一株双歧杆菌质粒聚合酶基因的PCR扩增和鉴定

目的PCR扩增人双歧杆菌天然质粒的聚合酶基因。方法用改良型MRS双歧杆菌选择培养基,从人新鲜粪便分离长双歧杆菌,PCR扩增长双歧杆菌质粒聚合酶(Bifidobacterium plasmid polymerase,BPP)基因,对BPP基因检测阳性的PCR产物通过序列分析,进行鉴定。结果人长双歧杆菌天然质粒的聚合酶基因PCR扩增后,经1.0%琼脂糖凝胶电泳,测得BPP基因的相对分子质量约为1.9 kb。通过BLAST序列比对分析与GenBank中相应基因同源性为96%。结论成功克隆了1株双歧杆菌天然质粒的聚合酶基因,为构建与双歧杆菌宿主质粒相适应的载体奠定了基础。     

双歧杆菌质粒的检测及其对抗生素敏感性

本文应用ＬｅＢＬａｎｃ法提取６种２０株双歧杆菌菌株的质粒,发现共有４个种的６株菌株存在着１～３个质粒,多数质粒的分子量小于６．０ｋｂ。存在质粒的双歧杆菌包括分离自人的短双歧杆菌、青春双歧杆菌、两歧双岐杆菌和婴儿双歧杆菌。用固体培养基滤纸片抑菌圈法测定双歧杆菌对１５种常用抗生素的敏感性,结果表明,测试的２０株双歧杆菌菌株对所检测的１５种抗生素的敏感性无规律可循,而且质粒消除实验表明质粒的存在与所检测的抗生素抗性无相关性。     

1株新分离的人两歧双歧杆菌耐药性研究

目的研究新分离的人两歧双歧杆菌对11种抗生素的耐药性。方法采用琼脂扩散纸片法测定人两歧双歧杆菌对11种抗生索的敏感性,通过在双歧杆菌培养基中添加不同浓度的抗生素来确定MIC值。结果该菌株对β-内酰胺类、大环内脂类和利福平非常敏感．对氨基糖苷类、黄胺类表现出较强抗性。结论该菌株对11种抗生索的药物敏感性与国内外其他文献所报道的结果一致。但该菌株带有一22kb大小的天然质粒。需进一步研究质粒与其耐药性的关系。     

猪源双歧杆菌的分离与鉴定

运用双歧杆菌选择性培养基,从１～４周龄乳猪的粪便中共分离纯化到５２个菌株。通过染色镜检、生化反应、代谢产物分析、抗生素敏感性试验研究表明这些菌株均与双歧杆菌属特征相符。根据糖发酵试验初步鉴定结果,其中４５个菌株为小猪双歧杆菌,５株为猪双歧杆菌,２株为嗜热双歧杆菌。部分菌株的急性毒性试验表明受试菌株对小白鼠无任何毒性副反应。     

反相高效液相色谱法测定色盐杆菌新种ST307的DNA G+C mol%含量

目的:使用反相高效液相色谱法测定色盐杆菌新种ST307的DNA G+C mol%含量.方法:以Escherichia coli DH5α为标准菌株,采用90%重蒸水10%甲醇为流动相,检测波长260nm,流速1ml·min-1,在Venusil MP C18柱上对四种碱基进行分离.结果:DNA碱基分离效果好,以外标法计算得到标准菌株DH5α的DNA G+C mol%含量为50.3%,待测菌株ST307的DNA G+C mol%含量为60.5%.结论:采用反向高效液相色谱法测定色盐杆菌的DNA G+C mol%含量准确可靠.     

人源双歧杆菌的分离纯化及鉴定

利用改良的乳酸细菌（MRS）培养基从健康人群的粪便中进行双歧杆菌的分离筛选。结果表明：在添加有5-溴-4-氯-3-吲哚-β-D-半乳糖苷（X-Gal）的MRS培养基上双歧杆菌的菌落呈典型的乳白色并带有蓝色,其他菌株的菌落呈白色或深蓝色。实验共分离出10株疑似菌株,经生理生化试验和Biolog自动微生物分析系统鉴定,结果发现其中4株为双歧杆菌,这4株菌中有3株为两歧双歧杆菌,1株为长双歧杆菌。和传统MBS培养基相比较,改良MRS培养基能显著提高筛选效率。     

