适用于分离人肠道中双歧杆菌的选择性培养基

【目的】比较并评价5种双歧杆菌选择性培养基对人源双歧杆菌的分离效果,试图筛选出一种适用于人肠道中双歧杆菌分离培养的选择性培养基。【方法】采集6份健康人粪便样品稀释涂布于5种选择性培养基上,厌氧培养后计数菌落并挑选单菌落进行鉴定。同时提取样品中细菌宏基因组DNA,应用变性梯度凝胶电泳技术(Denaturing Gel Gradient Electrophoresis,DGGE)和荧光定量PCR技术(Quantitative Polymerase Chain Reaction,q-PCR)揭示样品中双歧杆菌种类和数量,并以此为依据,客观评价上述5种选择性培养基的分离效果。【结果】BSM培养基和BLM培养基上双歧杆菌的计数结果与q-PCR的定量结果最为接近,并显著高于其它3种培养基。BLM培养基上分离到双歧杆菌的种类与DGGE图谱多样性分析的结果最为接近。【结论】BLM培养基是一种适用于人肠道中双歧杆菌分离培养的选择性培养基。     

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。     

水貂粪便中双歧杆菌的分离与鉴定

目的运用TPY培养基,从健康水貂的粪便中分离培养、筛选出2个肠道菌株。方法细菌培养、菌落形态观察、染色镜检、分离纯化、生化试验和药敏试验。结果分离培养出的2株菌株为双歧杆菌,其中1株为长双歧杆菌,另1株为青春双歧杆菌;双歧杆菌对氯霉素极其敏感,对阿米卡星耐药。结论本实验为毛皮特种经济动物微生态制剂的研究工作奠定了基础。     

一株携带质粒的人两歧双歧杆菌的分离与鉴定

目的:分离携带天然质粒的人双歧杆菌.方法:用自制的改良型Blb双歧杆菌选择培养基,从人新鲜粪便分离双歧杆菌,对初步质粒检测阳性的单菌落通过糖发酵试验、(G C)mol%测定和16S rDNA序列分析,进行菌株鉴定.结果:筛选到一株携带天然质粒的人双歧杆菌,编号B200304,在1.0%琼脂糖凝胶上,测得质粒的相对分子质量约为22 kb.通过对该菌株的形态学观察和糖发酵试验等生理生化特征研究,证明该菌株为两歧双歧杆菌(Bifidobacterium bifidum);HPLC法测得其(G C)mol%为55.6,16SrDNA序列分析进一步证实该菌株为两歧双歧杆菌.结论:分离得到一株携带天然质粒的人两歧双歧杆菌新菌株.     

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。     

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。     

培养基及培养工艺对双歧杆菌产量的影响

目的：对双歧杆菌生产培养基进行筛选,提高双歧杆菌的产量。方法：使用保蒲培养基,采用发酵罐培养工艺。结果：使用保蒲培养基较西红柿原汁培养基可使双歧杆菌的产量提高3.06-5.36倍。用保蒲培养基采用发酵罐培养工艺较立瓶静止培养工艺双歧杆菌的产量可提高4.91-54.8倍;发酵罐培养工艺较用西红柿原汁培养基、立瓶培养工艺产量提高17.33-154.29倍。结论：用保蒲培养基发酵罐培养可大大提高双歧杆菌的产量。     

对双歧杆菌DM9227株的实验研究——Ⅰ.双歧杆菌DM9227株的试管内生物拮抗作用的研究

本文对由健康婴儿分离的双歧杆菌DM9227株进行了试管内的生物拮抗试验。将双歧杆菌DM9227株分别与金黄色葡萄球菌、产毒性大肠杆菌及侵袭性大肠杆菌以一定的比例等量混合接种于PYG液体培养基中进行厌氧培养。试验证明双歧杆菌DM9227株能明显抑制上述3种细菌的生长繁殖,显示出较强的生物拮抗作用。拮抗机制可能与双歧杆菌DM9227株能产生一定量的醋酸和乳酸,降低培养基的pH,从而抑制该3种菌的生长有关。     

