利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究了5种高温和中温白酒大曲细菌群落结构,通过优势条带切胶鉴定确定了大曲中优势细菌种属信息。结果表明,Weissella cibaria、Lactobacillus helveticus、L.fermentum、L.panis等乳酸菌普遍存在于5种大曲中,Ther-moactinomyces sanguinis仅存在于高温曲酱曲中,同时DGGE检测到了传统方法未能分离鉴定的Staphylococcus xylosus、Klebsiella oxytoca。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。PCR-DGGE技术是一种能够快速有效地研究白酒大曲细菌群落结构的技术。     

圈养大熊猫肠道乳酸菌分离及益生特性

【背景】大熊猫数量稀少、繁殖困难,在其生长过程中极易感染消化系统疾病甚至死亡。【目的】分析圈养大熊猫肠道可培养乳酸菌的群落结构与功能,筛选具有益生特性的乳酸菌菌株,为大熊猫消化系统疾病的预防和肠道微生物研究提供理论参考及菌种资源。【方法】采用3种培养基分离大熊猫粪便中的乳酸菌,通过革兰氏染色镜检和过氧化氢试验对分离的菌株进行初步鉴定,基于BOXA1R-PCR图谱遗传多样性选取代表菌株进行16S rRNA基因测序分析并进行主成分分析(principal component analysis, PCA),同时分析乳酸菌菌株安全性和益生特性。【结果】通过初步鉴定共分离获得58株乳酸菌,根据BOXA1R-PCR结果挑选20株菌进行测序,结果显示20株菌分属于明串珠菌属(Leuconostoc)、肠球菌属(Enterococcus)、魏斯氏菌属(Weissella)和链球菌属(Streptococcus)这4个属,主成分分析结果表明不同年龄段的大熊猫肠道乳酸菌群落结构存在差异。20株乳酸菌均不溶血,17株乳酸菌对11种抗生素均敏感;11株菌耐pH值2.0酸性条件,14株菌对0.3%的胆盐具有良好...     

新疆额尔齐斯河流域冷水鱼肠道耐低温乳酸菌的分离筛选及其遗传差异

[目的]从新疆阿勒泰地区额尔齐斯河流域冷水鱼肠道中分离耐低温的乳酸菌,挖掘乳酸菌的物种和遗传多样性,为低温乳酸菌生物技术研发提供优良菌种.[方法]利用MRS、Elliker两种不同培养基从9种冷水鱼肠道中,分离筛选出耐低温菌,测定细菌最适生长温度并进行常规生理生化实验.根据16S rRNA基因序列初步确定耐低温菌的系统进化地位,并采用BOX、(GTG)5、ERIC三种不同的引物进行rep-PCR,对16S rRNA基因高度同源性的菌株进一步区分.[结果]分离得到78株耐冷菌,最终确定有24株为耐低温乳酸菌,菌株的最适生长温度在15-24℃之间.系统发育分析结果表明:24株耐低温乳酸菌隶属于6个属,其中肉杆菌属(Carnobacterium)3株,乳球菌属(Lactococcus)9株,肠球菌属(Enterococcus)7株,环丝菌属(Brochothrix)1株,魏斯氏菌属(Weissella)2株,链球菌属(Streptococcus)2株.指纹图谱聚类分析树状图显示乳球菌属(Lactococcus)和肠球菌属(Enterococcus)存在种及菌株水平上的遗传差异,前者包括4个种,后者2个种.[结论]新疆额尔齐斯河流域冷水鱼肠道中分离的耐低温乳酸菌以球菌为主,没分离到常见的乳杆菌,需进一步完善分离培养的方法,深入挖掘和研究其物种多样性和遗传多样性.     

培养和非培养方法分析发酵白菜卤乳酸菌的多样性

[目的]为了探明发酵白菜体系中乳酸菌的多样性和优势乳酸菌.[方法]采用培养和非培养的16SrRNA基因同源性分析发酵白菜卤中乳酸菌的多样性.[结果]从培养平板分离得到的90株乳酸菌鉴定为Lactobacillus属和Leuconostoc属.通过对115个16s rRNA基因序列对比发现非培养样品的乳酸菌为Lactobacillu属,Pediococcus属Leuconostoc 属,Weissella 属.[结论]非培养样品的乳酸菌多样性较培养方法丰富.两种方法分析的结果显示上Lactoacillus plantarum为发酵白菜体系中优势乳酸菌之一.     