婴幼儿粪便中双歧杆菌的分离及其菌株特性研究

为筛选具有潜在益生功能的双歧杆菌,本研究采用改良MRS培养基,从婴幼儿粪便中分离到5株菌株:AR499,AR667,AR668,AR669和AR610。通过16S r DNA测序分别鉴定为两歧双歧杆菌,短双歧杆菌,动物双歧杆菌,长双歧杆菌和假小链双歧杆菌。对其耐酸耐胆盐能力和黏附性能进行测试,结果表明,菌株AR499耐酸性较好,菌株AR610有较强的耐胆盐能力,菌株AR499的自动聚集能力和细胞表面疏水性都较高。实验表明,菌株AR499可作为一株耐受性和黏附性能较好的益生菌进一步深入研究,以期应用于优良双歧杆菌产品的开发。     

鸡源双歧杆菌定向选育和发酵参数研究

目的从肉仔鸡肠道中筛选出耐酸、耐胆盐和耐消化酶的优良双歧杆菌,研究其生长特性,并优化其发酵参数,为转化生产力提供理论依据。方法通过无菌采样并分离得到多株双歧杆菌,对分离获得的双歧杆菌进行形态学、生化特性研究,然后采用牛津杯法,测定90株双歧杆菌对大肠埃希菌和沙门菌的抑制作用,采用改良MRS培养基,模拟鸡胃肠道逆环境,对其耐消化道特性进行研究,筛选出优良双歧杆菌,再进行生长特性研究及发酵参数优化。结果从肉仔鸡肠道分离出90株双歧杆菌,初步挑选出23株作为候选菌株,抑菌试验测得双歧杆菌B1、B2和B3具有良好的抑菌效果,然后经过耐受消化道逆环境试验,发现B2菌株的耐受能力最好,初步鉴定双歧杆菌B2为小鸡双歧杆菌,并将其定名为Bifidobacterium pullorum B2,对其生长特性的研究发现经18 h发酵细菌总数可以从8.3×105CFU/mL升高到1.3×109CFU/mL,运用优化的发酵培养基进行中试试验,发酵后的活菌数可达1.41×1010CFU/mL。结论本实验从肉仔鸡肠道中分离筛选并初步鉴定了Bifidobacterium pullorum B2,优化了制备Bifidobacterium pullorum B2发酵液的发酵条件,降低了生产成本。     

一株油藏嗜热厌氧杆菌的分离、鉴定及代谢产物特征

[目的]了解油藏环境中细菌的生理生化特性及代谢产物.[方法]采用Hungate厌氧操作技术从胜利油田罗801区块油层采出水中分离到一株厌氧杆菌SC-2.采用生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株的系统发育地位,用气相色谱分析其代谢产物.[结果]菌株SC-2为严格厌氧的革兰氏阴性杆菌,菌体大小为0.38 um×1.7um-3.9um,单生、成对或成串生长,产端生芽孢.温度生长范围40℃-75℃(最适温度70℃);pH范围5.5-9.5(最适pH 6.5);NaCl浓度范围0%～5%(最适NaCl浓度0%).能够利用葡萄糖、麦芽糖、甘露糖、木糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、丙酸、H2、CO2及少量的乳酸.菌株SC-2的(G C)mol%含量为30.8%,与Thermoanaerobacter mathranii subsp.mathranii的16S rDNA序列相似性为99.85%.菌株利用葡萄糖产乙酸、乙醇的最佳初始pH为8.0;酵母粉能刺激生长并显著提高发酵葡萄糖的产酸、产醇率;培养基中添加4%(V/V)的乙醇能明显抑制菌体生长.[结论]菌株SC-2是从特殊生境(油层采出水)中分离到的一株嗜热、耐盐的厌氧菌,其发酵葡萄糖产生的代谢产物有利于改善油藏中的微环境.菌株SC-2与T.mathranii subsp.mathranii 11426T的最适pH和最大耐受NaCl浓度有所不同,且二者的(G C)mol%含量差异较大.     

