奶牛源乳酸菌的分离、鉴定及其微生态制剂的研究
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摘要
为筛选出能用于防治奶牛子宫内膜炎的益生性乳酸菌菌株并以其为原材料制作微生态制剂,采集产后30～50 d健康奶牛的子宫颈口处分泌物,利用MRS选择性培养基厌氧培养方式进行乳酸菌初步筛选。纯化得到的单株菌株进行染色和镜检,将典型的乳酸菌进行生化鉴定。提取其DNA,扩增16S rDNA全序列并测序,将所得序列与NCBI核酸数据库进行同源性比对,明确各分离菌株种属归属。将鉴定得到的单株乳酸菌过滤以及离心获取上清,得到的上清进行抑菌实验。采用琼脂扩散法检测对大肠埃希菌的抑菌活性。以得到的良好抑菌能力的菌株为原材料进行混合抑菌,进行微生态制剂最优比例的探究,并研究这些菌株的生长曲线和产酸能力。结果显示,经染色镜检、生化鉴定和16S rDNA测序分析和单株抑菌实验得到6株较好的抑菌能力的乳酸菌,其中鼠李糖乳杆菌、魏斯式细菌和粪肠球菌的混合上清抑菌效果最好,具有较强的生长和产酸能力。研究得到了可用于防治奶牛子宫内膜炎益生菌株,可进一步用于临床试验探究。
In order to screen probiotic lacto-bacteria strains to be used to control cow endometritis and use them as raw materials to make probiotics. Excretion from the cervix exit of healthy cows 30-50 days postpartum were used as material, and MRS selective medium was used to carry out initial lacto-bacteria screening. The purified strains were stained and microscopically examined for biochemical identification of typical lacto-bacteria. The DNA was extracted and the full sequence of 16 S rDNA was amplified and sequenced. The homology comparisons between the obtained sequences and the NCBI nucleic acid database were carried out to clarify the species and genus attribution of each isolate. The identified lacto-bacteria were filtered and centrifuged to obtain its supernatant, and the resulting supernatant was subjected to a bacteriostasis experiment. Agar diffusion method was adopted to test the antibacterial activity against E.coli. In order to obtain strains with good antibacterial ability as raw materials and carry out mixed bacteriostatic, and find out the optimal proportion of microecological preparations, as well as the growth curve and acid production ability of these strains. The results showed that six strains of lactic acid bacteria with good antibacterial activity were obtained by staining microscopy, biochemical identification, 16 S rDNA sequencing analysis and antibacterial test of each single strain. Among them, the antibacterial effects of supernatant of mixed Lactobacillus mucosae, Weissella cibaria and Enterococcus faecalis were the best, and possessing comparative growth and acid production ability. It can be concluded that the probiotic strains can be used to control cow endometritis were obtained, and can be used for further exploration in clinical experiments.
In order to screen probiotic lacto-bacteria strains to be used to control cow endometritis and use them as raw materials to make probiotics. Excretion from the cervix exit of healthy cows 30-50 days postpartum were used as material, and MRS selective medium was used to carry out initial lacto-bacteria screening. The purified strains were stained and microscopically examined for biochemical identification of typical lacto-bacteria. The DNA was extracted and the full sequence of 16 S rDNA was amplified and sequenced. The homology comparisons between the obtained sequences and the NCBI nucleic acid database were carried out to clarify the species and genus attribution of each isolate. The identified lacto-bacteria were filtered and centrifuged to obtain its supernatant, and the resulting supernatant was subjected to a bacteriostasis experiment. Agar diffusion method was adopted to test the antibacterial activity against E.coli. In order to obtain strains with good antibacterial ability as raw materials and carry out mixed bacteriostatic, and find out the optimal proportion of microecological preparations, as well as the growth curve and acid production ability of these strains. The results showed that six strains of lactic acid bacteria with good antibacterial activity were obtained by staining microscopy, biochemical identification, 16 S rDNA sequencing analysis and antibacterial test of each single strain. Among them, the antibacterial effects of supernatant of mixed Lactobacillus mucosae, Weissella cibaria and Enterococcus faecalis were the best, and possessing comparative growth and acid production ability. It can be concluded that the probiotic strains can be used to control cow endometritis were obtained, and can be used for further exploration in clinical experiments.
引文
[1] 杨娟. 奶牛子宫内膜炎研究进展[J]. 农业开发与装备, 2015,(5):150,125.
