甘肃和四川省牦牛奶制品中乳酸菌的多样性研究
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摘要
本研究从我国甘肃省和四川省6个县27个地区的不同牧民家庭采集牦牛奶及传统发酵牦牛奶制品(酸牦牛奶、曲拉、乳清和酥油)共152份,对其中蕴含的乳酸菌采用传统纯培养方法进行了分离,利用16S rDNA序列分析等方法对分离株进行了鉴定并分析了其中的乳酸菌多样性：选取有代表性的传统发酵牦牛奶制品样品,采用非培养的宏基因组学方法(PCR-DGGE和焦磷酸测序)对其中的微生物多样性进行了进一步的研究分析。结论如下：
1.采用BCP平板计数法检测了所有样品中乳酸菌活菌总数,结果显示乳酸菌活菌数变化范围在4.00lgcfu/mL～9.15lg cfu/mL,且大多数样品乳酸菌数在6.00lgcfu/mL以上。同时采用选择性培养基(MRS和M17)并结合形态观察对样品中的乳酸菌进行了分离,获得乳酸菌分离株533株。
2.通过形态观察和16S rDNA序列分析法对所有的乳酸菌分离株进行了鉴定,对16S rDNA序列相似性高的菌株采用种特异性PCR、PCR-RFLP、DGGE等技术进行了鉴定,最终将所有分离的乳酸菌鉴定到种或亚种水平,建立了鉴定乳酸菌的分子生物学平台。533株乳酸菌分离株属于6个属24个不同的种和亚种,其中乳杆菌属(Lactobacillus)246株,占分离乳酸菌总数46.15%；其次为明串珠菌属(Leuconostoc)156株,占29.27%；链球菌属(Streptococcus)67株,占12.57%；乳球菌属(Lactococcus)50株,占9.38%；肠球菌属(Enterococcus)11株,占2.64%；魏斯氏菌属(Weissella)3株,占0.56%：在微生物的多样性上,瑞士乳杆菌(24.2%)和肠膜明串珠菌肠膜亚种(20.64%)是甘肃和四川牦牛奶及传统发酵牦牛奶制品中的优势菌种。不同地区不同种类的牦牛奶制品中乳酸菌组成既有相似性也有差别,如：瑞士乳杆菌是甘肃地区的传统发酵酸牦牛奶、曲拉、乳清中的优势菌：而嗜热链球菌和瑞士乳杆菌分别是四川酸牦牛奶和曲拉的优势乳酸菌。
3.建立了甘肃和四川地区牦牛奶制品中乳酸菌的16S rDNA基因序列数据库,将分离得到的533株乳酸菌的16S rDNA序列全部上传到GenBank数据库,登录号为HM058517-HM058580, HM058582-HM0059029, HM217942-HM217944, HM217948, HM217953, HM217956, HM217962, HM217964, HM217967, HM217971-HM217975, M217984-HM217986. HM217994-HM217999.
4.采用非培养的PCR-DGGE技术分析研究了四川有代表性的10份曲拉中的微生物多样性。结果显示曲拉样本中包含了9种以上乳酸菌(Lb. helveticus, Lb. plantarum, Lb. fermentum, Lb. paralimentarius,Lb, sanfranciscensis, Leu. citreum, Lb. curvatus, E. durans, Lac. lactis)。
5.利用高通量的454焦磷酸测序技术分析了甘肃和四川有代表性的酸牦牛奶样本中微生物多样性。结果显示酸牦牛奶中包含了多种复杂微生物,根据16SrDNA序列比对后所有细菌被划分到Firmicutes (厚壁菌门)、Actinobacieria(放线菌门)、Bacteroidetesi.拟杆菌门),Deinococcus-Thermus (异常球菌-栖热菌门),Proteobacteria(变形菌门)和未知的Environmental_samples(环境样本)等6门37属；Firmicutes的乳酸菌(Lactobacillus, Streptococcus, Lactococcus, Leuconosioc)在酸牦牛奶样本中占比例最高；且甘肃和四川地区牦牛奶样本中细菌主成分具有明显的地域差异。这些大量的数据让我们对酸牦牛奶中微生物的菌群结构有了全新的认识。
6.纯培养方法与宏基因组学方法均证实传统发酵牦牛奶制品中微生物种类丰富多样,而且乳酸菌是优势菌,其中乳杆菌最多。所采用的三种分析多样性的方法各具优势,纯培养方法只能分析到可培养菌,优点是获得的纯培养菌株：可以用于下步发酵剂的筛选；PCR-DGGE和焦磷酸测序技术都能基于宏基因组学水平揭示菌群结构的。DGGE直观快速地反映出样品中优势菌群的构成；通过最新的焦磷酸测序技术得到的OUT数比PCR-DGGE多,更全面的反映出菌群的组成和丰度。
本文系统地研究了中国甘肃和四川地区牦牛奶及各种传统发酵牦牛奶制品中乳酸菌的组成,这为益生菌和发酵剂的筛选提供了宝贵的资源,同时为工业化生产传统发酵乳制品的发酵剂的设计提供了基础数据。
One hundred and fifty-two samples of raw yak milk and traditional fermented yak milk products were collected from twenty-seven different areas of six counties in Gansu and Sichuan province, China. The lactic acid bacteria (LAB) were isolated and identified from these samples using traditional pure culture method and16S rDNA sequencing analysis, respectively. Meanwhile, the microbiological biodiversity of some representative samples were analyzed by metagenomics methods, such as PCR-DGGE and454pyrophosphate sequencing. The conclusions were showed as follow:
The LAB counts in five kinds of yak milk products were ranged from4.00lg cfu/mL to9.15lg cfu/mL, and most of them were up to6.00lg cfu/mL. Five hundred and thirty-three LAB isolates were isolated from these samples by traditional pure culture method using MRS and M17broth.