长白山温泉无氧芽孢杆菌的分离鉴定

[目的]对长白山温泉中嗜热微生物进行分离鉴定,并了解其生理生化特性.[方法]采用橄榄油富集培养基,稀释平板涂布法对长白山温泉样品进行分离得到一株嗜热菌CBS-5;在电子和光学显微镜下观察菌体形态和芽孢;应用生理生化试验、16S rDNA序列分析以及(G C)mol%含量等方法对菌株特性进行鉴定.[结果]菌株CBS-5为革兰氏阳性菌,无鞭毛,产端生芽孢,最适生长温度为65℃,最适pH7.7左右,能以蔗糖、麦芽糖和乳糖等作为唯一碳源生长,具有酯酶和过氧化氢酶活性,对卡那霉素、红霉素和硫酸新霉素等抗生素均无抗性.Tm法测定该菌的(G C)mol%含量为41.9%.脂肪酸成分分析表明在CBS-5中iso-15:0的含量最高,为24.20%,与无氧芽孢杆菌属成员一致.以该菌的16S rDNA序列为基础构建了系统发育树;16S rDNA序列同源性比对表明该菌与无氧芽孢杆菌属各种之间的同源性在95.1%-98.5%之间.[结论]菌株CBS-5(=JCM 15484)是一株嗜热无氧芽孢杆菌,具有产酶活性,对于研究和开发化工、食品和环境保护方面的工业用酶具有重要价值.     

The effects of Bifidobacterium bifidum OFR9, a strain resistant to antituberculosis and antileprosy agents,on fecal flora in mice

Since Bifidobacterium bifidum, one of the strains of medical preparations used for human intestinal disorders, is sensitive to rifampicin (RFP) and fluoroquinolones, its therapeutic effect cannot be guaranteed when it is administered concomitantly with these antibiotics. To develop new strains of B. bifidum that are resistant to these drugs, B. bifidum RFR61, which is highly resistant to RFP, was selected by the N-methyl-N'-nitrosoguanidine (MNNG) mutation method. Then, B. bifidum OFR9 was selected in vitro from B. bifidum RFR61 by serial passage to increasing concentrations of ofloxacin (OFLX) on a solid medium. The minimal inhibition concentrations (MIC) of RFP and fluoroquinolones for B. bifidum OFR9 were >256 mg/ml and 16-256 &mgr;g/ml, respectively. We investigated the effects of B. bifidum OFR9 on the fecal bacterial flora of mice administered with both antibiotics and B. bifidum OFR9. The results showed that the concurrent use of B. bifidum OFR9 and antibiotics prevented the decrease of bifidobacteria, and quickly restored the flora to normal as compared with the use of antibiotic or parent strain therapy alone. The survival of Shigella organisms in mouse feces rapidly decreased, and were removed within two days as a result of the oral administration of B. bifidum OFR9.     

Rhodanobacter fulvus sp. nov., a beta-galactosidase-producing gammaproteobacterium

A taxonomic study was carried out on a bacterial strain designated as Jip2T isolated from a soil sample mixed with rotten rice straw. It was a Gram-negative, aerobic, motile, and rod-shaped bacterium. It grew well on nutrient agar medium and utilized a fairly narrow spectrum of carbon source. The G+C content of the genomic DNA was 65.3 mol%. The major ubiquinone was Q-8. The major fatty acids were branched fatty acids, especially large amounts of iso C15:0 and iso C17:1 w9c were detected in the cells grown on TSA agar for 24 h. Comparative 16S rDNA study showed a clear affiliation of this bacterium to the genus Rhodanobacter. The 16S rDNA sequence of strain Jip2T showed 96.4% sequence similarity to that of Rhodanobacter lindaniclasticus RP5575T. On the basis of phenotypic characteristics and 16S rDNA sequence analysis, strain Jip2T is clearly distinct from Rhodanobacter lindaniclasticus. We propose the name Rhodanobacter fulvus sp. nov. for strain Jip2T (=IAM 15025T=KCTC 12098T).     

深海放线菌08A4的鉴定及其抗真菌活性产物研究

从南海深海分离得到1株放线菌08A4,其发酵产物具有抗植物病原真菌活性,分离纯化得到3个化合物,通过1H-NMR初步鉴定为抗霉素类物质。结合形态学鉴定方法与16S rDNA序列分析方法,鉴定该菌株为微白黄链霉菌(Streptomyces albidoflavus)。     

Complete genome sequence of Bifidobacterium bifidum S17

Here, we report on the first completely annotated genome sequence of a Bifidobacterium bifidum strain. B. bifidum S17, isolated from feces of a breast-fed infant, was shown to strongly adhere to intestinal epithelial cells and has potent anti-inflammatory activity in vitro and in vivo. The genome sequence will provide new insights into the biology of this potential probiotic organism and allow for the characterization of the molecular mechanisms underlying its beneficial properties.     