专一性PCR和变性梯度胶电泳协助从焦化废水处理装置中分离优势功能菌Thauera属菌株

【目的】在专一性PCR和变性梯度胶电泳(DGGE)的协助下,从废水处理装置的微生物群落中分离较难分离的功能菌Thauera。【方法和结果】本研究首先使用Thauera特异性PCR-DGGE的方法鉴定了焦化废水处理装置反硝化池生物膜中的Thauera在6种培养基、好氧/厌氧条件下的生长情况。挑选Thauera多样性较高的培养基1/10 NB与MMQ在好氧条件下进行分离培养。然后使用Thauera特异性PCR方法确定分离得到的菌落是否呈阳性,并使用DGGE的方法检验其是否为纯菌。使用不同培养基对含有Thauera的混合菌落进行进一步纯化,DGGE检测发现MMP培养基对混合细菌菌落Q20中的Thauera有明显的富集作用。经过Thauera特异性PCR结合DGGE检测对Thauera属细菌进行追踪,将混合菌落在MMP培养基上多次划线,最终分离得到纯菌。通过这种方法,从反硝化池样品中分离获得了3株在样品中最为主要的Thauera菌株。【结论】以特异性分子标记为导向分离培养细菌,不仅提高了分离效率及细菌筛选的灵敏度,还能协助分离常规方法难以分离的细菌。     

酱香型与清香型白酒发酵过程中乳酸菌菌群的差异性分析

【目的】为认识乳酸菌在中国白酒酿造过程中的作用与影响, 分析、比较了酱香型与清香型白酒发酵过程中乳酸菌的菌群结构及其差异。【方法】运用PCR-DGGE技术分析酱香型与清香型白酒发酵过程中乳酸菌群的演变规律。并利用传统微生物分离筛选方法进一步确定酱香型白酒发酵中的主要乳酸菌种。【结果】DGGE图谱表明, 白酒发酵过程中的主要乳酸菌种是乳杆菌。但两种香型白酒发酵过程中乳酸菌群组成及动态变化均呈现出明显的差异。清香型白酒酒醅中Lactobacillus fuchuensis是优势菌种, 而酱香型白酒发酵中检测到多种含量较高的乳酸菌种。利用MRS培养基从酱香型白酒酒醅中共筛选获得5种乳酸菌种。通过两种方法, 确定Lactobacillus homohiochii是酱香型白酒发酵过程中含量最高的乳酸菌。【结论】深入研究白酒发酵过程中乳酸菌的组成及分布规律, 对于更好地认识中国白酒酿造中主要的细菌类群——乳酸菌的作用, 具有重要的理论意义和实践价值。     

广西传统发酵米粉中乳酸菌的分离鉴定

对广西传统米粉发酵液中的乳酸菌进行分离筛选,获得6株纯培养优势菌株。通过形态学鉴定及16S rDNA序列分析,结果表明其中4株乳酸菌属于戊糖片球菌(Pediococcus pentosaceus),另外2株鉴定为植物乳杆菌(Lactobacillus plantarum)。这2种乳酸菌均为对人类及动物安全的益生菌,该结果将为传统发酵米粉中有益微生物资源的挖掘和利用奠定基础。     

培养基灭菌方式对细菌分离效率的影响

自然界蕴含大量未/难培养微生物,分离这些微生物对理论研究和资源开发具有重要意义。本研究使用高压灭菌和过滤除菌方式制备培养基,采用稀释涂布方法,从红树林灰泥样品中分离获得123株细菌,通过16S rRNA基因序列分析对其进行鉴定,进而探究培养基灭菌方式对细菌分离效果的影响。结果表明：过滤除菌培养基生长的单菌落数目（339±82）个显著多于高压灭菌培养基生长的单菌落数目（179±65）个;两种培养基分离细菌的群落结构在门、科和属分类水平上总体相似,但优势类群的数目和少数类群存在差异;过滤除菌培养基分离细菌的Shannon Wiener’s指数、均匀度、新种率、基因多样性均高于高压灭菌培养基,而其与近缘模式菌株相似度的平均值和中位数则低于高压灭菌培养基。因此,过滤除菌培养基分离获得细菌的多样性、均匀性和新颖性均高于高压灭菌培养基。本研究首次探究培养基灭菌方式对细菌分离效果的影响,具有更高分离效率的过滤除菌培养基为未/难培养微生物菌株资源获取提供了借鉴。     

