内蒙古地区传统制作奶豆腐和乌日莫中乳酸菌多样性及分布特征

【背景】传统制作奶豆腐和酸性奶油(乌日莫)是内蒙古农牧地区最喜爱的食品,蕴含着十分丰富的乳酸菌资源,亟待开发利用。【目的】通过解析内蒙古农牧地区传统自制奶豆腐和乌日莫样品中乳酸菌多样性及分布特征,为优良菌株选育与利用提供资源和理论基础。【方法】采用稀释涂布法分离纯化乳酸菌,测定菌株16S rRNA基因序列鉴定种属关系,阐明乳酸菌系统发育、遗传分化及菌群结构。【结果】传统自制样品中共分离得到乳酸菌81株,主要归属于乳酸片球菌(Pediococcus acidilactici)、戊糖片球菌(Pediococcus pentosaceus)、短乳杆菌(Lactobacillus brevis)、瑞士乳杆菌(Lactobacillus helveticus)、副干酪乳杆菌(Lactobacillus paracasei)、食二酸乳杆菌(Lactobacillus diolivorans)、奥塔基乳杆菌(Lactobacillus otakiensis)、植物乳杆菌(Lactobacillus plantarum)、开菲尔乳杆菌(Lactobacillus kefir)、乳酸乳球菌(Lactoc...     

具高抗氧化能力乳酸菌菌株的筛选与鉴定

目的筛选抗氧化能力强的乳酸菌菌株并进行菌种鉴定。方法通过清除DPPH自由基、清除超氧阴离子自由基、还原能力等实验,筛选出高抗氧化能力的乳酸菌菌株。运用16SrDNA序列分析方法,对抗氧化能力强的菌株进行鉴定。结果本研究筛选得到的抗氧化能力较强的菌株有长双歧杆菌NQ1501、La8、La5和保加利亚乳杆菌NQ2508,经16SrDNA鉴定和系统进化分析,La5鉴定为发酵乳杆菌(Lactobacillus fermentum),La8鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。结论本研究筛选出四株具有高抗氧化活性的乳酸菌,分别为长双歧杆菌NQ1501、鼠李糖乳杆菌La8、发酵乳杆菌La5和保加利亚乳杆菌NQ2508。     

作为宿主系统的几株乳酸菌的表型特征

目的：研究乳酸菌载体－宿主系统。方法：采用涂片染色和形态特征观察,用鉴别生化实验如过氧化氢酶实验,碳水化合物发酵产酸实验,精氨酸水解实验及抗生素抗性实验等对含有pMG36e质粒的乳酸菌MG1363,乳球菌IL1403和乳杆菌ATCC4356进行研究鉴定。结果：乳酸乳球菌乳脂亚种MG1363,乳酸乳球菌乳酸亚种IL1403含有质粒pMG36e的MG1363致及嗜酸乳杆菌ATCC4356其表型特征分别与伯杰氏手册中相应细菌特征一致,质粒pMG36e含有红霉素抗性基因,结论：此乳酸菌宿主一载体系统可用载体来源的红霉素抗性进行筛选,用于外源基因在乳酸菌中克隆和表达的研究。     

基于冰核蛋白的乳酸菌表面展示系统的构建

[目的]验证来源于丁香假单胞菌的冰核蛋白在乳酸乳球菌表面展示外源蛋白的可能性.[方法]以绿色荧光蛋白(Green Fluorescence Protein,GFP)基因gfp为报告基因,以冰核蛋白基因的N末端和NC端作为展示单元,构建乳酸菌表面展示载体pHZ101和pHZ102,并转化大肠杆菌(Escherichia coli JM109和乳酸乳球菌(Lactococcus lactis)MG1363.[结果]荧光显微镜观察显示重组大肠杆菌和乳酸乳球菌均能检测到绿色荧光.Western blot结果表明GFP蛋白在重组大肠杆菌和乳酸乳球菌中均得到表达,并且INPN-GFP蛋白多数滞留于乳酸乳球菌细胞质内,而INPNC-GFP蛋白则大部分定位于乳酸乳球菌的细胞膜上.[结论]以上结果表明丁香假单胞菌的冰核蛋白能引导外源蛋白定位于乳酸乳球菌的细胞膜上,为乳酸菌表面展示系统的构建提供了新的方向.     

