新疆特色干酪中乳酸菌的分离鉴定

从新疆不同牧区采集工艺不同的干酪制品,对其中的乳酸菌进行分离纯化、生理生化性质试验和16S rRNA分析.结果表明,分离、纯化出的104株乳酸菌种,有82株为乳杆菌属(Lactobacillus),12株为肠球菌属(Enterococcus),10株为魏斯氏菌属(Weissella).利用16S rRNA序列同源分析和系统发育树分析对具有不同生理生化特性的代表菌株进行了分子鉴定,鉴定结果为TNM-2与干酪乳杆菌(Lactobacillus casei)、Y5-4与食窦魏斯氏菌(Weissella cibaria)、NS2-2与植物乳杆菌(Lactobacillus plantarum)的同源性达到100％,NM-2与瑞士乳杆菌(Lactobacillus helveticus)、Y1-1与马乳酒乳杆菌(Lactobacillus kefiranofaciens)、WG与耐久肠球菌(Enterococcus durans)的同源性达到99％.     

川西高原牦牛酸奶子乳酸菌遗传多样性及系统发育研究

采用16S rDNA基因的限制性酶切片段长度多态性分析(16S rDNA-PCR-RFLP)和16S～23S rDNA基因间区(ISR)的酶切多态性分析(ISR-PCR-RFLP)技术,研究分离自川西高原牦牛酸奶子的81株乳酸菌的遗传多样性,并结合16S rDNA序列分析技术研究16S rDNA-PCR-RFLP的各类群代表菌株的系统发育关系。结果表明：供试菌株共有23种16S rDNA遗传型,供试菌株按61%相似系数可分为4个类群,按81%相似系数,可分为16个类群,结合序列分析结果,乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)细菌为主要类群;16S～23S rDNA基因的ISR扩增产物的酶切分析表明,供试菌株共有19种ISR遗传型,按60%相似系数,供试菌株可分为4个类群,按80%相似系数,供试菌株可划分为14个类群。     

新疆地区不同酸奶中优势乳酸菌的分离与鉴定

在乳制品工业中乳酸菌被认为是重要的益生菌之一。本文对来自新疆的酸驼奶、酸牛奶、酸马奶、酸羊奶以及俄罗斯酸奶中的优势乳酸菌进行分离,结合16S rRNA序列同源性分析和生理生化试验,鉴定出几种优势乳酸杆菌,即发酵乳杆菌、干酪乳杆菌、植物乳杆菌、戊糖乳杆菌。     

河豚鱼16S rRNA基因部分DNA序列分析及应用

应用16S rRNA基因聚合酶链式反应（polymerase chain reaction,PCR）扩增通用引物,对3 属13 种福建省搜集的河豚鱼样品与9 个未知物种的河豚鱼样品的16S rRNA基因序列中的部分片段进行PCR扩增与脱氧核糖核酸（deoxyribonucleic acid,DNA）碱基序列测定,各物种序列长度在611～614 bp之间。应用DNAMAN V6软件进行样品间DNA序列同源性比对分析,建立样品间系统关系同源树。供试13 个样品被划分为4 个类群组,群间的同源率为87%,群内同源率为94%～100%。根据序列同源性分析结果,9 个未知种名的样品被归类到3 个类群组中,推测9 个未知种名的样品为东方鲀属或腹刺鲀属。探讨了16S rRNA基因部分DNA序列测试及同源性分析技术在河豚鱼种属鉴别中应用的可能性。     

自然发酵乳中粪肠球菌和屎肠球菌的4种鉴定方法的比较

采用传统生理生化鉴定方法,16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),对分离于自然发酵乳中的9株粪肠球菌和6株屎肠球菌进行鉴定,并对4种鉴定方法进行比较和评价。结果表明,16S-23S rRNA间区序列多态性分析技术和DGGE技术不但可以快速、精确地区分粪肠球菌和屎肠球菌,而且能够将粪肠球菌和屎肠球菌种内的不同基因亚型区分开,而传统生理生化鉴定方法和16S rRNA基因序列分析技术较以上两种方法区分效果略差。     

