大熊猫粪便乳酸菌的分离鉴定
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摘要
大熊猫作为国家保护动物,其健康问题备受瞩目。为了维护大熊猫的肠道健康,本研究从大熊猫肠道内分离出适宜于大熊猫肠道环境的乳酸菌菌株,有望将其制成熊猫肠道微生物制剂,从而改善大熊猫肠道菌群环境。从雅安市宝兴县蜂桶寨自然保护区选取圈养与野生大熊猫的粪便,通过体外培养分离出9个菌株。分离菌株经过革兰氏染色镜检、过氧化氢产气、菌落形态观察等方法与技术初步鉴定为乳酸菌。对这9株乳酸菌进行耐酸试验、耐胆盐试验、抑菌能力试验和产酸能力等测试,筛选出了3个适应性较强,有望制成调节大熊猫肠道内环境平衡作用的微生态菌剂的菌株。16S rRNA基因序列分析表明:分离菌株J1、J2和J4分别为融合魏斯氏菌(Weissella confusa),海氏肠球菌(Enterococcus heynei)和非解乳糖链球菌(Streptococcus alactolyticus),有望被应用于大熊猫肠道微生态制剂的研究。
As a national protected animal,giant panda has attracted much attention in its health problems. In order to maintain the intestinal health of giant panda,a method of improving its intestinal microflora environment by using microbial agents is proposed in this paper. Lactic acid bacteria suitable for the intestinal environment of giant pandas were isolated from the intestines of giant pandas,which are expected to be applied in panda intestinal microbial preparations.Nine strains were isolated from the feces of captive and wild giant pandas from Fengtongzhai Nature Reserve,Baoxing County,Ya'an City. The isolated strains were identified as lactic acid bacteria by Gram staining microscopy,hydrogen peroxide gas production and colony morphology observation. In addition,the 9 strains of lactic acid bacteria were tested for acid tolerance,bile salt tolerance,bacteriostasis and acid production. Three strains with strong adaptability were screened. Sequence analysis based on 16 S rRNA gene showed that strains J1,J2 and J4 were Weissella confusa,Enterococcus heynei and Streptococcus alactolyticus,respectively,which were expected to be applied in the study of intestinal microecological agents of giant pandas.
As a national protected animal,giant panda has attracted much attention in its health problems. In order to maintain the intestinal health of giant panda,a method of improving its intestinal microflora environment by using microbial agents is proposed in this paper. Lactic acid bacteria suitable for the intestinal environment of giant pandas were isolated from the intestines of giant pandas,which are expected to be applied in panda intestinal microbial preparations.Nine strains were isolated from the feces of captive and wild giant pandas from Fengtongzhai Nature Reserve,Baoxing County,Ya'an City. The isolated strains were identified as lactic acid bacteria by Gram staining microscopy,hydrogen peroxide gas production and colony morphology observation. In addition,the 9 strains of lactic acid bacteria were tested for acid tolerance,bile salt tolerance,bacteriostasis and acid production. Three strains with strong adaptability were screened. Sequence analysis based on 16 S rRNA gene showed that strains J1,J2 and J4 were Weissella confusa,Enterococcus heynei and Streptococcus alactolyticus,respectively,which were expected to be applied in the study of intestinal microecological agents of giant pandas.
引文
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[13]Zhou Z Y,Zhou X X,Zhong Z J,et al.Investigation of antibacterial activity of Bacillus spp.isolated from the feces of giant panda and characterization of their anti-microbial gene distributions[J].World J Microbiol Bio-technol,2014,30(12):3129-3136.
[14]Fan C,Li S J,Li C L,et al.Isolation,identification and cellulase production of a cellulolytic bacterium from in-testines of giant panda[J].Acta Microbiol Sin,2012,52(9):1113-1121.樊程,李双江,李成磊,等.大熊猫肠道纤维素分解菌的分离鉴定及产酶性质[J].微生物学报,2012,52(9):1113-1121.