葡萄藤叶青贮中乳酸菌的分离与鉴定

为了筛选出性状优良的乳酸菌菌株,本试验以实验室纯培养方式从葡萄藤叶自然发酵的青贮中分离鉴定乳酸菌。经过菌落形态、细胞形态、生理生化鉴定和16S r DNA基因序列以及系统发育树的分析,从葡萄藤叶青贮中分离出5株乳酸菌,其中菌株P-1. 2和P-2. 2为戊糖乳杆菌(Lactobacillus pentosus),菌株P-1. 7为戊糖片球菌(Pediococcus pentosaceus),菌株P-2. 1为粪肠球菌(Enterococcus faecium),菌株P-2. 3为短乳杆菌(Lactobacillus brevis)。测定产酸速率和生长速率,发现菌株P-2. 1的产酸能力和生长性能最好。     

乳酸菌表面蛋白对免疫细胞的诱导增殖及粘附抑制效应

目的研究表面蛋白在乳酸菌体外粘附和激活免疫细胞中的作用。方法应用5mol/L氯化锂结合盐酸胍提取植物乳杆菌LpYZU09、干酪乳杆菌LcYZU02、鼠李糖乳杆菌LrGG、发酵乳杆菌LfYZU15及戊糖片球菌PpYZU32的表面蛋白,并分析提取物对乳酸菌粘附鼠肠上皮细胞、巨噬细胞和脾细胞的抑制作用及诱导增殖效应。结果表面蛋白对3种细胞粘附菌体均具有显著抑制效应,抑制作用具有细胞和菌株差异性,其中菌株LrGG表面蛋白对巨噬细胞粘附5种菌体普遍显示了较强的抑制作用,抑制率为38.7%~76.0%。不同菌株表面蛋白对肠上皮细胞的诱导增殖指数为0.05~0.35,对巨噬细胞为0.05~0.42,对脾细胞为0.02~0.40,诱导效应具有菌株和剂量依赖性,菌株LrGG的诱导增殖指数显著高于其他四种。结论乳酸菌表面的蛋白类因子在粘附和激活免疫细胞中发挥了重要作用。     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

基于16SrDNA序列片段探讨织纹螺的系统发生关系

通过对细肋织纹螺、红带织纹螺、半褶织纹螺、拟半褶织纹螺、纵肋织纹螺和秀丽织纹螺等6种织纹螺属Nassarius动物线粒体16SrDNA的部分序列以及GeneBank中西格织纹螺和秀丽织纹螺2条同源序列的分析,使用MAGE软件构建了系统发生树(NJ树和ME树),探讨了这7种织纹螺种间以及亚属间的系统发生关系。结果表明,本研究所涉及的织纹螺16SrDNA序列能较好的反映织纹螺种间和亚属间的亲缘关系,16SrDNA可用于织纹螺近缘种类的系统学研究;系统树均支持将拟半褶织纹螺作为一独立种的观点;Reticunassa亚属的种和Varicinassa亚属的种亲缘关系相对前者与Zeuxis亚属的种近。     

ICR小鼠肠道中乳酸菌组成的克隆文库研究

目的对健康雄性ICR小鼠肠道内乳酸菌的组成结构进行研究。方法收集10只健康雄性ICR小鼠的新鲜粪便样品,提取粪便样品中微生物的总DNA,采用乳酸菌类群特异性引物（Lac1）和细菌通用性引物（1391r）的组合扩增16SrRNA基因并构建乳酸菌特异性克隆文库,研究小鼠肠道内各种乳酸菌的组成和比例。结果克隆文库的分析结果表明罗伊乳杆菌（Lactobacillus reuteri）和约氏乳杆菌（Lactobacillus johnsonii）为ICR小鼠肠道内的优势种,其他还包括鼠乳杆菌（Lactobacillus murinus）、阴道乳杆菌（Lactobacillus vaginalis）、肠乳杆菌（Lacto-bacillus intestinalis）等乳酸菌以及一个潜在的乳酸菌新种。结论健康ICR小鼠肠道内乳酸菌的多样性较高;L.re-uteri种可能具有较高的菌株水平多样性。     

Molecular phylogenies of plants and Frankia support multiple origins of actinorhizal symbioses

Molecular phylogenetic trees were reconstructed from nucleotide sequences of nifH and 16S rDNA for Frankia and of rbcL for actinorhizal plants. Comparison of Frankia phylogenetic trees reconstructed using nifH and 16S rDNA sequences indicated that subgroupings of both trees correspond with each other in terms of plant origins of Frankia strains. The results suggested that 16S rDNAs can be utilized for coevolution analysis of actinorhizal symbioses. Frankia and plant phylogenetic trees reconstructed using 16S rDNA and rbcL sequences were compared. The comparison by tree matching and likelihood ratio tests indicated that although branching orders of both trees do not strictly correspond with each other, subgroupings of Frankia and their host plants correspond with each other in terms of symbiotic partnership. Estimated divergence times among Frankia and plant clades indicated that Frankia clades diverged more recently than plant clades. Taken together, actinorhizal symbioses originated more than three times after the four plant clades diverged.     

