成人粪便中长双歧杆菌长亚种的分离鉴定及多位点序列分型分析

为了对成人粪便中分离的长双歧杆菌长亚种进行多位点序列分型分析,采用改良MRS培养基从健康成人粪便中分离的长双歧杆菌长亚种,通过生理生化试验结合16S rDNA和热应激蛋白60(heat-shock protein,hsp60)同源性分析鉴定分离株,利用同源性分析及多位点序列分型(multilocus sequence typing,MLST)技术进一步分析长双歧杆菌长亚种的基因多态性。从12个健康成人体内分离得到24株厌氧的细菌菌株,经形态学观察、生理生化试验、16S rDNA及hsp60同源性分析发现,其中14株分离株为长双歧杆菌长亚种(Bifidobacterium longum subsp. longum)。MLST结果表明:14株长双歧杆菌长亚种可分为10种基因型,且均与Bifidobacterium longum subsp. longum ATCC 15707为不同基因型。健康成人粪便中分离的长双歧杆菌长亚种具有较大的基因多态性。     

应用DGGE方法构建健康人粪便乳杆菌标准指纹图谱

从健康人的新鲜粪便样品中分离乳杆菌,鉴定属种后构建一种能够快速鉴定未知属种乳杆菌的标准指纹图谱.首先通过形态学观察及K2O2酶等生理生化试验对所分得菌株进行初步鉴定,然后利用16S rDNA序列同源性分析方法,对所分得菌株进行16S rDNA基因扩增与测序,测序结果与GenBank中该属内茵株的16S rDNA基因序列进行同源性分析,从而准确鉴定所分得菌株.最后,利用变性梯度凝胶电泳(DGGE)方法构建标准指纹图谱.结果表明,从健康人新鲜粪便中分离得到8株乳杆茵,经鉴定,其中4株为植物乳杆菌,1株为卷曲乳杆菌,3株为发酵乳杆菌.所得标准指纹图谱中试验菌株大体被分成4类,与16S rDNA基因序列同源性分析结果相吻合.通过传统方法可以从健康成人的新鲜粪便中分离得到乳杆菌,传统方法与分子生物学方法相结合可以将其准确鉴定到种的水平.变性梯度凝胶电泳(DGGE)方法可以在种的水平上作为快速鉴定乳杆菌的一种有效工具.     

新疆喀什地区维吾尔族母乳源双歧杆菌分离筛选及其益生特性分析

采用多重聚合酶链式反应指纹图谱结合16S rRNA基因测序技术对新疆喀什地区维吾尔族母乳分离的双歧杆菌进行鉴定和遗传差异分析,并检测常规生理生化和糖代谢表型特征,同时测试菌株对6 种常见病原菌和3 种母乳源条件致病菌的抑菌性能和对胃肠液的耐受性。结果显示,15 份母乳样品中共分离15 株双歧杆菌,测序结果将菌株归属于3 个种以及2 个亚种,包括8 株假小链双歧杆菌（Bifidobacterium pseudocatenulatum）、2 株短双歧杆菌（B. breve）、2 株长双歧杆菌长亚种（B. longum subsp. longum）和3 株长双歧杆菌婴儿亚种（B. longum subsp. infantis）。抑菌实验表明,15 株测试菌株中,隶属于B. pseudocatenulatum的4 株菌MY92、MY75-1、MY72、MY81的抑菌谱更广,抑菌能力更强;胃肠液耐受性实验表明菌株MY92无论在模拟胃液还是模拟肠液中存活率均最高,分别达到20.37%和0.302%。基于以上描述特性,MY92作为一株有效的益生菌株,具有潜在的利用价值,为后期进一步作为防止婴幼儿腹泻辅助制剂的开发提供参考。     

优良醋酸菌种AC2005的鉴定

将醋酸杆菌AC2005 16S rDNA片段转入载体pMD18-T,并转化大肠埃希氏杆菌(Escherichia coli).经测序和序列分析显示,醋酸杆菌AC2005 16S rDNA片段与巴斯德醋杆菌16S rDNA片段同源性均达95%.根据醋酸杆菌AC2005 16S rDNA的生理生化特征培养特征,形态特征结果将醋酸杆菌AC2005归属为巴斯德醋酸杆菌罗旺亚种(Acetobactor.pasteurianus subsp.lovaniensis).     