新疆伊犁地区原牛乳中乳酸菌的多样性分析

【目的】对新疆伊犁地区原牛奶中乳酸细菌的遗传多样性进行分析。【方法】采用菌落培养、Rep-PCR(Repetitive genomic fingerprinting)指纹图谱和16S r RNA基因序列分析相结合的方法研究牛乳内乳酸菌的遗传多样性。【结果】从5份原牛乳中分离出乳酸菌29株,基因序列分析和系统进化分析显示29株乳酸菌隶属于5个属,分别为:Lactococcus、Lactobacillus、Leuconostoc、Pediococcus和Enterococcus。优势属为Leuconostoc(27.6%),其次为Lactococcus(24.0%)。【结论】新疆伊犁地区原牛乳中乳酸菌多样性丰富,为开发新疆地区益生乳酸菌提供了丰富的活性资源。     

秦岭地区野生细鳞鲑肠道微生物多样性及产酶活性分析

为探究秦岭地区野生细鳞鲑(Brachymystax lenok)肠道细菌组成多样性,筛选出产胞外酶菌株,利用传统分离培养并分子鉴定的方法和基于16S r RNA基因克隆的现代分子生物技术相结合测定秦岭野生细鳞鲑肠道细菌菌群多样性并构建系统发育树,利用淀粉酶、蛋白酶、纤维素酶及脂肪酶4种胞外酶筛选培养基筛选出产上述酶的细菌。细菌传统分离培养并分子鉴定法从细鳞鲑肠道获得18个属的细菌类群,分别归属于变形菌门、拟杆菌门和厚壁菌门,其中,气单胞菌属(Aeromonas)为优势菌群。基于16S r RNA基因克隆的现代分子方法获得22个属的细菌类群,分别归属于变形菌门、拟杆菌门、厚壁菌门和放线菌门,其中,鞘氨醇杆菌属(Sphingomonas)为优势菌群。4种胞外酶筛选获得53株细菌产胞外酶,其中21株可在低温(10℃)环境下产胞外酶。结果表明,传统分离培养法与基于16S r RNA基因克隆的现代分子生物技术相结合能够更有效全面地分析细鳞鲑鱼肠道微生物的多样性,并且细鳞鲑肠道微生物具有一定的产酶活性。     

Genomic diversity of Oenococcus oeni from different winemaking regions of Portugal

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium that plays an important role in the elaboration of wine. It is often added as a starter culture to carry out malolactic conversion. Given the economic importance of this reaction, the taxonomic structure of this species has been studied in detail. In the present work, phenotypic and molecular approaches were used to identify 121 lactic acid bacteria strains isolated from the wines of three winemaking regions of Portugal. The strains were differentiated at the genomic level by M13-PCR fingerprinting. Twenty-seven genomic clusters represented by two or more isolates and 21 single-member clusters, based on an 85% similarity level, were recognized by hierarchic numerical analysis. M13-PCR fingerprinting patterns revealed a high level of intraspecific genomic diversity in O. oeni. Moreover, this diversity could be partitioned according to the geographical origin of the isolates. Thus, M13-PCR fingerprint analysis may be an appropriate methodology to study the O. oeni ecology of wine during malolactic fermentation as well as to trace new malolactic starter cultures and evaluate their dominance over the native microbiota.     

Lactic acid bacteria associated with wine grapes from several Australian vineyards

