两种来源乳杆菌的分离鉴定及遗传多态性研究
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摘要
乳杆菌属(Lactobacillus)是乳酸菌中最大的一个属,也是乳酸菌中对食品工业最为重要、最常见和应用最广泛的一个属。作为益生菌,乳杆菌以其优良的益生作用而受到广泛的关注。我国蔬菜资源丰富,酸菜作为一种传统的乳酸发酵蔬菜制品,其汁液中富含乳酸菌活菌,其中尤以短乳杆菌和植物乳杆菌占优势。另外,乳酸菌也是猪消化道前段的优势菌群,自然存在于猪肠道正常菌群的乳酸杆菌有:嗜酸乳杆菌、罗伊氏乳杆菌、短乳杆菌、纤维二糖乳杆菌、发酵乳杆菌、唾液乳杆菌,其中最主要的是嗜酸乳杆菌和罗伊氏乳杆菌。
乳杆菌的传统鉴定方法主要是依赖乳杆菌的表型特征,尽管这些方法依然是目前鉴定乳杆菌的主要方法,但更多的研究显示,对乳杆菌准确分类还需要进行基因水平上的鉴定。单纯的依靠表型特征试验易造成乳杆菌鉴定结果不准确,故必须结合分子生物学鉴定结果才能对乳杆菌进行准确鉴定。本研究的主要研究内容为分离植物源和动物源的乳杆菌,并对其进行鉴定和遗传多态性分析。
本研究主要取得如下研究结果:
1.乳杆菌的分离纯化及菌种保存
以自然发酵的优良东北酸菜和猪肠道内容物为样品,以改良MRS培养基为分离培养基,厌氧条件培养,对这两种来源的乳杆菌进行分离、纯化以及菌株保藏。并通过形态学特征和过氧化氢酶试验,初步确定从酸菜样品和猪肠道样品中分别分离得到乳杆菌39株和29株。
2.乳杆菌的生理生化鉴定
通过明胶液化试验、吲哚试验、H_2S产生试验、硝酸盐还原试验进一步验证所分离的菌株,结果显示从酸菜样品和猪肠道样品分离得到的菌株中分别有34和25株属于乳杆菌属:对分离自酸菜的L1～L10株,以及分离自猪肠道的L10～L20株进行糖发酵实验,通过它们对单糖、二糖、多糖以及糖苷和糖醇类等碳水化合物的利用情况初步确定其种。
3.利用16S rDNA的序列聚类分析鉴定乳杆菌
使用一对乳酸菌特异性引物扩增分离株L1～L20的16S rDNA序列,得到大小为1500bp左右的16S rDNA片段,然后进行测序。将分离株的测序结果与参比菌株的序列进行比对,得出他们之间的相似百分比,同时结合糖发酵结果确定这20株菌为:干酪乳杆菌1株、短乳杆菌4株、棒状乳杆菌1株、植物乳杆菌3株和米酒乳杆菌1株:罗伊氏乳杆菌4株、约氏乳杆菌2株、嗜酸乳杆菌2株、发酵乳杆菌1株、唾液乳杆菌1株。
4.利用PCR-RFLP技术研究乳杆菌的遗传多态性
用3种限制性内切酶XbaⅠ、MspⅠ和AluⅠ分别消化分离株L1～L20的16S rDNA,其中MspⅠ酶和AluⅠ酶产生丰富的酶切图谱,将L1～L20分成了10个遗传型,表明分离株具有遗传多态性特征。
5.利用PCR-DGGE技术区分不同种的乳杆菌
使用一对乳酸菌的特异性引物扩增L1～L20的V7～V8高可变区,变性梯度凝胶电泳的结果证明这种方法能够区分不同种的分离株,其结果与传统方法以及16s rDNA的序列聚类分析的结果相一致。
本研究从两种来源样品中分离得到59株乳杆菌,并将其中的20株分离菌株鉴定到种水平,为进一步研究和应用奠定了基础:另外还分析了几种分子生物学方法在乳杆菌鉴定和区分中的作用,为以后系统化鉴定乳杆菌提供了依据。
Lactobacillus is the biggest genus in the lactic acid bacteria,it's so important that it's widely used in food industry.Be probiotics,lactobacilli have received many attentions because of its probiotic function.As a traditional lactic acid fermentation vegetables production,sauerkraut is rich in living lactic acid bacteria in its juice;especially the Lactobacillus brevis and Lactobacillus plantarum are predominant.Besides,lactic acid bacteria are the dominance flora in the digestive tract of pig.The lactobacilli present normally in the digestive tract of pig contain:Lactobacillus acidophilus,Lactobacillus reuteri,Lactobacillus brevis,Lactobacillus fermentum and Lactobacillus salivarius,in which the Lactobacillus acidophilus and Lactobacillus reuteri are dominance.