[15]Zhou D Q.Microbiology experiment course[M].Bei-jing:Higher Education Press,2006:275-276.周德庆.微生物学实验教程[M].北京:高等教育出版社,2006:275-276.
[16]Huang X L,Xin M X.Guidance for microbiology exper-iments[M].Beijing:Higher Education Press,2008.黄秀梨,辛明秀.微生物学实验指导[M]..北京:高等教育出版社,2008.
[17]Wu H H,Duan L F,Zhang Y Y,et al.Isolation and identification of lactic acid bacteria from panda feces and their cholesterol-lowering effects[J].Heilongjiang Ani-mal Sci Vet Med,2011(6):14-18.吴慧昊,段龙飞,张园园,等.熊猫粪便中乳酸菌的分离鉴定与降胆固醇作用研究[J].黑龙江畜牧兽医,2010(6):14-18.
[18]Li L L,Wang Y T,Yang X,et al.Isolation and charac-terization of lactic acid bacteria from swine[J].J North-west A&F Univ Nat Sci Ed 2016,44(2):1-7.李琳琳,王雅婷,杨欣,等.猪源乳酸菌的分离及其生物学性能研究[J].西北农林科技大学学报,2016,44(2):1-7.
[19]Sun F L,Liu J X,Xi D,et al.Pathogens of intestinal diseases in giant panda[J].J Econ Animal,2002,6(2):20-23.孙飞龙,刘敬贤,席丹,等.大熊猫肠道疾病致病菌[J].经济动物学报,2002,6(2):20-23.
[20]Zhou Z Y,Zhong Z J,Zhou X X,et al.Giant panda in-testinal bacteria:a review[J].Microbiology,2016,43(6):1366-1371.周紫峣,钟志军,周潇潇,等.大熊猫肠道菌群的研究进展[J].微生物学通报,2016,43(6):1366-1371.
[2]Zhou Z Y,Zhong Z J,Zhou X X,et al.Giant panda in-testinal bacteria:a review[J].Microbiol China,2016,43(6):1366-1371.周紫峣,钟志军,周潇潇,等.大熊猫肠道菌群的研究进展[J].微生物学通报,2016,43(6):1366-1371.
[3]Bi Y Z,Xie Q M.Development trends of animal intesti-nal microorganisms and microecological agents[J].Chi-na Poult,2015,37(4):1-4.毕英佐,谢青梅.动物肠道微生物及微生态制剂发展动态[J].China Poultry,2015,37(4):1-4.
[4]Wang Y.Estimation of giant panda's intestinal flora structure in intestine based on ERIC-PCR and 16S rD-NA-RFLP technologies[D].Ya'an:Sichuan Agricultur-al University,2011.王燚.基于ERIC-PCR和16S rDNA-RFLP技术对亚成体大熊猫肠道菌群结构的研究[D].雅安:四川农业大学,2011.
[5]Bateup J,Dobbinson S,Munro K,et al.Molecular analysis of the composition of lactobacillus populations inhabiting the stomach and caecum of pigs[J].Microb Ecol Heal Dis,1998,10(2):95-102.
[6]Zhang M,Pan G L,Liang W L,et al.Isolation and identification of probiotics from giant pandas in Qinling mountains[J].Prog Vet Med,2016,37(1):125-129.张曼,潘广林,梁武龙,等.秦岭大熊猫肠道益生菌的分离鉴定[J].动物医学进展,2016,37(1):125-129.
[7]Guo Q Y.Screening of lactic acid bacteria and study on the application effects isolated from calf[D].Harbin:Harbin Northeast Agricultural University,2010.郭奇苑.犊牛源酸菌的筛选及其应用效果研究[D].哈尔滨:东北农业大学,2010.
[8]Yang Y J,Fu W J,Liu X H,et al.Screening and iden-tification of lactic acid bacteria producing high-efficient bacteriocin[J].Chin J Animal Sci,2013,49(19):72-76.杨亚晋,付文娟,刘旭辉,等.产高效细菌素乳酸菌的筛选及鉴定[J].中国畜牧杂志,2013,49(19):72-76.