新疆维吾尔族肠道中高产胞外多糖双歧杆菌的筛选及其抗氧化活性

使用Wilkins-Chalgren厌氧菌琼脂和BSM培养基作为筛选平板,结合重复基因外回文序列-聚合酶链式反应和16S rRNA序列分析,对新疆维吾尔族婴儿及其母亲粪便中的双歧杆菌(Bifidobacterium)进行分离鉴定,并筛选出高产胞外多糖的双歧杆菌,测定其多糖的抗氧化活性以及菌株的耐受性和黏附性。结果显示,20份粪便样品中共分离出52株双歧杆菌,其中假小链双歧杆菌(B. pseudocatenulatum)14株,假长双歧杆菌(B. pseudolongum)8株,两歧双歧杆菌(B.bifidum)9株,短双歧杆菌(B.breve)7株,长双歧杆菌婴儿亚种(B.longumsubsp.infantis)5株,动物双歧杆菌乳亚种(B. animal subsp. lactis)6株以及长双歧杆菌(B. longum)3株。经过表型初筛和苯酚-硫酸法复筛,共筛选出7株高产胞外多糖的双歧杆菌,37℃发酵36 h后胞外多糖产量均可达400 mg/L以上。抗氧化活性实验结果表明7株双歧杆菌所产的胞外多糖对过氧化氢自由基、超氧阴离子自由基、羟自由基和1,1-二苯基-2-三硝基苯肼自由基均有一定的清除能力。此外,菌株BF66-16相较于其他几株双歧杆菌具有较强的胃肠液耐受性以及黏附性,因此来源于婴儿粪便的BF66-16可以作为潜在的抗氧化菌株应用于制药和食品工业中。     

四川牦牛奶和曲拉中九株乳酸菌的分子鉴定

为准确鉴定分离自我国四川鲜牦牛奶和曲拉中9株乳酸菌到种水平,作者运用16S rDNA序列分析、recA基因特异扩增和hsp60-RFLP等多种分子技术对分离自我国四川地区鲜牦牛奶和曲拉中的9株乳酸菌进行分类鉴定。结果证实,16S rDNA序列分析法可将9株乳酸菌初步归类为植物乳杆菌群(4株),肠膜明串珠菌(4株),瑞士乳杆菌(1株)。由于16S rDNA序列分析法不能区分植物乳杆菌和戊糖乳杆菌,为了进一步鉴定植物乳杆菌群中的4株菌,继续采用recA基因特异扩增和hsp60-RFLP技术对其细分,结果表明recA基因特异扩增和hsp60-RFLP的方法均能很好地把植物乳杆菌群中的4株菌鉴定到种水平,且均为植物乳杆菌。     

高黏附力双歧杆菌的筛选与鉴定

利用体外细胞培养法,从实验室现有的10株双歧杆菌中筛选具有较强粘附能力的菌株。采用革兰氏染色法和平板计数法,评价了这10株双歧杆菌对HT-29细胞的粘附性能,通过测定这10株双歧杆菌的16S rRNA基因序列(16S rDNA)进行分类学鉴定。结果显示,WBBI01,WBBI02,WBIN03,WBBI06具有极强的黏附力,其黏附指数分别达1.97×103,2.17×103,3.57×103,1.88×103。经16S rDNA测序鉴定WBBI01,WBIN03,WBBI06均与两歧双歧杆菌S17有极高的同源性,而WBBI02属于长双歧杆菌。结果表明,双歧杆菌黏附性能具有明显的种属特异性,不同种属双歧杆菌的粘附能力相差极大,以两歧双歧杆菌的黏附性能最强,婴儿长双歧杆菌为最弱。     

长双歧杆菌PCR快速定向筛选方法

传统筛种方法具有随机性、盲目性,而PCR筛菌,增加了目的性和效率.在基因序列数据库中查找益生双歧杆菌的16S rDNA共有序列,找出它们共有的特异性序列,设计了PCR引物.平板培养分离,初步筛选双歧杆菌菌落.根据细菌16S rDNA和23S rDNA两侧高度保守区域设计通用引物,提取菌落基因组DNA,扩增16S-23S rDNA间区序列,并送测序.通过序列同源性比对分析,鉴定、筛选到人体长双歧杆菌.     