The traditional identify method of the lactobacilli is depends on the phenotypic characteristics of the lactobacilli,although it's still the main methods now,more research indicate that the accurate classification of lactobacilli needs the identification on gene level.Test on phenotypic characteristics only can easily cause the inaccuracy of the identify result,so combining with the molecular biology identification,the result can be trust.The main contents of this study are isolation lactobacilli of animal origin and plant origin,identification of them,and analysis the genetic polymorphism of them.
This research mainly included the following five parts:
1.The isolation,purification and preservation of lactobacilli
Take the wild fermentation sauerkraut and pig digestive tract contents as the simple,the modified MRS medium as the isolation medium,cultivate under anaerobic conditions,lactobacilli of these two source were isolated,purification and preservation.According to the morphological characteristics and catalase test,39 strains and 29 strains were identified as lactobacilli.
2.The physiological and biochemical identification of lactobacilli
According to the gelatin liquefaction test,indole test,H_2S creation and nitrate reduction test, we discovered that 34 and 25 strains isolated from these two sources belong to Lactobacillus.And confirm the species according to the fermentation experiment about carbohydrate.
3.Identify of lactobacilli by 16S rDNA sequence cluster analysis
Amplify the 16S rDNA sequence of the isolation strains L1～L20 using a pair of primer which amplify lactic acid bacteria only,obtaining a fragment length 1500bp around,and then the 16S rDNA sequence was alignments with type strains blast on the GengBank.The result can be obtained according to the homology.Finally,1 Lactobacillus casei strain,4 Lactobacillus brevis strains,1 Lactobacillus coryniformis strain,3 Lactobacillus plantarum strains,1 Lactobacillus sakei strain;4 Lactobacillus reuteri strains,2 Lactobacillus johnsonii strains,2 Lactobacillus acidophilus strains,1 Lactobacillus fermentum strain,1 Lactobacillus salivarius strain were identified.
4.Genetic polymorphism analysis of the lactobacilli by the PCR-RFLP technique
Using three restriction enzyme:XbaⅠ,MspⅠand AluⅠdigest the 16S rDNA of isolation strains L1～L20,the MspⅠand AluⅠproduce an abundant enzyme map,divided L1～L20 into 10 groups,present the polymorphism,reflect the genetic polymorphism of isolation strains.
5.Discriminate lactobacilli of different species by PCR-DGGE
Amplify the V7～V8 hypervariable region of the isolation strains L1～L20 using a pair of primer which amplify lactic acid bacteria only,the result of the DGGE showed that these isolation strains can be discriminated by this method;the result is matched with the traditional method and 16S rDNA sequence cluster analysis.
59 strains were isolated from two source,part of them were analysis and identified,these laid the foundation for them to be used in different domain;besides,the role of some molecular biology methods in identification and discrimination of lactobacilli was analysis,which provides basis for the systematized identification of lactobacilli.
乳杆菌的传统鉴定方法主要是依赖乳杆菌的表型特征,尽管这些方法依然是目前鉴定乳杆菌的主要方法,但更多的研究显示,对乳杆菌准确分类还需要进行基因水平上的鉴定。单纯的依靠表型特征试验易造成乳杆菌鉴定结果不准确,故必须结合分子生物学鉴定结果才能对乳杆菌进行准确鉴定。本研究的主要研究内容为分离植物源和动物源的乳杆菌,并对其进行鉴定和遗传多态性分析。
本研究主要取得如下研究结果:
1.乳杆菌的分离纯化及菌种保存
以自然发酵的优良东北酸菜和猪肠道内容物为样品,以改良MRS培养基为分离培养基,厌氧条件培养,对这两种来源的乳杆菌进行分离、纯化以及菌株保藏。并通过形态学特征和过氧化氢酶试验,初步确定从酸菜样品和猪肠道样品中分别分离得到乳杆菌39株和29株。
2.乳杆菌的生理生化鉴定
通过明胶液化试验、吲哚试验、H_2S产生试验、硝酸盐还原试验进一步验证所分离的菌株,结果显示从酸菜样品和猪肠道样品分离得到的菌株中分别有34和25株属于乳杆菌属:对分离自酸菜的L1～L10株,以及分离自猪肠道的L10～L20株进行糖发酵实验,通过它们对单糖、二糖、多糖以及糖苷和糖醇类等碳水化合物的利用情况初步确定其种。
3.利用16S rDNA的序列聚类分析鉴定乳杆菌
使用一对乳酸菌特异性引物扩增分离株L1～L20的16S rDNA序列,得到大小为1500bp左右的16S rDNA片段,然后进行测序。将分离株的测序结果与参比菌株的序列进行比对,得出他们之间的相似百分比,同时结合糖发酵结果确定这20株菌为:干酪乳杆菌1株、短乳杆菌4株、棒状乳杆菌1株、植物乳杆菌3株和米酒乳杆菌1株:罗伊氏乳杆菌4株、约氏乳杆菌2株、嗜酸乳杆菌2株、发酵乳杆菌1株、唾液乳杆菌1株。
4.利用PCR-RFLP技术研究乳杆菌的遗传多态性
用3种限制性内切酶XbaⅠ、MspⅠ和AluⅠ分别消化分离株L1～L20的16S rDNA,其中MspⅠ酶和AluⅠ酶产生丰富的酶切图谱,将L1～L20分成了10个遗传型,表明分离株具有遗传多态性特征。
5.利用PCR-DGGE技术区分不同种的乳杆菌
使用一对乳酸菌的特异性引物扩增L1～L20的V7～V8高可变区,变性梯度凝胶电泳的结果证明这种方法能够区分不同种的分离株,其结果与传统方法以及16s rDNA的序列聚类分析的结果相一致。