Five hundred and thirty three isolates were identified to species and/or subspecies by phenotypic characterization and16S rRNA gene sequence analysis. For the LAB species with very closely related16S rRNA gene sequence, species-specific PCR, PCR-RFLP and DGGE technologies were used. Meanwhile, the platform for LAB identification by molecular biology methods was established. All the isolates were classified into twenty-four species and/or subspecies, which belonged to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus. Enterococcus and Weissella). The distributions of the isolates by genus were as follows:246strains of lactobacilli (46.15%of total),156strains of leuconostoc (29.27%),67strains of Streptococci (12.57%),50strains of lactococci (9.38%),11strains of enterococci (2.64%) and three strains of weissella (0.56%). Meanwhile, according to the results of LAB biodiversity analysis, we can concluded that Lb. helveticus (129strains,24.2%of total) and Leuconostoc mesenteroides subsp. mesenteroides (110strains,20.64%) could be considered as the predominant LAB species in traditional fermented yak milk products in Gansu and Sichuan provinces. The composition of LAB in different types of yak milk products in different regions expressed differences. Lb. helveticus was the dominant bacteria in kurut, qula and whey samples in Gansu province. However. Streptococcus thermophilus and Lb. helveticus are the predominant LAB in kurut and qula in Sichuan province, respectively.
The16S rRNA gene sequence database of LAB in yak milk products in Gansu and Sichuan povinces were established. The16S rRNA gene sequences of533LAB strains were deposited to GenBank database with the accession numbers as HM058517-HM058580, HM058582-HM0059029, HM217942-HM217944, HM217948, HM217953, HM217956, HM217962, HM217964, HM217967, HM217971-HM217975, M217984-HM217986, HM217994-HM217999.
Microbial diversity in ten representative qula samples from Sichuan province were analyzed using metagenomic method PCR-DGGE. The result showed that a complex microflora including more than nine kinds of LAB species(Lb. helveticus, Lb. plantarum, Lb. fermentum, Lb. paralimentarius, Lb. sanfranciscensis, Leu. citreum, Lb. curvatus, E. durans and Lac. lactis) was exised in these sampls.
Microbial diversity in seven representative kurut samples collected from Gansu and Sichuan province were comprehensively examined by high-throughput parallel454Pyrosequencing technology. The results showed that an abundant and complex microflora was presented in these kurut samples. The kurut microbiota was far more diverse than previous studies, and thirty seven genera species belonging to six phyla(Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Proteobacteria, Environmental samples) were identified after16S rDNA sequencing analysis. Firmicutes(Lactobacillus, Streptococcus, Lactococcus, Leuconostoc) possessed the highest ratio in these kurut samples. Moreover, the main bacterial composition of these kut samples in Gansu and Sichuan province expressed obvious regional differences. These data gave us the opportunity to renew the understanding of the microbial diversity in kurut samples.
The pure culture method and metagenomics methods all confirmed that the microorganisms in traditional fermented yak milk products were diverse and LAB could be considered as the predominant bacteria, among which Lactobacillus was the preponderant genus. The three methods used for microbial diversity analysis have their specific advantag Traditional method can only analyse the culturable bacteria, and its advantage was the obtained pure culture could be used for further starter culture selection. PCR-DGGE can intuitively reflect the the composition of the dominant microorganisms in these samples.454Pyrosequencing analysis enables us to analyze an increased number of samples at a time, to obtain more reads in a single run and to perform indepth analyses for studies of comparative microbial ecology.
