基于纯培养方法和PacBio三代测序技术研究蒙古国传统酸马奶中乳酸菌多样性
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摘要
通过纯培养方法结合PacBio三代测序技术对采自蒙古国的12份传统酸马奶中的乳酸菌多样性进行研究。应用传统的纯培养技术从12份传统酸马奶样品中分离出62株乳酸菌,通过16S rRNA基因序列分析和系统进化关系研究,62株乳酸菌被鉴定为5个属,6个种,分别是:25株瑞士乳杆菌、20株植物乳杆菌、7株产马乳酒乳杆菌、5株屎肠球菌、3株嗜热链球菌和2株粪肠球菌。采用PacBio SMRT测序技术研究其中8份样品中乳酸菌的多样性,宏基因组测序得到的优势菌为乳酸菌,同时也检测到一些痕量的其它种属细菌。共检测到5个乳酸菌的属,分别是:乳杆菌属、乳球菌属、链球菌属、明串珠菌属和肠球菌属,20个乳酸菌的种,即:瑞士乳杆菌、乳酸乳球菌、副乳链球菌、棉子糖乳球菌、肠膜阴串珠球菌和产马乳酒乳杆菌。宏基因组测序相对含量表明:蒙古国酸马奶样品中乳酸菌优势属为乳杆菌属(95.56%),优势种为瑞士乳杆菌(94.64%),纯培养方法和宏基因组三代测序方法都表明瑞士乳杆菌为蒙古国传统酸马奶中的优势菌种。宏基因组测序技术除检测到纯培养获得的菌株外,还获得14个低丰度乳酸菌,如棉籽糖乳球菌、肠膜明串珠球菌和开菲尔乳杆菌等,这些菌在酸马奶中含量较少。通过PacBio SMRT测序技术可以全面了解酸马奶中乳酸菌的多样性,为通过纯培养方法获得低丰度乳酸菌提供指导。
Biodiversity of lactic acid bacteriain 12 traditional koumiss samples collected from Mongolia were studied by using culture-dependent method and Pacific Biosciences(PacBio) single-molecule, real-time sequencing technology(SMRT) analysis in the present study. 62 strains of lactic acid bacteria were isolated from 12 traditional koumiss samples by using pure culture method. Based on the 16 S rRNA gene sequence and phylogenetic relationship analysis, these strains were identified as Lactobacillus helveticus(25 strains), Lactobacillus plantarum(20 strains), Streptococcus thermophilus(3 strains) and Enterococcus faecium(5 strains), Lactobacillus kefiranofaciens(7 strains) and Enterococcus faecalis(2 strains). In addition, we used PacBio SMRT sequencing technology found 5 genera of lactic acid bacteria, including Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and Enterococcus, and 20 species of lactic acid bacteria,such as Lactobacillus helveticus, Lactococcus lactis, Streptococcus parauberis, Leuconostoc mesenteroides and Lactobacillus kefiranofaciens and so on. The dominant genus and speciesrespectively were Lactobacillus(94.64%) and Lactobacillus helveticus(94.64%) in these traditional koumiss samples. The culture-dependent method and PacBio SMRT sequencing technology both verified Lactobacillus helveticus was the dominant species in 8 traditional koumiss samples in the results of the study. In addition, PacBio SMRT sequencing technology also obtained traces of 14 low-abundance lactic acid bacteria such as Lactococcus raffinolactis, Leuconostoc mesenteroides, and Lactobacillus kefiri and so on. The diversity of lactic acid bacteria in koumiss can be comprehensively understood by PacBio SMRT sequencing technology, which could provide guidance for obtaining low abundance lactic acid bacteria through pure culture method.