新疆地区盐湖的中度嗜盐菌16S rDNA全序列及DNA同源性分析

通过数值分类和16S rDNA PCR-RFLP分析,对分离自新疆地区的中度嗜盐革兰氏阴性菌进行研究,发现了一个新类群。在此基础上,进行了中心株AI－3的16S rDNA全序列分析,并与中度嗜盐菌已知种和相关种进行比较,得到系统发育树状图。在此树状图中,大多数参比菌株聚在一起,其16S rDNA全序列的同源性在96％以上,而AI－3与参比菌株的16S rDNA全序列相比,其相似性低于75％。但是,AI－3与Alcanivorax borkumensis^[1]的16S rDNA全序列的相似性为96％,与Halobacillus litoralis的16S rDNA全序列的相似性为99％,三者构成一个独立的发育分支。这说明在系统发育上,AI－3与参比菌株属于不同的分支,是一个新的类群。在新类群内,菌株之间的DNA同源性大于70％,而中心株AI－3与标准菌株伸长盐单胞菌（Halomonas elongata)的DNA同源性为44％,表明新分离的菌株可能构成一个新种群。     

Phylogenetic analysis of enteric species of the family Enterobacteriaceae using the oriC-locus

Phylogenetic analysis of 38 enteric species belonging to the Enterobacteraceae family was carried out using the non-coding locus oriC, the chromosomal replication origin. The oriC loci were amplified with conserved oligonucleotides and the PCR fragments were sequenced directly. The results establish a phylogenetic tree for the classification of different species of the genera Escherichia, Shigella, Salmonella, Enterobacter, Citrobacter, Klebsiella, Raoultella, Kluyvera, Cedecea and Buttiauxella. Functional important protein-binding sites located in oriC are well conserved throughout the enteric group. More over, due to a high overall divergence value phylogenetic trees were robust and well supported by bootstrap analysis. In comparison with 16S rDNA analysis, the oriC sequences indicated a greater evolutionary divergence for bacteria. We propose that the oriC locus might be a suitable phylogenetic marker for the identification and classification of bacteria, in particular for closely related species.     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from a fermented locust bean product manufactured in West Africa

Twenty-five Bacillus strains capable of producing gamma-polyglutamic acid (PGA) were isolated from fermented locust bean products manufactured in the savanna area of Ghana. To clarify the phylogeny of these PGA-producing strains, phylogenetic analyses based on sequences of 16S rDNA, rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) genes were performed. A phylogenetic tree based on 16S rDNA indicated that ten isolates were clustered in the same group of Bacillus subtilis. Another ten isolates were located in the cluster of B. amyloliquefaciens, and the remaining isolates were identified as B. pumilus (three isolates) and B. licheniformis (two isolates), respectively. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. Thirty-four strains in 27 species belonging to the genus Bacillus and its neighbors were also investigated for PGA production. It was found that PGA was produced by B. amyloliquefaciens NBRC 14141 and NBRC 15535(T), B. atrophaeus NBRC 15539(T), B. licheniformis NBRC 12107, B. mojavensis NBRC 15718(T), B. pumilus NBRC 12094, B. subtilis NBRC 16449, and Lysinibacillus sphaericus NBRC 3525. Except for L. sphaericus, the above Bacillus species are very closely related in phylogeny, indicating that PGA-producing Bacillus strains constitute a cluster.     

Molecular characterization of dichloromethane-degrading Hyphomicrobium strains using 16S rDNA and DCM dehalogenase gene sequences

A phylogenetic analysis of 6 strains of dichloromethane (DCM) utilizing bacteria was performed. Based on the almost complete 16S rDNA sequence determination, all strains clustered together and showed high sequence similarity to Hyphomicrobium denitrificans, except for the strain MC8b, which is only moderately related to them and probably represents a distinct species. The 16S rDNA-based phylogenetic tree was compared to the one obtained from the DNA sequence data of the dcmA gene coding DCM dehalogenase, the key enzyme of DCM utilization. The topology of the two trees is in good agreement and may suggest an ancient origin of DCM dehalogenase, but also raises questions about the original role of the enzyme.     

西藏根瘤菌新表观群的DNA同源性及16S rDNA全序列分析

在前期数值分类工作的基础上,对7株与Rhizobium关系较密切的分离自西藏部分地区豆科植物Trigonellaspp.和Astragalusspp.的根瘤菌所形成的独立表观群,通过DNA同源性测定及16S rDNA全序列分析进行了分类地位的进一步确定。结果表明:该独立表观群菌株的(G C)mol%为59.5%~63.3%,群内菌株间DNA同源性在74.3%~92.3%之间,中心菌株XZ2-3与相关Rhizobium种之间的DNA同源性在0%~47.4%之间,是不同于Rhizobium内各种的新DNA同源群。另外,16S rDNA全序列分析结果也表明,中心菌株XZ2-3占居Rhizobium系统发育分支中的一个独立亚分支,其与临近R.leguminosarumUSDA2370T和R.etliCFN42T之间的序列相似性分别为96.55%和96.62%。根据国际系统细菌学委员会提出的细菌种属分类标准,该独立表观群构成了一个不同于Rhizobium内各种的新种群。该研究结果丰富了现有根瘤菌分类系统,将为国际上现有Rhizobium的14个种中再增添一个新的分类单元。     