AIMS: The detection and isolation of lactic acid bacteria by enrichment methods from wine grapes cultivated in vineyards located in New South Wales, Australia. METHODS AND RESULTS: Enrichment cultures in de Man, Rogosa and Sharpe (MRS) broth, MRS + ethanol (5%), MRS broth supplemented with 15% (v/v) tomato juice (MRST), pH 5.5 and 3.5 and autoenrichment in grape juice homogenate were used to detect lactic acid bacteria on wine grapes. Bacteria were isolated from enrichment cultures by plating onto MRS and MRST agar and identified by 16S rDNA sequence analysis and phenotypical methods. A molecular method, PCR-denaturing gradient gel electrophoresis (DGGE) was also used to examine the bacteria that developed in enrichment cultures. Species of Lactobacillus, Enterococcus, Lactococcus and Weissella were detected in enrichments by plating and PCR-DGGE. Other bacteria (Sporolactobacillus, Asaia, Bacillus ssp.) were also found in some enrichment cultures. The principal malolactic bacterium, Oenococcus oeni, was not isolated. CONCLUSIONS: The incidence and populations of lactic acid bacteria on wine grapes were very low. Damaged grape berries showed a greater presence of these bacteria than undamaged berries. The diversity of bacterial species isolated from the grapes was greater than those previously reported and represented both lactic acid bacteria and nonlactic acid bacteria. Some of these bacteria (i.e. Lactobacillus lindneri, Lactobacillus kunkeei) could be detrimental to wine production. Oenococcus oeni was not found on grapes, but its recovery could be obscured by overgrowth from other species. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria are significant in wine production because they conduct the malolactic fermentation and cause stuck or sluggish alcoholic fermentation and wine spoilage. This study investigates wine grapes as a potential source of these bacteria.     

Isolation and identification of lactic acid bacteria from Tarag in Eastern Inner Mongolia of China by 16S rRNA sequences and DGGE analysis

Tarag is a characteristic fermented dairy product with rich microflora (especially lactic acid bacteria), developed by the people of Mongolian nationality in Inner Mongolia of China and Mongolia throughout history. One hundred and ninety-eight samples of Tarag were collected from scattered households in Eastern Inner Mongolia, and total of 790 isolates of lactic acid bacteria (LAB) were isolated by traditional pure culture method. To identify these isolates and analyze their biodiversity, 16S rRNA gene sequences analysis and PCR-DGGE were performed respectively. The results showed that 790 isolates could be classified as 31 species and subspecies. Among these isolates, Lactobacillus helveticus (153 strains, about 19.4%), Lactococcus lactis subsp. lactis (132 strains, about 16.7%) and Lactobacillus casei (106 strains, about 11.0%) were considered as the predominated species in the traditional fermented dairy products (Tarag) in Eastern Inner Mongolia. It was shown that the biodiversity of LAB in Tarag in Inner Mongolia was very abundant, and this traditional fermented dairy product could be considered as valuable resources for LAB isolation and probiotic selection.     

Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods

Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 10¹ colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.     

In Vivo PCR-DGGE Analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Populations in Red Wine

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety “Primitivo di Puglia”), a polymerase chain reaction– denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30–60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.     

Inventory and monitoring of wine microbial consortia

The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.     

Microbiology of the malolactic fermentation: Molecular aspects

Abstract Malolactic fermentation conducted by lactic acid bacteria follows alcoholic fermentation during winemaking, and several positive effects make it indispensable for most wines. Research has focused on the growth and physiology of lactic acid bacteria in wine; resulting in the design of malolactic starter cultures. Future work on these starters will concentrate on aromatic changes as additional criteria for strain selection. Although the main features of the malolactic enzyme and its gene are known, the detailed mechanism of the malolactic reaction remains unclear. Cloning and expression of this activity in enological strains of Saccharomyces cereuisiae might be one of the next most important advances in the control of malic acid degradation in wine.     

Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non-Saccharomyces yeasts (Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced.     

Plasmid contents of lactic acid bacteria isolated from different types of sour doughs

The plasmid contents of 13 lactic acid bacteria isolated from different types of sour doughs were examined and compared with the plasmid contents of 11 culture collection strains and one commercial pure starter culture for sour doughs. In addition, plasmid analysis was used as a tool to study the stability of a starter culture during sour dough fermentation in a bakery.The tested strains varied in plasmid content from no plasmid up to six plasmids, with molecular weights from 1.5 to 43 MDal. In most cases, the wild-type strains contained a higher number of plasmids than the culture collection strains. The ability of the strains to ferment different carbohydrates was also investigated, but no obvious correlations between the fermentation patterns and the plasmid patterns could be observed. During the fermentation of the bakery sour dough, strains other than the inoculated starter culture gradually became dominant in the microflora. These new strains contained 1–3 plasmids, contrary to the plasmidless starter culture, and they also fermented more carbohydrates than the starter culture.