Biodiversity of lactic acid bacteriain 12 traditional koumiss samples collected from Mongolia were studied by using culture-dependent method and Pacific Biosciences(PacBio) single-molecule, real-time sequencing technology(SMRT) analysis in the present study. 62 strains of lactic acid bacteria were isolated from 12 traditional koumiss samples by using pure culture method. Based on the 16 S rRNA gene sequence and phylogenetic relationship analysis, these strains were identified as Lactobacillus helveticus(25 strains), Lactobacillus plantarum(20 strains), Streptococcus thermophilus(3 strains) and Enterococcus faecium(5 strains), Lactobacillus kefiranofaciens(7 strains) and Enterococcus faecalis(2 strains). In addition, we used PacBio SMRT sequencing technology found 5 genera of lactic acid bacteria, including Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and Enterococcus, and 20 species of lactic acid bacteria,such as Lactobacillus helveticus, Lactococcus lactis, Streptococcus parauberis, Leuconostoc mesenteroides and Lactobacillus kefiranofaciens and so on. The dominant genus and speciesrespectively were Lactobacillus(94.64%) and Lactobacillus helveticus(94.64%) in these traditional koumiss samples. The culture-dependent method and PacBio SMRT sequencing technology both verified Lactobacillus helveticus was the dominant species in 8 traditional koumiss samples in the results of the study. In addition, PacBio SMRT sequencing technology also obtained traces of 14 low-abundance lactic acid bacteria such as Lactococcus raffinolactis, Leuconostoc mesenteroides, and Lactobacillus kefiri and so on. The diversity of lactic acid bacteria in koumiss can be comprehensively understood by PacBio SMRT sequencing technology, which could provide guidance for obtaining low abundance lactic acid bacteria through pure culture method.
引文
[1]温永平,侯强川,张和平.自然发酵酸马奶对人体肠道菌群的影响-基于PacBio SMRT测序技术[J].中国乳品工业,2017,45(2):4-7.
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[10]乌日娜,张和平,孟和毕力格.酸马奶中乳杆菌Lb casei.Zhang和ZL12-1的16S rDNA基因序列及聚类分析[J].中国乳品工业,2005,33(6):4-9.
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[13]YU J,GAO W,QIN M J,et al.Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan,China[J].The Journal of General and Applied Microbiology,2012,58(3):163-172.
[14]ALTSCHUL S F,MADDEN T L,SCH魧FFER AA,et al.Gapped BLAST and PSI-BLAST:A new generation of protein database search programs[J].Nucleic Acids Research,1997,25(17):3389-3402.
[15]YU J,WANG W H,MENGHE B L G,et al.Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia[J].Journal of Dairy Science,2011,94(7)3230-3241.
[16]KUMAR S,STECHER G,TAMURA K.MEGA7:Molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870-1874.
[17]孟和毕力格,乌日娜,王立平,等.不同地区酸马奶中乳杆菌的分离及其生物学特性的研究[J].中国乳品工业,2004,32(11):6-11.
[18]ISHIIS.Microbiol study of traditional fermented dairy products Haruha and Buriat in Mongolia[J].Milk Science,2001,50:25-30
[19]孙天松,王俊国,张列兵,等.中国新疆地区酸马奶中乳酸菌生物多样性研究[J].微生物学通报,2007,34(3):451-454.
[20]FANG L,LI-HUI D U,PENG D U,et al.Biodiversity of lactic acid bacteria in home-made koumiss from Inner Mongolia[J].Food Science,2008,29(2):218-224
[21]JIN H,MO L,PAN L,et al.Using PacBio sequencing to investigate the bacterial microbiota of traditional Buryatian cottage cheese and comparison with Italian and Kazakhstan artisanal cheeses[J].Journal of Dairy Science,2018,10(8):6885-6896
[22]OKI K,DUGERSUREN J,DEMBEREL S,et al Pyrosequencing analysis of the microbial diversity of airag,khoormog and tarag,traditional fermented dairy products of Mongolia[J].Bioscience of Microbiota,Food and Health,2014,33(2):53-64.
[23]其木格苏都.自然发酵酸马奶细菌多样性及其基因动态变化研究[D].呼和浩特:内蒙古农业大学,2017.
[24]SADAT-MEKMENE L,JARDIN J,CORRE C,et al.Simultaneous presence of PrtH and PrtH2 proteinases in Lactobacillus helveticus strains improves breakdown of the pure alphas1-casein[J].Appl Environ Microbiol,2011,77(1):179-186.