基于16S rRNA基因与相关基因序列分析格氏乳球菌亲缘关系

通过PCR扩增7株分离自传统发酵乳制品中的格氏乳球菌的dnaA、rpoB、recA基因,将扩增产物测序,测得的序列构建系统发育树,进行分类鉴定.同时,比较了这三个基因位点与16S rRNA基因揭示的格氏乳球菌种内菌株间以及其与乳酸乳球菌种间的亲缘关系的远近.结果表明,dnaA、rpoB、recA基因能够有效的鉴定出格氏乳球菌,且比16S rRNA基因更清晰地揭示了格氏乳球菌菌株间以及其与乳酸乳球菌种间的亲缘关系,特别是将3个管家基因联合起来,亲缘关系更明确,验证了多个管家基因是研究细菌亲缘关系的有力工具.     

新疆伊犁牧区传统手工奶酪中微生物多样性及其功能分析

为探究新疆伊犁牧区传统手工奶酪中的可培养乳酸菌和细菌多样性,首先采用Illumina MiSeq高通量测序技术对细菌16S rRNA基因的V1～V3可变区进行测序分析,结果显示：14 份奶酪中共存在493 种细菌属和780 种细菌种,其中奶酪中的优势细菌属和优势乳酸菌属为雷尔氏菌属和乳杆菌属,优势乳酸菌种为瑞士乳杆菌,且奶酪中还存在致病菌。在此基础上,利用传统培养法从MRS培养基中筛选出68 株可培养疑似乳酸菌,经生理生化实验及16S rDNA序列同源性分析技术对其进行属种鉴定。结果表明：68 株疑似乳酸菌分别为短乳杆菌、融合魏斯氏菌、屎肠球菌、粪肠球菌、耐久肠球菌、鸟肠球菌、棉籽糖肠球菌、植物乳杆菌、瑞士乳杆菌,其中融合魏斯氏菌为奶酪中的优势乳酸菌种。在乳酸菌的属种鉴定上,传统培养法能够更准确地将乳酸菌鉴定到种水平。奶酪微生物基因COG功能预测结果显示：奶酪中的微生物富含与氨基酸转运和代谢有关的基因。将各个样品的微生物群落组成与功能组成进行比较,发现各个样品的细菌群落组成虽存在明显差异,但其功能组成却具有高度相似性。     

基于高通量测序分析不同品种水牛生乳中细菌多样性

目的 利用16S rRNA高通量测序研究广西水牛研究所种畜厂内摩拉、尼里-拉菲及三品杂3品种水牛生鲜乳中的细菌多样。方法 提取当天采集的生水牛乳样本细菌总DNA,PCR扩增细菌的16S rDNA,纯化PCR扩增目的片断,将PCR产物与pMD18-T载体连接,构建其菌群的16S rDNA文库。用Miseq对其16S rRNA基因的V3-V4可变区进行测序以及BLAST比对。结果 3品种27个样本共获得1223个OUTs, M、N、S独有的OTU分别为295、41和42,共有OTU为511。物种组成与丰度差异显示：3品种优势菌门为：变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)。优势菌属为：金黄杆菌属(Chryseobacterium)、不动杆菌属(Acinetobacter)、乳球菌属(Lactococcus)、假单胞菌属(Pseusomonas)。样本层级聚类显示：3品种样本间菌落组成相似度较高。PCA与PLS-DA分析表明：3品种样本组内菌群多样性差异不显著,但组间菌群丰度差异明显。结论 摩拉、尼里-拉菲及三品杂水牛生鲜乳中细菌菌群均呈现较丰富的多样性,其共有的优势菌群为金黄杆菌(Chryseobacterium)、不动杆菌(Acinetobacter)、乳球菌(Lactococcus)、假单胞菌(Pseusomonas)。各品种间菌群组成种类整体相似,但其主要菌落丰富度差异明显。     

