太行鸡肠道益生菌的分离与鉴定
|
摘要
为了挖掘鸡源益生菌的菌种资源,试验采用肠道内容物分离纯化、生长曲线和产酸曲线绘制、抑菌性能测定、生化试验、基因测序、同源性分析、遗传进化树构建的方法对太行鸡肠道益生菌的种属和生物学特性进行了研究。结果表明:从散养太行鸡肠道分离、纯化了6株纯化益生菌,将其编号为J1～J6,其中J1～J4增殖速度快,2～10 h可达到对数生长期;6株菌均有一定的产酸能力,其pH值最低可达到3.78;6株菌对大肠杆菌和沙门氏杆菌的抑菌圈直径均大于14.67 mm,大肠杆菌对J3极度敏感,沙门氏杆菌对J1、J2极度敏感;生化试验显示,J1～J6依次为罗伊氏乳杆菌、粪肠球菌、枯草芽孢杆菌、鼠李糖乳杆菌、粪肠球菌、蜡样芽孢杆菌;PCR扩增得到大小为1 500 bp的目的条带,菌株序列与GenBank比对的结果与生化试验结果一致;各菌株与其GenBank标准菌株的同源性为96%～99%;J2、J5与粪肠球菌标准株遗传距离较远,与标准株相比有显著遗传变异,其余菌株均与标准株遗传距离较近,其中J1和J4遗传距离最近,但J3、J6与其余菌株遗传距离均较远。说明鸡源益生菌J1、J2和J4具备作益生菌饲料添加剂菌种的特性。
In this study, the species and biological characteristics of probiotics in Taihang chicken were initially identified by instinal seperation and purfication, growth curve and acid production curve picturing,bacteriostatic dectetion,biochemical testing, gene sequencing, homology analyze, and biological phylogenetic tree construction. The results showed that 6 purified probiotics were separated from intestinal tract of Taihang chicken, numbered J1 to J6.What's more, J1 to J4 replicated rapidly whose logarithmic growth phase in 2-10 h, and were able to produce acid to a pH of 3.78. In addition, all isolates were able to inhibit the growth of E.coli and Salmonella, as evidenced by the observation that the diameter of the zone of inhibition were all longer than 14.67 mm, they all inhibited the growth of E. coli and Salmonella. E.coli was highly sensitive to J3 and Salmonella to J1, J2.The biochemical test showed that the 6 strains were Lactobacillus reuteri, Enterococcus faecalis(E. faecalis), Bacillus subtilis, Lactobacillus rhamnosus, E. faecalis, and Bacillus cereus in turn.A 1 500 bp target band was obtained by PCR amplification. The results of strain sequence and GenBank pair were consistent with those of biochemical experiments. The homology of the strains and its GenBank standard strain is 96% to 99%.The results of biological phylogenetic tree construction showed that J2, J5 and E. faecalis standard strains shared a long genetic distance, and there were genetic variations compared with the standard strain. The other strains were close to the standard strain. Among them, J1 shared high homology with J4, but J3 and J6 shared further distant genetic distances from other strains.It indicates that J1, J2 and J4 of chicken source probiotics matches the characteristics of probiotics feed additives.
In this study, the species and biological characteristics of probiotics in Taihang chicken were initially identified by instinal seperation and purfication, growth curve and acid production curve picturing,bacteriostatic dectetion,biochemical testing, gene sequencing, homology analyze, and biological phylogenetic tree construction. The results showed that 6 purified probiotics were separated from intestinal tract of Taihang chicken, numbered J1 to J6.What's more, J1 to J4 replicated rapidly whose logarithmic growth phase in 2-10 h, and were able to produce acid to a pH of 3.78. In addition, all isolates were able to inhibit the growth of E.coli and Salmonella, as evidenced by the observation that the diameter of the zone of inhibition were all longer than 14.67 mm, they all inhibited the growth of E. coli and Salmonella. E.coli was highly sensitive to J3 and Salmonella to J1, J2.The biochemical test showed that the 6 strains were Lactobacillus reuteri, Enterococcus faecalis(E. faecalis), Bacillus subtilis, Lactobacillus rhamnosus, E. faecalis, and Bacillus cereus in turn.A 1 500 bp target band was obtained by PCR amplification. The results of strain sequence and GenBank pair were consistent with those of biochemical experiments. The homology of the strains and its GenBank standard strain is 96% to 99%.The results of biological phylogenetic tree construction showed that J2, J5 and E. faecalis standard strains shared a long genetic distance, and there were genetic variations compared with the standard strain. The other strains were close to the standard strain. Among them, J1 shared high homology with J4, but J3 and J6 shared further distant genetic distances from other strains.It indicates that J1, J2 and J4 of chicken source probiotics matches the characteristics of probiotics feed additives.