[25]CHEN Y,LIU W,XUE J,et al.Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect of fermented milk of strain H9[J].Journal of Dairy Science,2014,97(11):6680-6692.
[26]KIMOTO-NIRA H,AOKI R,MIZUMACHI K,et al.Interaction between Lactococcus lactis and Lactococcus raffinolactis during growth in milk:Development of a new starter culture[J].Journal of Dairy Science,2012,95(4):0-2185.
[27]NHO S W,HIKIMA J,CHA I S,et al.Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis[J].Journal of Bacteriology,2011,193(13):3356.
[28]CHEN W M,YANG S H,YOUNG C C,et al Rheinheimeratilapiae sp.nov.isolated from a freshwater culture pond[J].International Journal of Systematic and Evolutionary Microbiology,2013,63(4):1457-1463.
[29]RASOLOFO E A,ST-GELAIS D,LAPOINTE G,et al.Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk[J].International Journal of Food Microbiology,2010,138(1/2):108-118.
[30]BOUBENDIR A,SERRAZANETTI D I,HAMIDECHIM A,et al.Changes in bacterial populations in refrigerated raw milk collected from a semi-arid area of Algeria[J].Annals of Microbiology,2016,66(2):777-783.
[31]VITHANAGE N R,YEAGER T R,JADHAV S R,et al.Comparison of identification systems for psychrotrophic bacteria isolated from raw bovine milk[J].International Journal of Food Microbiology,2014,189:26-38.
[32]HANTSISZACHAROV E,HALPERN M.Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits[J].Applied&Environmental Microbiology,2007,73(22):7162-7168.
[33]COOREVITS A,DE JONGHE V,VANDROEMMEJ,et al.Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms[J].Syst Appl Microbiol,2008,31(2):126-140.
[2]白阿茹娜,布仁满达,敖其,等.蒙医酸马奶疗法的研究进展及其临床应用[J].中国民族医药杂志,2016,22(7):67-68.
[3]陆东林,刘朋龙.酸马奶的营养价值和医疗保健作用[J].新疆畜牧业,2018,33(6):4-10.
[4]白曙明,特木其乐,敖其尔.浅谈酸马奶(策格)的临床应用[J].中国民族医药杂志,2018,24(1):62-64.
[5]乌云,马媛媛.蒙食策格-酸马奶疗法[J].家庭医学,2014,下半月(1):12-13.
[6]赵勇.应用分子生物学技术解析不同处理状况下土壤微生物群落结构的变化[D].南京:南京农业大学,2005.
[7]BRASLAVSKY I,HEBERT B,KARTALOV E,et al.Sequence information can be obtained from single DNA molecules[J].Proceedings of the National A-cademy of Sciences of the United States of America,2003,100(7):3960-3964.
[8]WU R N,ZHAO X W,ZHAO C Y,et al.Identification of lactic acid bacteria in suancai,a traditional Northeastern Chinese fermented food,and salt response of Lactobacillus paracasei LN-1[J].Annals of Microbiology,2014,64(3):1325-1332.
[9]ZHU H,QU F,ZHU L H.Isolation of genomic DNAs from plants,fungi and bacteria using benzyl chloride[J].Nucleic Acids Research,1993,21(22):5279-5280.
[10]乌日娜,张和平,孟和毕力格.酸马奶中乳杆菌Lb casei.Zhang和ZL12-1的16S rDNA基因序列及聚类分析[J].中国乳品工业,2005,33(6):4-9.
[11]SUN Z H,LIU W J,GAO W,et al.Identification and characterization of the dominant lactic acid bacteria from kurut:The naturally fermented yak milk in Qinghai,China[J].The Journal of General and Applied Microbiology,2010,56(1):1-10.
[12]杨彦荣,任艳,德亮亮,等.传统奶酪样品中乳酸菌的分离鉴定[J].中国乳品工业,2015,43(9):19-22.
[13]YU J,GAO W,QIN M J,et al.Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan,China[J].The Journal of General and Applied Microbiology,2012,58(3):163-172.