Phylogeny of gamma-polyglutamic acid-producing Bacillus strains isolated from fermented soybean foods manufactured in Asian countries

Natto-like fermented soybean products are manufactured and consumed in many Asian countries. In this study, we isolated thirty-four Bacillus strains capable of producing gamma-polyglutamic acid (PGA) from natto in mountainous areas of South Asia and Southeast Asia and from soils in Japan. To elucidate the phylogeny of these PGA-producing strains, phylogenetic trees based on sequences of 16S rDNA, housekeeping genes of rpoB (RNA polymerase beta-subunit) and fus (elongation factor G) were constructed. A phylogenetic tree based on 16S rDNA sequences showed that twenty-one isolates were clustered in the same group of B. subtilis. The other thirteen isolates were located in the cluster of B. amyloliquefaciens. Phylogenetic trees based on the partial sequences of rpoB and fus genes were similar to the phylogeny based on 16S rDNA sequences. The results of the present study indicate that PGA-producing strains isolated from local natto in Asian countries and soil in Japan can be divided into two species, B. subtilis and B. amyloliquefaciens.     

产甾体皂甙华重楼内生菌的筛选与鉴定

从华重楼(Paris polyphyllavar.chinensisFranch)的地下块茎中分离筛选得到2株可能产生甾体皂甙的内生菌(SS01和SS02),薄层层析检测菌株SS01、SS02的发酵产物分别有3条和2条层析带与重楼总皂甙的层析带迁移率相当。形态和生理生化特征初步表明SS01和SS02分属于肠杆菌科(Enterobacteriaceae)和芽孢杆菌属(Bacillussp.)细菌。扩增、测序得到SS01和SS02的部分16S rDNA序列,GenBank接收号分别为AY842143和AY842144。用Blastn调出与菌株16SrDNA同源的序列,用Clustal W进行多重序列对比,用软件Phylip按Neighbor-Joining方法构建16SrDNA系统发育树。菌株SS01和SS02分别与Cedecea davisae DSM4568、Paenibacillus daejeonensis处于同一分支,相似性分别为98.9%和97.7%,将它们鉴定为Cedecea davisae SS01和Paenibacillus daejeonensis SS02。     

Phylogenetic congruence of armored scale insects (Hemiptera: Diaspididae) and their primary endosymbionts from the phylum Bacteroidetes

Insects in the sap-sucking hemipteran suborder Sternorrhyncha typically harbor maternally transmitted bacteria housed in a specialized organ, the bacteriome. In three of the four superfamilies of Sternorrhyncha (Aphidoidea, Aleyrodoidea, Psylloidea), the bacteriome-associated (primary) bacterial lineage is from the class Gammaproteobacteria (phylum Proteobacteria). The fourth superfamily, Coccoidea (scale insects), has a diverse array of bacterial endosymbionts whose affinities are largely unexplored. We have amplified fragments of two bacterial ribosomal genes from each of 68 species of armored scale insects (Diaspididae). In spite of initially using primers designed for Gammaproteobacteria, we consistently amplified sequences from a different bacterial phylum: Bacteroidetes. We use these sequences (16S and 23S, 2105 total base pairs), along with previously published sequences from the armored scale hosts (elongation factor 1alpha and 28S rDNA) to investigate phylogenetic congruence between the two clades. The Bayesian tree for the bacteria is roughly congruent with that of the hosts, with 67% of nodes identical. Partition homogeneity tests found no significant difference between the host and bacterial data sets. Of thirteen Shimodaira-Hasegawa tests, comparing the original Bayesian bacterial tree to bacterial trees with incongruent clades forced to match the host tree, 12 found no significant difference. A significant difference in topology was found only when the entire host tree was compared with the entire bacterial tree. For the bacterial data set, the treelengths of the most parsimonious host trees are only 1.8-2.4% longer than that of the most parsimonious bacterial trees. The high level of congruence between the topologies indicates that these Bacteroidetes are the primary endosymbionts of armored scale insects. To investigate the phylogenetic affinities of these endosymbionts, we aligned some of their 16S rDNA sequences with other known Bacteroidetes endosymbionts and with other similar sequences identified by BLAST searches. Although the endosymbionts of armored scales are only distantly related to the endosymbionts of the other sternorrhynchan insects, they are closely related to bacteria associated with eriococcid and margarodid scale insects, to cockroach and auchenorrynchan endosymbionts (Blattabacterium and Sulcia), and to male-killing endosymbionts of ladybird beetles. We propose the name "Candidatus Uzinura diaspidicola" for the primary endosymbionts of armored scale insects.