乌骨鸡肠乳杆菌的分离鉴定及体外抑菌试验  

Isolation and Identification of Lactobacillus from Black-bone Fowl and Inhibitory Tests in Vitro

作　　者：刘小兰[1] 邓舜洲[1] 康绍珠 曹刚 高蕊 刘可心 钟霆霆 黄靖文 朱芝秀[1] LIU Xiao-lan;DENG Shun-zhou;KANG Shao-zhu;CAO Gang;GAO Rui;LIU Ke-xin;ZHONG Ting-ting;HUANG Jing-wen;ZHU Zhi-xiu(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045

出　　处：《江西农业大学学报》2020年第6期1195-1203,共9页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31460666);江西省科技厅项目(20122BBF60082);江西省教育厅项目(GJJ150416);江西省现代农业产业技术体系建设专项(JXARS-03)。

摘　　要：【目的】寻找能在禽类生产实践中替代抗生素的益生菌,以提高乌骨鸡生产性能,减少抗生素的使用,对乌骨鸡肠道黏膜进行细菌分离鉴定及体外抑菌试验。【方法】利用常规理化法做生理生化试验;PCR扩增测序法进行分子生物学鉴定;牛津杯琼脂扩散法做体外抑菌试验;灌喂乳酸菌法做动物安全性试验。【结果】分离菌株生长迅速、产酸强,有明显透明溶钙圈,为革兰氏阳性短杆菌;能发酵乳糖、七叶苷等,不分解木糖、阿拉伯糖;甲基红(M-R)试验结果为阳性,H2S、氧化酶、乙酰甲基甲醇(V-P)等均为阴性,生理生化特性与乳酸杆菌基本一致;PCR扩增在约1500 bp处出现目的条带,16S rDNA基因测序显示分离菌与唾液乳酸杆菌的同源性最高;分离菌对新生霉素高度敏感,对诺氟沙星耐药;对大肠杆菌、链球菌等病原菌均表现明显的抑菌作用;分离菌发酵液呈酸性时才具有抑菌活性,蛋白酶、高温对分离菌发酵液抑菌活性影响不大;动物试验中小鼠均生长良好,无一死亡。【结论】分离菌均为唾液乳杆菌,具备成为益生菌的优势;分离菌对多种病原菌具有抑制作用,与诺氟沙星等配合使用,可防治由大肠杆菌、链球菌等多种病原指示菌引起的疾病。[Objective]In order to find probiotics that can replace antibiotics in poultry production to improve the production performance of black bone chicken and reduce the use rate of antibiotics,bacteria were isolated and identified from the intestinal mucosa of silky fowl and bacteriostatic test was conducted in vitro.[Methods]In this experiment,physiological and biochemical tests were conducted by conventional physical and chemical methods,molecular biological identification was conducted by PCR amplification and sequencing,in vitro antibacterial test was conducted by Oxford cup agar diffusion method,animal safety test was conducted by feed Lactobacillus.[Results]Isolated strains grew rapidly and produced strong acid.They were gram positive Brevibacteria with obvious transparent calcium dissolving circle.They could ferment lactose and aescin,but did not decompose xylose and arabinose.The result of M-R test was positive while H2S,oxidase and V-P tests were negative.The physiological and biochemical characteristics of isolates were basically consistent with those of Lactobacillus.The result of PCR amplification showed that the target band was about 1500 bp and the 16S rDNA gene sequencing showed that the isolated bacteria had the highest homology with Lactobacillus sali⁃varius.The isolates were highly sensitive to neomycin and resistant to norfloxacin and had obviously antibacterial activity against Escherichia coli,Streptococcus and other pathogenic bacteria only when the fermentation broth of the isolated bacteria was acidic,protease and high temperature had little effect on the antibacterial activity.In the animal experiment,the mice all grew well and none died[.Conclusion]The isolated bacteria were all Lacto⁃bacillus salivarius,which have the advantage of being probiotics.The isolated bacteria can inhibit many kinds of pathogenic bacteria and prevent and treat diseases caused by various pathogenic indicator bacteria such as Escherichia coli and Streptococcus when used in conjunction with norfloxacin.

关 键 词：唾液乳酸杆菌 分离鉴定 体外抑菌 药敏试验 

分 类 号：S831[农业科学—畜牧学]