[14]ALTSCHUL S F,MADDEN T L,SCH魧FFER AA,et al.Gapped BLAST and PSI-BLAST:A new generation of protein database search programs[J].Nucleic Acids Research,1997,25(17):3389-3402.
[15]YU J,WANG W H,MENGHE B L G,et al.Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia[J].Journal of Dairy Science,2011,94(7)3230-3241.
[16]KUMAR S,STECHER G,TAMURA K.MEGA7:Molecular evolutionary genetics analysis version 7.0for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870-1874.
[17]孟和毕力格,乌日娜,王立平,等.不同地区酸马奶中乳杆菌的分离及其生物学特性的研究[J].中国乳品工业,2004,32(11):6-11.
[18]ISHIIS.Microbiol study of traditional fermented dairy products Haruha and Buriat in Mongolia[J].Milk Science,2001,50:25-30
[19]孙天松,王俊国,张列兵,等.中国新疆地区酸马奶中乳酸菌生物多样性研究[J].微生物学通报,2007,34(3):451-454.
[20]FANG L,LI-HUI D U,PENG D U,et al.Biodiversity of lactic acid bacteria in home-made koumiss from Inner Mongolia[J].Food Science,2008,29(2):218-224
[21]JIN H,MO L,PAN L,et al.Using PacBio sequencing to investigate the bacterial microbiota of traditional Buryatian cottage cheese and comparison with Italian and Kazakhstan artisanal cheeses[J].Journal of Dairy Science,2018,10(8):6885-6896
[22]OKI K,DUGERSUREN J,DEMBEREL S,et al Pyrosequencing analysis of the microbial diversity of airag,khoormog and tarag,traditional fermented dairy products of Mongolia[J].Bioscience of Microbiota,Food and Health,2014,33(2):53-64.
[23]其木格苏都.自然发酵酸马奶细菌多样性及其基因动态变化研究[D].呼和浩特:内蒙古农业大学,2017.
[24]SADAT-MEKMENE L,JARDIN J,CORRE C,et al.Simultaneous presence of PrtH and PrtH2 proteinases in Lactobacillus helveticus strains improves breakdown of the pure alphas1-casein[J].Appl Environ Microbiol,2011,77(1):179-186.
[25]CHEN Y,LIU W,XUE J,et al.Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect of fermented milk of strain H9[J].Journal of Dairy Science,2014,97(11):6680-6692.
[26]KIMOTO-NIRA H,AOKI R,MIZUMACHI K,et al.Interaction between Lactococcus lactis and Lactococcus raffinolactis during growth in milk:Development of a new starter culture[J].Journal of Dairy Science,2012,95(4):0-2185.
[27]NHO S W,HIKIMA J,CHA I S,et al.Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis[J].Journal of Bacteriology,2011,193(13):3356.
[28]CHEN W M,YANG S H,YOUNG C C,et al Rheinheimeratilapiae sp.nov.isolated from a freshwater culture pond[J].International Journal of Systematic and Evolutionary Microbiology,2013,63(4):1457-1463.
[29]RASOLOFO E A,ST-GELAIS D,LAPOINTE G,et al.Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk[J].International Journal of Food Microbiology,2010,138(1/2):108-118.
[30]BOUBENDIR A,SERRAZANETTI D I,HAMIDECHIM A,et al.Changes in bacterial populations in refrigerated raw milk collected from a semi-arid area of Algeria[J].Annals of Microbiology,2016,66(2):777-783.
[31]VITHANAGE N R,YEAGER T R,JADHAV S R,et al.Comparison of identification systems for psychrotrophic bacteria isolated from raw bovine milk[J].International Journal of Food Microbiology,2014,189:26-38.
[32]HANTSISZACHAROV E,HALPERN M.Culturable psychrotrophic bacterial communities in raw milk and their proteolytic and lipolytic traits[J].Applied&Environmental Microbiology,2007,73(22):7162-7168.
[33]COOREVITS A,DE JONGHE V,VANDROEMMEJ,et al.Comparative analysis of the diversity of aerobic spore-forming bacteria in raw milk from organic and conventional dairy farms[J].Syst Appl Microbiol,2008,31(2):126-140.
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