[2] 严作廷, 王东升, 张世栋,等. 奶牛子宫内膜炎的防治[J]. 中国畜牧兽医文摘, 2011, 27(6):97-98.
[3] 陈宏亮. 微生态制剂治疗奶牛子宫内膜炎临床试验初报[J]. 今日畜牧兽医:奶牛, 2015,(2):67-68.
[4] 严作廷, 王东升, 王旭荣,等. 我国奶牛主要疾病研究进展[J]. 中国草食动物科学, 2011, 31(6):69-72.
[5] 王孝武, 王旭荣, 杨志强,等. 奶牛子宫内膜炎研究进展[J]. 动物医学进展, 2014, 35(7):98-102.
[6] 孙翠焕, 冀宝营, 朱巍巍,等. 藏灵菇中Enterococcus faecalis RQ15的分离及鉴定[J]. 微生物学杂志, 2013, 33(1):89-92.
[7] 王凡, 王建设, 刘婷,等. 一株猪源屎肠球菌的分离、鉴定及益生功能初步研究[J]. 中国畜牧兽医, 2012, 39(7):231-234.
[8] 李嘉祺, 赵文婧, 张茜,等. 东北酸菜发酵中几株乳酸菌的筛选[J]. 微生物学杂志, 2010, 30(6):68-70.
[9] 田丰松,王军,刘畅,等.健康奶牛阴道乳酸菌分离鉴定及作为候选益生菌株的体外评价[J].中国预防兽医学报,2016,38(6):463-466.
[10] 朱芬花, 冯秉福, 罗永珍,等. 健康奶牛阴道乳酸菌的分离与鉴定[J]. 畜牧与兽医, 2017, 49(9):107-110.
[11] 李向楠. 奶牛生殖道内益生乳酸菌的分离、 筛选及功能特性研究[D]. 长春:吉林农业大学, 2014.
[12] 赵静, 李向楠, 王军,等. 奶牛生殖道内益生乳酸菌的筛选及分子鉴定[J].中国兽医学报, 2014, 34(6):986-990.
[13] 孙雯娟, 张波, 李大魁,等. 益生菌制剂的发展现状与临床应用进展[J]. 中国医院药学杂志, 2015, 35(9):850-857.
[14] 薛洋洋. 微生态制剂治疗奶牛子宫内膜炎的试验研究[D]. 大庆:黑龙江八一农垦大学, 2014.
[15] 姚火春. 兽医微生物学实验指导[M]. 北京:中国农业出版社, 2002.
[16] 刘冬梅,李理,杨晓泉,等. 用牛津杯法测定益生菌的抑菌活力[J]. 食品研究与开发, 2006, 27(3):110-111.
[17] 史国英, 余功明, 胡春锦. 广西传统发酵米粉中乳酸菌的分离鉴定[J]. 微生物学杂志, 2012, 32(5):54-57.
[18] 孟军, 刘淑英, 黄丽红,等. 动物微生态制剂猪肠源乳球菌的分离与鉴定[J]. 微生物学杂志, 2006, 26(3):12-15.
[19] Sandra G, Alex B, Francesc F, et al.Protential of lactic acid bacteria at regulating Escherichia coli infection and inflammation of bovine endometrium[J]. Theriogenology, 2016, 85(4):625-637.
[20] Samot J, Lebreton J, Badet C. Adherence capacities of oral lactobacilli for potential probiotic purposes[J]. Anaerobe, 2011, 17(2): 69-72.
[21] Parolin C, Marangoni A, Laghi L, et al. Isolation of Vaginal lactobacilli and characterization of anti-candida activity[J].PLoS One, 2015, 10(6): e0131220.
[22] 杨龙龙, 蒋建军, 剡根强,等. 牛瘤胃乳酸菌的分离与鉴定[J]. 中国畜牧兽医, 2014, 41(8):145-148.
[23] 王秋菊, 崔一喆, 刘胜军,等. 笼养成年蛋鸡盲肠中乳酸菌的分离与鉴定[J]. 中国畜牧兽医, 2013, 40(8):112-115.
[24] 田丰松, 王军, 杨连玉,等. 不同乳酸菌分离株抗菌物质及其抗菌活性的体外检测及鉴定[J]. 中国预防兽医学报, 2016, 38(7):558-561.
[25] 魏利杰, 徐琳, 王锦卓. 乳酸杆菌代谢产物对阴道常见致病菌抑制作用的试验研究[J]. 吉林医学, 2013, 34(13):2475-2476.