[9]Guan L J,Li Y F,Cheng Y Q,et al.Screening and iso-lation of lactic acid bacteria producing gamma-aminobu-tyric acid[J].Food Sci Technol,2009,34(1):2-5.管立军,李艳芳,程永强,等.产γ-氨基丁酸乳酸菌的筛选及分离鉴定[J].食品科技,2009,34(1):2-5.
[10]Zhang X J,Shen J B,Mo X L,et al.Coevolution of ani-mals and their gut microbes[J].Chin Bull Life Sci,2014,26(2):158-160.张雪敬,沈佳斌,莫小立,等.动物及其肠道菌群的协同进化研究[J].生命科学,2014,26(2):158-160.
[11]Xiong Y,Xu Z W,Bai Y,et al.Influence of antibiotics and micro-ecologic preparation on intastinal flora of sub-mature giant panda[J].Chin J Vet Sci Technol,2000,30(7):34-35.熊焰,许志文,白喻,等.抗菌药和微生态制剂对亚成体大熊猫肠道菌群影响的试验[J]中国兽医科技,2000,30(7):34-35.
[12]Wang Q,He G X,Yu X M,et al.Clinical application of microecological agents in the treatment of panda diseas-es[J].Chin J Microecol,1998,10(6):349-350,353.王强,何光昕,余星明,等.微生态制剂在大熊猫疾病治疗中的临床应用研究[J].中国微生态学杂志,1998,10(6):349-350,353.
[13]Zhou Z Y,Zhou X X,Zhong Z J,et al.Investigation of antibacterial activity of Bacillus spp.isolated from the feces of giant panda and characterization of their anti-microbial gene distributions[J].World J Microbiol Bio-technol,2014,30(12):3129-3136.
[14]Fan C,Li S J,Li C L,et al.Isolation,identification and cellulase production of a cellulolytic bacterium from in-testines of giant panda[J].Acta Microbiol Sin,2012,52(9):1113-1121.樊程,李双江,李成磊,等.大熊猫肠道纤维素分解菌的分离鉴定及产酶性质[J].微生物学报,2012,52(9):1113-1121.
[15]Zhou D Q.Microbiology experiment course[M].Bei-jing:Higher Education Press,2006:275-276.周德庆.微生物学实验教程[M].北京:高等教育出版社,2006:275-276.
[16]Huang X L,Xin M X.Guidance for microbiology exper-iments[M].Beijing:Higher Education Press,2008.黄秀梨,辛明秀.微生物学实验指导[M]..北京:高等教育出版社,2008.
[17]Wu H H,Duan L F,Zhang Y Y,et al.Isolation and identification of lactic acid bacteria from panda feces and their cholesterol-lowering effects[J].Heilongjiang Ani-mal Sci Vet Med,2011(6):14-18.吴慧昊,段龙飞,张园园,等.熊猫粪便中乳酸菌的分离鉴定与降胆固醇作用研究[J].黑龙江畜牧兽医,2010(6):14-18.
[18]Li L L,Wang Y T,Yang X,et al.Isolation and charac-terization of lactic acid bacteria from swine[J].J North-west A&F Univ Nat Sci Ed 2016,44(2):1-7.李琳琳,王雅婷,杨欣,等.猪源乳酸菌的分离及其生物学性能研究[J].西北农林科技大学学报,2016,44(2):1-7.
[19]Sun F L,Liu J X,Xi D,et al.Pathogens of intestinal diseases in giant panda[J].J Econ Animal,2002,6(2):20-23.孙飞龙,刘敬贤,席丹,等.大熊猫肠道疾病致病菌[J].经济动物学报,2002,6(2):20-23.
[20]Zhou Z Y,Zhong Z J,Zhou X X,et al.Giant panda in-testinal bacteria:a review[J].Microbiology,2016,43(6):1366-1371.周紫峣,钟志军,周潇潇,等.大熊猫肠道菌群的研究进展[J].微生物学通报,2016,43(6):1366-1371.
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