This paper firstly systematically analyzed the microbial composition in raw yak milk and various traditional fermented yak milk products in Gansu and Sichuan province of China. These obtained LAB pure cultures maybe a valuable source for selection of probiotics and starter cultures in dairy industry. Meanwhile, these results provided the basic data for further industrilization of traditional fermented dairy products.
1.采用BCP平板计数法检测了所有样品中乳酸菌活菌总数,结果显示乳酸菌活菌数变化范围在4.00lgcfu/mL～9.15lg cfu/mL,且大多数样品乳酸菌数在6.00lgcfu/mL以上。同时采用选择性培养基(MRS和M17)并结合形态观察对样品中的乳酸菌进行了分离,获得乳酸菌分离株533株。
2.通过形态观察和16S rDNA序列分析法对所有的乳酸菌分离株进行了鉴定,对16S rDNA序列相似性高的菌株采用种特异性PCR、PCR-RFLP、DGGE等技术进行了鉴定,最终将所有分离的乳酸菌鉴定到种或亚种水平,建立了鉴定乳酸菌的分子生物学平台。533株乳酸菌分离株属于6个属24个不同的种和亚种,其中乳杆菌属(Lactobacillus)246株,占分离乳酸菌总数46.15%；其次为明串珠菌属(Leuconostoc)156株,占29.27%；链球菌属(Streptococcus)67株,占12.57%；乳球菌属(Lactococcus)50株,占9.38%；肠球菌属(Enterococcus)11株,占2.64%；魏斯氏菌属(Weissella)3株,占0.56%：在微生物的多样性上,瑞士乳杆菌(24.2%)和肠膜明串珠菌肠膜亚种(20.64%)是甘肃和四川牦牛奶及传统发酵牦牛奶制品中的优势菌种。不同地区不同种类的牦牛奶制品中乳酸菌组成既有相似性也有差别,如：瑞士乳杆菌是甘肃地区的传统发酵酸牦牛奶、曲拉、乳清中的优势菌：而嗜热链球菌和瑞士乳杆菌分别是四川酸牦牛奶和曲拉的优势乳酸菌。
3.建立了甘肃和四川地区牦牛奶制品中乳酸菌的16S rDNA基因序列数据库,将分离得到的533株乳酸菌的16S rDNA序列全部上传到GenBank数据库,登录号为HM058517-HM058580, HM058582-HM0059029, HM217942-HM217944, HM217948, HM217953, HM217956, HM217962, HM217964, HM217967, HM217971-HM217975, M217984-HM217986. HM217994-HM217999.
4.采用非培养的PCR-DGGE技术分析研究了四川有代表性的10份曲拉中的微生物多样性。结果显示曲拉样本中包含了9种以上乳酸菌(Lb. helveticus, Lb. plantarum, Lb. fermentum, Lb. paralimentarius,Lb, sanfranciscensis, Leu. citreum, Lb. curvatus, E. durans, Lac. lactis)。
5.利用高通量的454焦磷酸测序技术分析了甘肃和四川有代表性的酸牦牛奶样本中微生物多样性。结果显示酸牦牛奶中包含了多种复杂微生物,根据16SrDNA序列比对后所有细菌被划分到Firmicutes (厚壁菌门)、Actinobacieria(放线菌门)、Bacteroidetesi.拟杆菌门),Deinococcus-Thermus (异常球菌-栖热菌门),Proteobacteria(变形菌门)和未知的Environmental_samples(环境样本)等6门37属；Firmicutes的乳酸菌(Lactobacillus, Streptococcus, Lactococcus, Leuconosioc)在酸牦牛奶样本中占比例最高；且甘肃和四川地区牦牛奶样本中细菌主成分具有明显的地域差异。这些大量的数据让我们对酸牦牛奶中微生物的菌群结构有了全新的认识。
6.纯培养方法与宏基因组学方法均证实传统发酵牦牛奶制品中微生物种类丰富多样,而且乳酸菌是优势菌,其中乳杆菌最多。所采用的三种分析多样性的方法各具优势,纯培养方法只能分析到可培养菌,优点是获得的纯培养菌株：可以用于下步发酵剂的筛选；PCR-DGGE和焦磷酸测序技术都能基于宏基因组学水平揭示菌群结构的。DGGE直观快速地反映出样品中优势菌群的构成；通过最新的焦磷酸测序技术得到的OUT数比PCR-DGGE多,更全面的反映出菌群的组成和丰度。
本文系统地研究了中国甘肃和四川地区牦牛奶及各种传统发酵牦牛奶制品中乳酸菌的组成,这为益生菌和发酵剂的筛选提供了宝贵的资源,同时为工业化生产传统发酵乳制品的发酵剂的设计提供了基础数据。
One hundred and fifty-two samples of raw yak milk and traditional fermented yak milk products were collected from twenty-seven different areas of six counties in Gansu and Sichuan province, China. The lactic acid bacteria (LAB) were isolated and identified from these samples using traditional pure culture method and16S rDNA sequencing analysis, respectively. Meanwhile, the microbiological biodiversity of some representative samples were analyzed by metagenomics methods, such as PCR-DGGE and454pyrophosphate sequencing. The conclusions were showed as follow:
The LAB counts in five kinds of yak milk products were ranged from4.00lg cfu/mL to9.15lg cfu/mL, and most of them were up to6.00lg cfu/mL. Five hundred and thirty-three LAB isolates were isolated from these samples by traditional pure culture method using MRS and M17broth.