保健食品双歧杆菌和乳酸菌计数方法的优化

目的通过稀释液和培养基的选择对保健食品双歧杆菌和乳酸菌计数方法进行优化。方法对2个品种6个批次的益生菌类保健食品,分别采用2种稀释液和3种培养基进行双歧杆菌和乳酸菌计数,并比较结果。结果选择CYS缓冲液为最优稀释液,M-TOS培养基为双歧杆菌计数的最优培养基,BBL培养基为乳酸菌总数计数的最优培养基。结论优化方法所得菌数均明显优于国标方法,可以用于保健食品的双歧杆菌和乳酸菌计数。     

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。     

Stationary-phase acid and heat treatments for improvement of the viability of probiotic lactobacilli and bifidobacteria

AIMS: To investigate whether sublethal treatments of stationary-phase probiotic cultures enhance their survival during lethal treatments and to adapt these treatments to the fermenter-scale production of probiotic cultures. METHODS AND RESULTS: Conditions for acid and heat pretreatments were screened for three Lactobacillus and two Bifidobacterium strains. Strains were sublethally treated both at laboratory scale and at fermenter scale in a strain-specific manner and exposed to a subsequent lethal treatment. At laboratory scale viability improvement was detected in each strain. However, improvement was more pronounced in the Lactobacillus than in the Bifidobacterium strains. At fermenter scale three strains were tested: for the two Lactobacillus strains a marked improvement in viability was obtained whereas for the Bifidobacterium strain the improvement was either minor or not detected. CONCLUSIONS: Development of treatments for viability enhancement of probiotic strains is feasible, but strain-specific optimization is necessary to obtain notable improvements. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain-specific treatments were developed for the viability enhancement of stationary-phase probiotic cells both at laboratory and fermenter scale. These results can be utilised in the production of probiotic cultures with improved viability.     

Influence of pH and impeller tip speed on the cultivation of<Emphasis Type="Italic">Bifidobacterium pseudocatenulatum</Emphasis> G4 in a milk-based medium

Biomass production ofBifidobacterium pseudocatenulatum G4 in a milk-based medium was carried out in a 2- and 10-L stirred tank fermenters. The effects of impeller tip speed (0.28, 0.56, and 0.83 m/s) and pH control (6.0, 6.5, and 7.0) on the biomass production were investigated. The growth performance in the 2-L fermenter was significantly improved when the impeller tip speed was held constant at 0.56 m/s and the pH was controlled at 6.5. These conditions yielded a maximum biomass of 1.687×10⁹ cfu/mL, a maximum specific growth rate of 0.504 h⁻¹, a biomass productivity of 9.240×10⁷ cfu/mL·h, and a biomass yield of 9.791×10¹⁰ cfu/g lactose. The consumption of milk lactose resulted in the accumulation of 7.353 g/L acetic acid and 6.515 g/L lactic acid, with an acetic:lactic ratio of 1.129. Scale-up of the fermentation process to a 10-L fermenter based on a constant impeller tip speed of 0.56 m/s yielded reproducible results with respect to biomass production and cell viability.     

应用实时荧光定量PCR方法定量检测双歧杆菌对Caco-2细胞的黏附

【目的】采用实时荧光定量PCR的方法定量分析黏附于Caco-2细胞的双歧杆菌,并建立一种快速有效分离黏附于细胞的细菌的方法。【方法】采用Triton X-100溶液处理黏附于Caco-2细胞上的菌体,确定获得最佳分离效果的处理时间;建立实时荧光定量PCR定量检测双歧杆菌的方法,获得标准曲线,进行特异性、灵敏度、重复性评价;应用建立的方法分析11株双歧杆菌对Caco-2细胞的黏附能力。【结果】Triton X-100处理黏附于Caco-2细胞的双歧杆菌的最佳作用时间为10 min。实时荧光定量PCR定量检测双歧杆菌的方法重复性好、特异性强、灵敏度高;起始模板浓度范围在104?108 CFU/mL之间具有良好的线形关系,相关系数>99%,在该浓度范围线性方程为：y=?3.345 2x+37.637 0。应用建立的方法定量分析双歧杆菌的黏附能力,与直接镜检法相比差异不显著(P>0.05),检测时间由48 h缩短至4 h。【结论】Triton X-100分离处理结合实时荧光定量PCR方法是一种快速、有效的检测双歧杆菌对Caco-2细胞黏附能力的方法。     

Quantitative PCR with 16S rRNA-gene-targeted species-specific primers for analysis of human intestinal bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10(6) cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