长双歧杆菌婴儿亚种的快速区分

为建立一种有效、快速、低成本区分长双歧杆菌婴儿亚种的方法,对13株基因组测定的长双歧杆菌构建了基于同源基因的系统进化树,确认其中7株为长亚种,5株为婴儿亚种,1株为猪亚种。通过碳水化合物利用谱比较和特异性基因扩增的手段确认可有效用于快速区分长双歧杆菌婴儿亚种的方法。结果显示,同一亚种的菌株利用同一种碳水化合物的能力存在差异,表明无法根据碳水化合物的利用来区分长双歧杆菌的不同亚种;长双歧杆菌长亚种糖激酶基因和长双歧杆菌婴儿亚种唾液酸酶基因可有效用于长双歧杆菌婴儿亚种的快速区分,并用该方法对40株长双歧杆菌进行了亚种确认,其中5株为长双歧杆菌婴儿亚种。后随机选择了其中的4株菌进行基因组草图测序,经系统进化树构建,确认为长双歧杆菌婴儿亚种,并进一步证实特异性基因扩增可实现长双歧杆菌婴儿亚种的快速区分。     

双歧杆菌分子分型鉴定研究进展

综述了双歧杆菌属的分类学进展和16S rDNA、MLST、RAPD等分子生物学技术在双歧杆菌分型鉴定中的应用新进展,并分析了各类技术的原理、步骤和优缺点,展望了其发展趋势。     

婴儿源益生性双歧杆菌的筛选及肠道定殖性研究

本研究旨在从婴儿粪便中筛选出具有潜在益生特性的双歧杆菌,并探究其肠道定殖情况,为双歧杆菌的产品开发提供优良的菌株。采用MRS培养基对样品进行分离纯化,菌株经F6PPK检测及16S r DNA测序鉴定,之后进行模拟胃肠液、胆盐耐受性、对食源性致病菌(大肠杆菌、沙门氏菌、单增李斯特菌等)的抑制及对HT-29细胞的粘附能力测定,将筛选出的菌株进行动物实验,测定其肠道定殖能力。分离到的27株双歧杆菌,经分子生物学鉴定为7个不同的种:Bifidobacterium longum、Bifidobacterium breve、Bifidobacterium bifidum、Bifidobacterium pseudocatenulatum、Bifidobacterium infantis、Bifidobacterium animalis和Bifidobacterium adolescentis。体外实验表明,B.longum A9、B.breve A4、B.bifidum B6、B.longum C6、B.adolescentis F8和B.infantis H6等具有较强的潜在益生特性;动物实验表明,B.infantis H6和B.longum C6具有较强的肠道定殖能力。B.longum C6和B.infantis H6有望作为优良的益生性菌株,应用于双歧杆菌的产品开发。     

婴儿肠道菌群中乳酸菌的分离鉴定及其多样性分析

结合传统培养方法与基于PacBio Sequel平台的单分子实时(Single-molecule real-time,SMRT)三代测序技术,对中国婴儿肠道菌群中的乳酸菌进行分离鉴定并分析其多样性,同时探究抗生素治疗对婴儿肠道乳酸菌多样性的影响。采用稀释涂布法培养分离粪便样品菌落,运用生理生化试验及16S rDNA序列比对鉴定菌株,并分析不同菌株间的系统发育关系。同时提取整体粪便样品的基因组DNA进行16S rDNA全长SMRT测序,利用QIIME、Mothur等生物信息学分析软件进行细菌群落结构和多样性分析。结果表明:中国婴儿肠道菌群中乳酸菌丰度及多样性普遍较低,传统培养方法分离出7种共32株乳酸菌,经鉴定为Lactobacillus rhamnosus、Enterococcus faecalis、L. gasseri、L. plantarum、L. casei、Pediococcus pentosaceus与Bifidobacterium longum;SMRT测序技术共获得11种乳酸菌,分别为L. rhamnosus、E. faecalis、L. gasseri、L. casei、P. pentosaceus、L. paracasei、L. salivarius、L. paraplantarum、L. porcinae、B. pseudocatenulatum、B. longum。其中,抗生素组仅分离检测到2种乳酸菌,分别为L. porcinae和E. faecalis。传统培养方法与SMRT测序技术对婴儿肠道菌群中乳酸菌多样性的分析存在差异,且抗生素的使用会降低婴儿肠道菌群中乳酸菌的多样性。     