Five hundred and thirty three isolates were identified to species and/or subspecies by phenotypic characterization and16S rRNA gene sequence analysis. For the LAB species with very closely related16S rRNA gene sequence, species-specific PCR, PCR-RFLP and DGGE technologies were used. Meanwhile, the platform for LAB identification by molecular biology methods was established. All the isolates were classified into twenty-four species and/or subspecies, which belonged to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus. Enterococcus and Weissella). The distributions of the isolates by genus were as follows:246strains of lactobacilli (46.15%of total),156strains of leuconostoc (29.27%),67strains of Streptococci (12.57%),50strains of lactococci (9.38%),11strains of enterococci (2.64%) and three strains of weissella (0.56%). Meanwhile, according to the results of LAB biodiversity analysis, we can concluded that Lb. helveticus (129strains,24.2%of total) and Leuconostoc mesenteroides subsp. mesenteroides (110strains,20.64%) could be considered as the predominant LAB species in traditional fermented yak milk products in Gansu and Sichuan provinces. The composition of LAB in different types of yak milk products in different regions expressed differences. Lb. helveticus was the dominant bacteria in kurut, qula and whey samples in Gansu province. However. Streptococcus thermophilus and Lb. helveticus are the predominant LAB in kurut and qula in Sichuan province, respectively.
The16S rRNA gene sequence database of LAB in yak milk products in Gansu and Sichuan povinces were established. The16S rRNA gene sequences of533LAB strains were deposited to GenBank database with the accession numbers as HM058517-HM058580, HM058582-HM0059029, HM217942-HM217944, HM217948, HM217953, HM217956, HM217962, HM217964, HM217967, HM217971-HM217975, M217984-HM217986, HM217994-HM217999.
Microbial diversity in ten representative qula samples from Sichuan province were analyzed using metagenomic method PCR-DGGE. The result showed that a complex microflora including more than nine kinds of LAB species(Lb. helveticus, Lb. plantarum, Lb. fermentum, Lb. paralimentarius, Lb. sanfranciscensis, Leu. citreum, Lb. curvatus, E. durans and Lac. lactis) was exised in these sampls.
Microbial diversity in seven representative kurut samples collected from Gansu and Sichuan province were comprehensively examined by high-throughput parallel454Pyrosequencing technology. The results showed that an abundant and complex microflora was presented in these kurut samples. The kurut microbiota was far more diverse than previous studies, and thirty seven genera species belonging to six phyla(Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Proteobacteria, Environmental samples) were identified after16S rDNA sequencing analysis. Firmicutes(Lactobacillus, Streptococcus, Lactococcus, Leuconostoc) possessed the highest ratio in these kurut samples. Moreover, the main bacterial composition of these kut samples in Gansu and Sichuan province expressed obvious regional differences. These data gave us the opportunity to renew the understanding of the microbial diversity in kurut samples.
The pure culture method and metagenomics methods all confirmed that the microorganisms in traditional fermented yak milk products were diverse and LAB could be considered as the predominant bacteria, among which Lactobacillus was the preponderant genus. The three methods used for microbial diversity analysis have their specific advantag Traditional method can only analyse the culturable bacteria, and its advantage was the obtained pure culture could be used for further starter culture selection. PCR-DGGE can intuitively reflect the the composition of the dominant microorganisms in these samples.454Pyrosequencing analysis enables us to analyze an increased number of samples at a time, to obtain more reads in a single run and to perform indepth analyses for studies of comparative microbial ecology.
This paper firstly systematically analyzed the microbial composition in raw yak milk and various traditional fermented yak milk products in Gansu and Sichuan province of China. These obtained LAB pure cultures maybe a valuable source for selection of probiotics and starter cultures in dairy industry. Meanwhile, these results provided the basic data for further industrilization of traditional fermented dairy products.
引文
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