克鲁维酵母Y-85合成菊粉酶最适条件的研究

采用响应面方法对克鲁维酵母Ｙ－８５产菊粉酶培养基成份进行了优选,和正交试验相比,该法选出的最适培养基的酶发酵水平提高２８％。用１５Ｌ自控发酵罐进行产酶条件控制试验,并在１０００Ｌ罐上进行了５批次酶发酵中试,平均菊粉酶活性达６８．９ｕ／ｍｌ。     

Selective medium for isolation and enumeration of Bifidobacterium spp

A new method was developed for the isolation and enumeration of Bifidobacterium spp. from natural aquatic environments. The method was based on the utilization of a new medium, Bifidobacterium iodoacetate medium 25, and resuscitation techniques were used to isolate injured bifidobacteria. The new medium was tested with a nonselective reference medium on sewage and sewage-polluted surface waters. Relatively little colonial growth of any other bacterial genera occurred; when such colonies did grow, Bifidobacterium could be easily differentiated by its colonial morphology or, after Gram staining, by its typical bifidobacterial morphology.     

用生料制备功能性双歧醋生产工艺的实验室研究

目的将双歧杆菌、醋酸菌、酵母菌和粉碎的制醋原料及麸曲共同发酵,通过生料制醋的方法来制备功能性双歧醋。方法将粉碎的玉米与麸曲、酵母液、麸皮和水搅拌均匀,使其经过液态糖化和酒精发酵后,接入醋酸菌和双歧杆菌(二者比例为1∶1),同时加入辅料,进行醋酸发酵,当检测到醋酸酸度为5.0%~7.5%时,加入食盐终止发酵,经过过滤,除菌澄清得到功能性双歧醋。结果双歧醋的最终醋酸度为3.2%,外观红棕色,光泽度好,清澈透明,无沉淀和悬浮物。总菌数:醋酸菌为3.3×1011/m l,双歧杆菌为1.9×107/m l;活菌数:醋酸菌为1.7×1011/m l,双歧杆菌为6.8×106/m l;大肠菌群数3个/100 m l;致病菌:不得检出。结论双歧杆菌及其代谢物可以在双歧醋中存活,生料固态发酵制备双歧醋的方法可行。     

Selective medium for isolation and enumeration of Bifidobacterium spp.

A new method was developed for the isolation and enumeration of Bifidobacterium spp. from natural aquatic environments. The method was based on the utilization of a new medium, Bifidobacterium iodoacetate medium 25, and resuscitation techniques were used to isolate injured bifidobacteria. The new medium was tested with a nonselective reference medium on sewage and sewage-polluted surface waters. Relatively little colonial growth of any other bacterial genera occurred; when such colonies did grow, Bifidobacterium could be easily differentiated by its colonial morphology or, after Gram staining, by its typical bifidobacterial morphology.     

双歧杆菌属天然质粒系统分类

【背景】以往双歧杆菌质粒分类学研究较少,双歧杆菌属质粒系统分类方法缺失。【目的】建立双歧杆菌属天然质粒系统分类和鉴定方法,促进质粒在双歧杆菌生物学研究中的理解和应用。【方法】利用质粒复制起始蛋白进化树和基因组共线性分析方法,对目前所有已测序的双歧杆菌属天然质粒进行系统分类研究。【结果】双歧杆菌所有已知天然质粒可以划分为6个不同类型的质粒家族和3个独特的复合质粒,家族Ⅲ和家族Ⅵ质粒可进一步划分为不同的亚型类群。家族Ⅲ质粒是双歧杆菌属天然质粒的主要类型和优势家族。家族Ⅵ质粒成员亚型分类最丰富。在质粒家族水平,2种方法的分类结果完全一致。【结论】本文揭示了所有分析质粒之间的系统分类关系,建立了双歧杆菌属天然质粒系统的分类标准、方法和体系,可为今后双歧杆菌天然质粒分类和鉴定提供重要的理论参考和分类依据。     

利用球孢链霉菌发酵液消化双歧杆菌细胞壁提取质粒的研究

目的寻找一种经济适用的提取双歧杆菌质粒的方法。方法利用球孢链霉菌发酵产生的变溶菌素来消化双歧杆菌细胞壁,提取质粒,并与商业化溶菌酶法比较。结果球孢链霉菌发酵液与溶菌酶均成功提取到质粒。结论利用球孢链霉菌发酵液提取质粒经济简便。