本研究从两种来源样品中分离得到59株乳杆菌,并将其中的20株分离菌株鉴定到种水平,为进一步研究和应用奠定了基础:另外还分析了几种分子生物学方法在乳杆菌鉴定和区分中的作用,为以后系统化鉴定乳杆菌提供了依据。
Lactobacillus is the biggest genus in the lactic acid bacteria,it's so important that it's widely used in food industry.Be probiotics,lactobacilli have received many attentions because of its probiotic function.As a traditional lactic acid fermentation vegetables production,sauerkraut is rich in living lactic acid bacteria in its juice;especially the Lactobacillus brevis and Lactobacillus plantarum are predominant.Besides,lactic acid bacteria are the dominance flora in the digestive tract of pig.The lactobacilli present normally in the digestive tract of pig contain:Lactobacillus acidophilus,Lactobacillus reuteri,Lactobacillus brevis,Lactobacillus fermentum and Lactobacillus salivarius,in which the Lactobacillus acidophilus and Lactobacillus reuteri are dominance.
The traditional identify method of the lactobacilli is depends on the phenotypic characteristics of the lactobacilli,although it's still the main methods now,more research indicate that the accurate classification of lactobacilli needs the identification on gene level.Test on phenotypic characteristics only can easily cause the inaccuracy of the identify result,so combining with the molecular biology identification,the result can be trust.The main contents of this study are isolation lactobacilli of animal origin and plant origin,identification of them,and analysis the genetic polymorphism of them.
This research mainly included the following five parts:
1.The isolation,purification and preservation of lactobacilli
Take the wild fermentation sauerkraut and pig digestive tract contents as the simple,the modified MRS medium as the isolation medium,cultivate under anaerobic conditions,lactobacilli of these two source were isolated,purification and preservation.According to the morphological characteristics and catalase test,39 strains and 29 strains were identified as lactobacilli.
2.The physiological and biochemical identification of lactobacilli
According to the gelatin liquefaction test,indole test,H_2S creation and nitrate reduction test, we discovered that 34 and 25 strains isolated from these two sources belong to Lactobacillus.And confirm the species according to the fermentation experiment about carbohydrate.
3.Identify of lactobacilli by 16S rDNA sequence cluster analysis
Amplify the 16S rDNA sequence of the isolation strains L1～L20 using a pair of primer which amplify lactic acid bacteria only,obtaining a fragment length 1500bp around,and then the 16S rDNA sequence was alignments with type strains blast on the GengBank.The result can be obtained according to the homology.Finally,1 Lactobacillus casei strain,4 Lactobacillus brevis strains,1 Lactobacillus coryniformis strain,3 Lactobacillus plantarum strains,1 Lactobacillus sakei strain;4 Lactobacillus reuteri strains,2 Lactobacillus johnsonii strains,2 Lactobacillus acidophilus strains,1 Lactobacillus fermentum strain,1 Lactobacillus salivarius strain were identified.
4.Genetic polymorphism analysis of the lactobacilli by the PCR-RFLP technique
Using three restriction enzyme:XbaⅠ,MspⅠand AluⅠdigest the 16S rDNA of isolation strains L1～L20,the MspⅠand AluⅠproduce an abundant enzyme map,divided L1～L20 into 10 groups,present the polymorphism,reflect the genetic polymorphism of isolation strains.
5.Discriminate lactobacilli of different species by PCR-DGGE
Amplify the V7～V8 hypervariable region of the isolation strains L1～L20 using a pair of primer which amplify lactic acid bacteria only,the result of the DGGE showed that these isolation strains can be discriminated by this method;the result is matched with the traditional method and 16S rDNA sequence cluster analysis.
59 strains were isolated from two source,part of them were analysis and identified,these laid the foundation for them to be used in different domain;besides,the role of some molecular biology methods in identification and discrimination of lactobacilli was analysis,which provides basis for the systematized identification of lactobacilli.
引文
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