠道菌群检测中的应用进展[J].临床消化病杂志,2015,27(3):186-189.
[23]易旻,杨玉婷,李梦霖,等.一株耐高温纤维素降解菌的分离筛选及鉴定[J].吉首大学学报(自然科学版),2017,38(4):72-77.
[24]康纪婷,吴翔,甘炳成,等.纤维素酶活力测定方法[J].河北农业科学,2010,22(4):151-153.
[25]孙盈,田永强,赵丽坤.纤维素酶的CMC酶活测定条件的研究[J].食品工业科技,2013,35(2):68-71,74.
[2]黄国欣,梁自超,孙瑞涛,等.纤维素分解菌在反刍动物生产中的应用研究[J].中国奶牛,2017,35(4):5-8.
[3]熊冬梅,周红丽.纤维素降解菌群的研究进展[J].酿酒科技,2011,32(5):94-97.
[4]张盼,刘婉瑜,李晓秀,等.高效纤维素优势分解菌的筛选和鉴定[J].中国土壤与肥料,2017,54(2):149-156.
[5]杨伟平,孟凡旭,马丽,等.藏香猪源纤维素分解菌的分离鉴定及酶学特性分析[J].动物营养学报,2014,26(3):620-629.
[6]刘宇,李阳,尹珺伊,等.一株产纤维素酶的解淀粉芽孢杆菌的分离鉴定及其酶促反应适宜条件初步研究[J].家畜生态学报,2017,38(4):58-62.
[7]黄旺洲.高效纤维素分解菌群对猪粪和牛粪降解能力与除臭效果的影响[D].兰州:甘肃农业大学,2016.
[8]何伟聪,董晓慧,谭北平,等.益生菌对军曹鱼幼鱼生长性能、消化酶和免疫酶活性的影响[J].动物营养学报,2015,27(12):3 821-3 830.
[9]王永芬,赵志军,席磊,等.单一菌种与复合菌种益生菌制剂对蛋鸡生产效果研究[J].中国畜牧兽医,2011,38(11):28-32.
[10]戴求仲,王向荣,蒋桂韬,等.日粮中添加芽孢杆菌制剂对黄羽肉鸡生长性能及养分利用率的影响[J].家畜生态学报,2011,32(4):18-22.
[11]赫忠睿.日粮纤维对吉林白鹅消化功能及微生物多样性的影响[D].长春:吉林农业大学,2015.
[12]TEATHER R M,WOOD P J.Use of Congo red polysaccharide interactions complex formation between Congo red and polysaccharide in detection and assay of polysaccharide hydrolases[J].Methods Enzymol,1982,90(160):59-74.
[13]中华人民共和国农业部.饲料添加剂纤维素酶活力的测定分光光度法:NY/T912-2004[S].北京:中国农业出版社,2005.
[14]钱存柔,黄仪秀.微生物学实验教程[M].2版.北京:北京大学出版社,2013.
[15]刘箭.分子生物学及基因工程实验教程[M].2版.北京:中国科学出版社,2012.
[16]SIPOS R,SZ?KELY AJ,PALATINSZKY M,et al.Effect of primer mismatch,annealing temperature and PCR cycle number on 16SrRNA gene‐targetting bacterial community analysis[J].FEMS Microbiol,2007;60(2):341-350.
[17]LEE CK,JANG MY,PARK HR,et al.Cloning and characterization of xylanase in cellulolytic Bacillus sp strain JMY1isolated from forest soil[J].ApplBiolChem,2016,59(3):415-423.
[18]KASANA RC,SALWAN R,DHAR H,et al.A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine[J].CurrMicrobiol,2008,57(5):503-507.
[19]MISHRA VK,RAWAT V,RANA S,et al.Isolation and screening of cellulose degrading microorganisms and evaluation of its cellulolytic activity[J].J Pure ApplMicrobio,2015,9(2):1 033-1 038.
[20]MAKI M,LEUNG KT,QIN WS.The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosicbiomass[J].Int J BiolSci,2009,5(5):500-516.
[21]刘驰,李家宝,芮俊鹏,等.16SrRNA基因在微生物生态学中的应用[J].生态学报,2015,35(9):2 769-2 788.
[22]徐爱蕾,黄毅,王为.16SrRNA基因序列分析技术在肠道菌群检测中的应用进展[J].临床消化病杂志,2015,27(3):186-189.
[23]易旻,杨玉婷,李梦霖,等.一株耐高温纤维素降解菌的分离筛选及鉴定[J].吉首大学学报(自然科学版),2017,38(4):72-77.
[24]康纪婷,吴翔,甘炳成,等.纤维素酶活力测定方法[J].河北农业科学,2010,22(4):151-153.
[25]孙盈,田永强,赵丽坤.纤维素酶的CMC酶活测定条件的研究[J].食品工业科技,2013,35(2):68-71,74.