[2] 严作廷, 王东升, 张世栋,等. 奶牛子宫内膜炎的防治[J]. 中国畜牧兽医文摘, 2011, 27(6):97-98.
[3] 陈宏亮. 微生态制剂治疗奶牛子宫内膜炎临床试验初报[J]. 今日畜牧兽医:奶牛, 2015,(2):67-68.
[4] 严作廷, 王东升, 王旭荣,等. 我国奶牛主要疾病研究进展[J]. 中国草食动物科学, 2011, 31(6):69-72.
[5] 王孝武, 王旭荣, 杨志强,等. 奶牛子宫内膜炎研究进展[J]. 动物医学进展, 2014, 35(7):98-102.
[6] 孙翠焕, 冀宝营, 朱巍巍,等. 藏灵菇中Enterococcus faecalis RQ15的分离及鉴定[J]. 微生物学杂志, 2013, 33(1):89-92.
[7] 王凡, 王建设, 刘婷,等. 一株猪源屎肠球菌的分离、鉴定及益生功能初步研究[J]. 中国畜牧兽医, 2012, 39(7):231-234.
[8] 李嘉祺, 赵文婧, 张茜,等. 东北酸菜发酵中几株乳酸菌的筛选[J]. 微生物学杂志, 2010, 30(6):68-70.
[9] 田丰松,王军,刘畅,等.健康奶牛阴道乳酸菌分离鉴定及作为候选益生菌株的体外评价[J].中国预防兽医学报,2016,38(6):463-466.
[10] 朱芬花, 冯秉福, 罗永珍,等. 健康奶牛阴道乳酸菌的分离与鉴定[J]. 畜牧与兽医, 2017, 49(9):107-110.
[11] 李向楠. 奶牛生殖道内益生乳酸菌的分离、 筛选及功能特性研究[D]. 长春:吉林农业大学, 2014.
[12] 赵静, 李向楠, 王军,等. 奶牛生殖道内益生乳酸菌的筛选及分子鉴定[J].中国兽医学报, 2014, 34(6):986-990.
[13] 孙雯娟, 张波, 李大魁,等. 益生菌制剂的发展现状与临床应用进展[J]. 中国医院药学杂志, 2015, 35(9):850-857.
[14] 薛洋洋. 微生态制剂治疗奶牛子宫内膜炎的试验研究[D]. 大庆:黑龙江八一农垦大学, 2014.
[15] 姚火春. 兽医微生物学实验指导[M]. 北京:中国农业出版社, 2002.
[16] 刘冬梅,李理,杨晓泉,等. 用牛津杯法测定益生菌的抑菌活力[J]. 食品研究与开发, 2006, 27(3):110-111.
[17] 史国英, 余功明, 胡春锦. 广西传统发酵米粉中乳酸菌的分离鉴定[J]. 微生物学杂志, 2012, 32(5):54-57.
[18] 孟军, 刘淑英, 黄丽红,等. 动物微生态制剂猪肠源乳球菌的分离与鉴定[J]. 微生物学杂志, 2006, 26(3):12-15.
[19] Sandra G, Alex B, Francesc F, et al.Protential of lactic acid bacteria at regulating Escherichia coli infection and inflammation of bovine endometrium[J]. Theriogenology, 2016, 85(4):625-637.
[20] Samot J, Lebreton J, Badet C. Adherence capacities of oral lactobacilli for potential probiotic purposes[J]. Anaerobe, 2011, 17(2): 69-72.
[21] Parolin C, Marangoni A, Laghi L, et al. Isolation of Vaginal lactobacilli and characterization of anti-candida activity[J].PLoS One, 2015, 10(6): e0131220.
[22] 杨龙龙, 蒋建军, 剡根强,等. 牛瘤胃乳酸菌的分离与鉴定[J]. 中国畜牧兽医, 2014, 41(8):145-148.
[23] 王秋菊, 崔一喆, 刘胜军,等. 笼养成年蛋鸡盲肠中乳酸菌的分离与鉴定[J]. 中国畜牧兽医, 2013, 40(8):112-115.
[24] 田丰松, 王军, 杨连玉,等. 不同乳酸菌分离株抗菌物质及其抗菌活性的体外检测及鉴定[J]. 中国预防兽医学报, 2016, 38(7):558-561.
[25] 魏利杰, 徐琳, 王锦卓. 乳酸杆菌代谢产物对阴道常见致病菌抑制作用的试验研究[J]. 吉林医学, 2013, 34(13):2475-2476.