新疆特色果蔬中乳酸菌分离鉴定及其抑菌活性

利用MRS,M17等4种培养基对采自新疆不同地区的8种水果中的乳酸菌进行分离鉴定,根据16S rRNA基因序列确定菌株的系统进化关系,并利用牛津杯法筛选具有抑菌活性的乳酸菌。共分离疑似乳酸菌90株,rep-PCR指纹图谱分型选出15株代表菌株,经16S rRNA基因序列分析,它们分别隶属于乳杆菌属(Lactobacillus)8株、明串珠菌属(Leuconostoc)6株、乳球菌属(Lactococcus)1株,共3个属,8个种。利用牛津杯法筛选出8株对枯草芽孢杆菌、单增李斯特菌、金黄色葡萄球菌具有明显抑制作用的乳酸菌,为乳酸菌作为生物防腐剂应用到食品工业中奠定基础。     

pheS基因序列分析在干酪乳杆菌群种水平鉴定中的应用

对干酪乳杆菌群(Lactobacillus casei group)内5个种和4个亚种的23个代表菌株的苯丙氨酰-tRNA合成酶α亚基基因(pheS)序列和18个16S rRNA基因序列进行系统发育学分析,探索pheS基因序列分析在干酪乳杆菌群种水平鉴定中应用的可行性。分析结果表明,pheS基因序列群内种间的差异率在6.8%～34%之间,种内最大差异率可达3%,其变异率远高于16S rRNA基因,可作为该群内种水平鉴定的重要手段。     

植物乳杆菌及其近缘种部分看家基因的系统发育分析

本文研究了16S rRNA基因、pheS和pyr G部分基因序列对Lactobacillus plantarum(L.plantarum)及其近缘种的区分能力,采用分离自酸面团、酸牛奶和泡菜中的10株L.plantarum为研究对象,以其看家基因pheS和pyr G部分基因序列作为分子标记,结合已上传至Gene Bank的近缘种的相应序列构建系统发育树并与以16S rRNA基因构建的系统发育树进行比较。结果表明:基于16S rRNA基因序列不能区分L.plantarum及其近缘种,而看家基因pheS和pyr G部分基因序列能够很好的区分植物乳杆菌种及其近缘种,其中,pheS在植物乳杆菌种内分型时相较于pyr G基因效果更好,以及将pheS和pyr G部分基因序列串联使用后,试验菌株与参考菌株的分类关系更加明晰,因此,联合基因(pheS-pyr G)可作为16S rRNA基因的辅助工具用于L.plantarum及近缘种和植物乳杆菌种内分型的快速分类鉴定。     

Strain-level characterization of nonstarter lactic acid bacteria in Norvegia cheese by high-resolution melt analysis

The nonstarter lactic acid bacteria (NSLAB) constitute an important microbial group found during cheese ripening and they are thought to be fundamental to the quality of cheese. Rapid and accurate diagnostic tests for NSLAB are important for cheese quality control and in understanding the cheese ripening process. Here, we present a novel rapid approach for strain-level characterization through combined 16S rRNA gene and repetitive sequence-based high-resolution melt analysis (HRM). The approach was demonstrated through the characterization of 94 isolates from Norvegia, a Gouda-type cheese. The HRM profiles of the V1 and V3 variable regions of the 16S rRNA gene of the isolates were compared with the HRM profiles of 13 reference strains. The HRM profile comparison of the V1 and V3 regions of the 16S rRNA gene allowed discrimination of isolates and reference strains. Among the cheese isolates, Lactobacillus casei/paracasei (62 isolates) and Lactobacillus plantarum/Lactobacillus pentosus (27 isolates) were the dominant species, whereas Lactobacillus curvatus/Lactobacillus sakei were found occasionally (5 isolates). The HRM profiling of repetitive sequence-based PCR using the (GTG)(5) primer was developed for strain-level characterization. The clustering analysis of the HRM profiles showed high discriminatory power, similar to that of cluster analysis based on the gel method. In conclusion, the HRM approach in this study may be applied as a fast, accurate, and reproducible method for characterization of the NSLAB microflora in cheese and may be applicable to other microbial environments following selective plate culturing.     