引文
[1] BARRIERE S L.Clinical,economic and societal impact of antibiotic resistance[J].Expert Opin Pharmacother,2015,16(2):151-153.
[2] 王秋菊,崔一喆,刘洪辰,等.30周龄笼养海蓝褐壳蛋鸡盲肠中优良乳酸菌的筛选[J].中国微生态学杂志,2015,27(1):28-32,54.
[3] 翟云,托娅.益生菌的免疫调节作用及其相关应用研究进展[J].中国微生态学杂志,2018,30(2):235-239.
[4] 杨军,赵权,杨桂连.益生菌在禽类肠道疾病防治中的应用[J].黑龙江畜牧兽医,2015(09下):149-151.
[5] 解相霞.一株蛋鸡肠道枯草芽孢杆菌的分离鉴定与益生菌对鸡的应用效果[D].青岛:山东农业大学,2015.
[6] 齐博,武书庚,王晶,等.枯草芽孢杆菌对肉仔鸡生长性能、肠道形态和菌群数量的影响[J].动物营养学报,2016,28(6):1748-1756.
[7] MESSAOUDI S,MANAI M,KERGOURLAY G,et al.Lactobacillus salivarius:bacteriocin and probiotic activity[J].Food Microbiol,2013,36(2):296-304.
[8] 陆克文.新型猪用复合益生菌制剂的研制与开发[D].南京:南京农业大学,2011.
[9] 布坎南 R E,吉本斯 N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984.
[10] 凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999.
[11] 周珍辉,关文怡,向双云,等.健康成年鸡肠道乳酸菌的分离与初步鉴定[J].黑龙江畜牧兽医,2017(03下):186-187,284-285.
[12] 杨家军,钱坤,李庆岗,等.罗伊氏乳杆菌抑制致病性大肠杆菌感染效果的研究[J].中国畜牧兽医,2017,44(8):2431-2436.
[13] 王学静,陈立功,魏忠华,等.放养与笼养的太行鸡新城疫与禽流感免疫抗体监测[J].今日畜牧兽医,2015(7):46-48.
[14] 靳爱红.猪源益生菌分离鉴定及复合物筛选[D].邯郸:河北工程大学,2018.
[2] 王秋菊,崔一喆,刘洪辰,等.30周龄笼养海蓝褐壳蛋鸡盲肠中优良乳酸菌的筛选[J].中国微生态学杂志,2015,27(1):28-32,54.
[3] 翟云,托娅.益生菌的免疫调节作用及其相关应用研究进展[J].中国微生态学杂志,2018,30(2):235-239.
[4] 杨军,赵权,杨桂连.益生菌在禽类肠道疾病防治中的应用[J].黑龙江畜牧兽医,2015(09下):149-151.
[5] 解相霞.一株蛋鸡肠道枯草芽孢杆菌的分离鉴定与益生菌对鸡的应用效果[D].青岛:山东农业大学,2015.
[6] 齐博,武书庚,王晶,等.枯草芽孢杆菌对肉仔鸡生长性能、肠道形态和菌群数量的影响[J].动物营养学报,2016,28(6):1748-1756.
[7] MESSAOUDI S,MANAI M,KERGOURLAY G,et al.Lactobacillus salivarius:bacteriocin and probiotic activity[J].Food Microbiol,2013,36(2):296-304.
[8] 陆克文.新型猪用复合益生菌制剂的研制与开发[D].南京:南京农业大学,2011.
[9] 布坎南 R E,吉本斯 N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984.
[10] 凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999.
[11] 周珍辉,关文怡,向双云,等.健康成年鸡肠道乳酸菌的分离与初步鉴定[J].黑龙江畜牧兽医,2017(03下):186-187,284-285.
[12] 杨家军,钱坤,李庆岗,等.罗伊氏乳杆菌抑制致病性大肠杆菌感染效果的研究[J].中国畜牧兽医,2017,44(8):2431-2436.
[13] 王学静,陈立功,魏忠华,等.放养与笼养的太行鸡新城疫与禽流感免疫抗体监测[J].今日畜牧兽医,2015(7):46-48.
[14] 靳爱红.猪源益生菌分离鉴定及复合物筛选[D].邯郸:河北工程大学,2018.