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。     

In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteria

This study was conducted to investigate the catabolism and fermentation of human milk oligosaccharides (HMO) by individual strains of bifidobacteria. Oligosaccharides were isolated from a pooled sample of human milk using solid-phase extraction, and then added to a growth medium as the sole source of fermentable carbohydrate. Of five strains of bifidobacteria tested (Bifidobacterium longum biovar infantis, Bifidobacterium bifidum, Bifidobacterium longum biovar longum, Bifidobacterium breve, and Bifidobacterium adolescentis), B. longum bv. infantis grew better, achieving triple the cell density then the other strains. B. bifidum did not reach a high cell density, yet generated free sialic acid, fucose and N-acetylglucosamine in the media, suggesting some capacity for HMO degradation. Thin layer chromatography profiles of spent fermentation broth suggests substantial degradation of oligosaccharides by B. longum bv. infantis, moderate degradation by B. bifidum and little degradation by other strains. While all strains were able to individually ferment two monosaccharide constituents of HMO, glucose and galactose, only B. longum bv. infantis and B. breve were able to ferment glucosamine, fucose and sialic acid. These results suggest that as a potential prebiotic, HMO may selectively promote the growth of certain bifidobacteria strains, and their catabolism may result in free monosaccharides in the colonic lumen.     

Use of tuf gene-based primers for the PCR detection of probiotic Bifidobacterium species and enumeration of bifidobacteria in fermented milk by cultural and quantitative real-time PCR methods

Due to the increasing use of bifidobacteria in probiotic products, it is essential to establish a rapid method for the qualitative and quantitative assay of the bifidobacteria in commercial products. In this study, partial sequences of the tuf gene for 18 Bifidobacterium strains belonging to 14 species were determined. Alignment of these sequences showed that the similarities among these Bifidobacterium species were 82.24% to 99.72%. Based on these tuf gene sequences, 6 primer sets were designed for the polymerase chain reaction (PCR) assay of B. animalis subsp. animalis, B. animalis subsp. lactis, B. bifidum, B. breve, B. longum subsp. infantis, B. longum subsp. longum, and the genus of Bifidobacterium, respectively. These Bifidobacterium species are common probiotic species present in dairy and probiotic products. When each target Bifidobacterium spp. was assayed with the designed primers, PCR product with expected size was generated. In addition, for each target species, more than 70 bacterial strains other than the target species, including strains of other Bifidobacterium species, strains of Lactobacillus spp., Enterococcus spp., and other bacterial species, all generated negative results. PCR assay with primers specific to B. animalis subsp. lactis and B. longum subsp. longum confirmed the presence of these Bifidobacterium species in commercial yogurt products. In addition, for each product, enumeration of the bifidobacteria cells by culture method with BIM-25 agar and the quantitative real-time PCR showed similar cell counts. Such results indicated that within 15-d storage (4 °C) after manufacture, all the bifidobacteria cells originally present in yogurt products were viable and culturable during the storage.     

Effects of administration of Bifidobacterium animalis subsp. lactis GCL2505 on defecation frequency and bifidobacterial microbiota composition in humans

The aim of this study was to evaluate the changes in endogenous bifidobacteria and administered Bifidobacterium animalis subsp. lactis (B. lactis) GCL2505 (GCL2505) in the intestine after administration of GCL2505 by means of a randomized, placebo-controlled double-blind, cross-over study. An increase in the number of total bifidobacteria (the sum of B. bifidum, B. breve, B. longum subsp. longum, B. adolescentis, B. anglatum, B. catenulatum, B. pseudocatenulatum, B. dentium, B. longum subsp. infantis and B. lactis) in the feces were observed after administration of GCL2505 using species- and subspecies-specific real-time polymerase chain reaction analysis. However, the number of endogenous bifidobacteria species (excluding B. lactis) remained unchanged. B. lactis also became the predominant bifidobacterial species. Taking into account the number of GCL2505 administered, the findings further suggested that GCL2505 proliferated in the intestine. In addition, the defecation frequency increased during GCL2505 administration compared with the placebo. Moreover, a single administration study (n=17) clearly demonstrated that GCL2505 successfully reached the intestine before proliferating at least 10-fold. This is the first report to show an increase in intestinal bifidobacteria, with no changes to the endogenous species, and improvements in constipation following proliferation of administered bifidobacteria.     

Origin and identification of bifidobacteria strains isolated from meat and meat products

Forty-seven strains of bifidobacteria isolated from meat and meat products have been identified following phenotypic numerical analysis and DNA-DNA hybridization. Twenty-three strains were identified to the species B. thermophilum and 14 to B. pseudolongum subsp. pseudolongum. All others were also of animal origin, except for two strains -- B. longum and B. pseudocatenulatum -- that were of human origin. These strains were isolated from artificially contaminated meat by manual handling.