传统发酵成熟期豆瓣酱醅中的微生物群落分析

为了探明传统发酵成熟期豆瓣酱醅中的功能微生物种类,采用免培养法(基因克隆文库的构建技术)对发酵成熟酱醅中的细菌16SrRNA和真菌18SrRNA进行了分析。结果表明,成熟酱醅中存在的细菌分别为Staphylococcus属、Lactobacillus属和Tetragenococcus属,其与具有最高同源性的菌株的相似性分别为99%、98%、99%。成熟酱醅中发现的真菌共5个属,分别为Zygosac charomyces属、Rhizochaete属、Aspergillus属、Rhodotorula属和Phaseoleae environmental sample,与具有最高同源性的菌株的相似性均为99%。     

Rapid differentiation of lactic acid bacteria from autochthonous fermentation of Iberian dry-fermented sausages

The populations of lactic acid bacteria (LAB) in different types of Iberian dry-fermented sausages from central-west Spain were identified. A simple and rapid electrophoretic method of whole-cell protein profiles was evaluated, correlating it with 16S rRNA gene sequence analysis and biochemical identification by API 50 CHL. A total of 96 isolates were identified by SDS-PAGE showing stable profiles corresponding to 30-45 polypeptides in the range 95-8kDa that were clearly different for the different species and were grouped with those of the 9 reference strains used in this study. The SDS-PAGE method showed that the predominant species were Pediococcus acidilactici (48%) followed by Lactobacillus plantarum (23%) and Lactobacillus brevis (18%). The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. However, biochemical identifications by API 50 CHL showed different errors at the genus and species level. In sum, the SDS-PAGE analysis showed itself to be a rapid and accurate differentiation method for the most commonly encountered LAB isolates in dry-fermented sausages.     

食品检验中乳酸菌鉴定方法的探讨

目的评价GB 4789《食品安全国家标准食品微生物学检验》中乳酸菌的鉴定方法。方法选择5种双歧杆菌、4种乳酸杆菌和1种链球菌共10株乳酸菌,分别采用GB 4789标准中的方法、API生化鉴定系统、16S rRNA基因序列分析法和RiboPrinter全自动基因指纹图谱分析法进行鉴定。结果 GB 4789对乳酸杆菌和嗜热链球菌鉴定效果较好,对双歧杆菌的鉴定能力较弱;API鉴定乳酸菌一般至"属"水平;16S rRNA序列分析和RiboPrinter系统可鉴定乳酸菌至"种"水平,婴儿双歧杆菌的鉴定结果为长双歧杆菌婴儿亚种。结论建议GB 4789标准中增加分子生物学方法作为乳酸菌鉴定方法的补充,同时建议及时更新标准中菌株的分类和名称。     

贵州少数民族酸肉、酸鱼中乳酸菌的分离鉴定

采用传统微生物分离方法进行乳酸菌纯种分离,利用16S rRNA序列分析方法进行乳酸菌鉴定,从7个酸肉、酸鱼样品中共分离出14株乳酸菌,有乳杆菌属、环丝菌属、乳球菌属3个属,9个种.从其中鉴定出7株乳酸菌,分别是:植物乳杆菌(Lactobacillus plantarum)、消化乳杆菌(Lactobacillus alimentarius)、清酒乳杆菌(Lactobacillus sakei)、泡菜乳杆菌(Lactobacillus kinchi)、清酒乳杆菌亚种(肉)(Lactobacillus sakei subsp.carnosus)、草乳杆菌(Lactobacillus graminis)、弯曲乳杆菌(Lactobacillus curvatus).     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.