两歧双歧杆菌86321生长特性的研究

目的研究两歧双歧杆菌86321的生长特性,为该菌生理功能研究和高效发酵剂的研制提供理论依据。方法通过生长曲线、产酸量、最适厌氧方式、最适pH、最适培养温度及最适接种量等一系列实验,对两歧双歧杆菌86321进行生长特性的研究。结果两歧双歧杆菌86321在BL培养基中培养时间可缩短至16 h,最高活菌数的lg值达到9.5;其最适厌氧方式为自然厌氧法或密封法,装液量视实际情况而定;在pH7.08.0生长良好,最适初始pH为8.0;在3742℃生长良好,最适温度为37℃;综合总菌量和生产成本,确定最适接种量为7%（v/v）。结论用BL培养基可以大大提高两歧双歧杆菌86321的产量。细菌产量的高低和发酵速度的快慢与菌种活力、厌氧方式、培养温度及pH等因素密切相关。     

Novel Bifidobacterium promoters selected through microarray analysis lead to constitutive high-level gene expression

For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.     

Complete Genome Sequence of the Probiotic Bacterium Bifidobacterium bifidum Strain BGN4

Bifidobacterium bifidum, a common endosymbiotic inhabitant of the human gut, is considered a prominent probiotic microorganism that may promote health. We completely decrypted the 2.2-Mb genome sequence of B. bifidum BGN4, a strain that had been isolated from the fecal sample of a healthy breast-fed infant, and annotated 1,835 coding sequences.     

西瓜枯萎病高效拮抗菌XJUL-12的筛选与鉴定

目的:分离获得西瓜枯萎病高效拮抗菌,为研究西瓜枯萎病高效拮抗菌的拮抗机制奠定基础。方法:用碾碎法从新疆有毒植物麻(Urtica cannabina L.)、亚洲薄荷(Mentha asiatica Boris.)、阿尔泰藜芦(Veratrum lobelianum Bernh.)中分离内生菌,并用琼脂扩散法筛选出对西瓜枯萎病具有较强抗性的内生菌XJUL-12,通过PCR方法扩增XJUL-12的16S rDNA,并与GeneBank中已鉴定菌的16S rDNA序列对比,用N-J方法构建XJUL-12进化树,用Bootstraping法对其评估,同时结合其形态特征、生理生化检测、G C mol%含量对XJUL-12进行鉴定。结果:XJUL-12的16S rDNA序列与Bacillus subtilisstrain CGMCC1869同源性为99%,G C mol%含量为46.72mol%。结论:筛选获得的内生菌XJUL-12对西瓜枯萎病具有较强抗性,并将XJUL-12鉴定为枯草芽孢杆菌。     

蒙古族儿童源益生特性双歧杆菌的筛选及鉴定

【目的】双歧杆菌在人和动物胃肠道中发挥着重要的生理作用,然而双歧杆菌能否耐受胃酸、肠液及胆汁酸是影响活菌制剂益生效果的关键因素,本研究旨在从蒙古族儿童粪便中分离筛选出具有良好益生特性的双岐杆菌。【方法】本文采用双岐杆菌选择性培养基对样品进行分离纯化,并对菌株进行生理生化鉴定,以耐受人工胃肠液、耐受牛胆盐为手段对各菌株的益生特性进行评价,并且对B. animalisV9进行了16S rDNA分子生物学鉴定。【结果】本文从12位健康蒙古族儿童粪便中分离得到11株双歧杆菌,经传统生理生化试验鉴定为5株B. adolescentis：A1、H3、G4、A8、V10;3株B. longum：C6、C7、D11;1株B. pseudocatenlatum：B2;1株B. bifidum：G5;1株B. animalis：V9。B. animalis V9具有较强的耐酸性,在pH2.0的人工胃液中厌氧培养3h后存活率为92.4%,而其它10株双歧杆菌在此条件下的存活率均小于31.25%;B. animalis V9在pH2.0的人工胃液中厌氧培养3h后接入pH8.0的人工肠液中消化8h,存活率为99.7%,并且可以耐受0.3%的牛胆盐。进一步对V9菌株进行16S rDNA分子生物学鉴定,发现与Bifidobacterium animalis subsp. lactic BB12的同源性为99%。【结论】本研究结果显示B. animalis V9来源安全,并且具有良好耐酸、耐胆盐益生特性,有望在乳制品及保健类产品中得到广泛的应用。