利用16S rDNA序列及tuf-RFLP鉴定蒙古国发酵乳中的乳酸菌

运用16S rDNA序列分析和tuf-RFLP技术对采于蒙古国扎布汗省的25份发酵乳样中分离出的110株乳酸菌进行鉴定。首先将分离的110株乳酸菌的16S rRNA基因进行扩增,测序并构建系统发育树,初步鉴定为41株嗜热链球菌,40株瑞士乳杆菌,11株德氏乳杆菌保加利亚亚种,2株发酵乳杆菌,1株乳明串珠菌,2株肠膜明串肠膜亚种,1株乳酸乳球乳酸亚种和12株属于干酪族的菌株。由于干酪乳杆菌族的16S rDNA序列差异很小,故采用tuf-RFLP技术对这12株进行了进一步的验证,通过分离菌株与模式菌株tuf-RFLP图谱的比较分析,结果表明这12株菌均为干酪乳杆菌。     

辣白菜中乳酸菌的分离鉴定

从沈阳市7 个区25 份朝鲜族家庭制作的传统发酵辣白菜中分离出81 株乳酸菌疑似菌株,初步鉴定34 株为杆菌,47 株为球菌。进一步采用16S rDNA序列分析对81 株菌进行分子鉴定,通过序列分析进行属种鉴定。结果表明：81 株菌均为乳酸菌,分别来自2 个属6 个种,45 株为屎肠球菌,25 株为植物乳杆菌,4 株为干酪乳杆菌,3 株为戊糖乳杆菌,2 株为短乳杆菌,2 株为坚强肠球菌。研究结果为我国东北辣白菜中乳酸菌作进一步研究奠定了基础。     

水牛乳中可培养乳酸菌多样性分析

采用传统分离培养方法,从三品杂交生水牛奶混合样品中,分离出105株乳酸菌,通过形态、生理生化、API细菌鉴定系统及16S rDNA基因序列分析方法对各菌株属种进行鉴定。16S rRNA序列分析结果显示,105株菌共分为5个属8个种,呈现较为丰富的乳酸菌多样性,具体数量分布为乳酸乳球菌21株,植物乳杆菌19株,格氏乳球菌17株,乳明串珠菌13株,食窦魏斯氏菌11株,肠膜明串珠菌8株,类肠膜魏斯氏菌6株,嗜热链球菌5株,糊精乳杆菌5株。由此可知,水牛乳中可培养乳酸菌优势菌群的主次关系为：乳酸乳球菌（Lactococcus lactis）＞植物乳杆菌（Lactobacillus plantaru）＞格氏乳球菌（Lactococcus garvieae）＞乳明串珠菌（Leuconostoc lactis）＞食窦魏斯氏菌（Weissella cibaria）,此为后续开发水牛乳中优势乳酸菌资源提供了良好的理论基础。     

东北酸菜中乳酸菌的分离鉴定与耐酸性菌株的筛选

为筛选出高耐酸性乳酸菌,通过形态学观察和随机扩增多态性DNA标记分析技术,对自然发酵酸菜中分离纯化的乳酸菌菌株进行初步鉴别,随后利用16S rDNA序列同源性分析进行种属鉴定,并筛选在pH 3.0条件下存活率较高的菌株。结果表明,72 株分离乳酸菌包括62 株乳杆菌和10 株乳球菌,其中有21 株菌的指纹图谱不相同,经鉴定,分别为乳肠球菌（Enterococcus lactis）、弯曲乳杆菌（Lactobacillus curvatus）、米曲霉乳杆菌（L. oryzae）、短乳杆菌（L. brevis）、副干酪乳杆菌（L. paracasei）、棒状乳杆菌（L. coryniformis）。pH 3.0条件下存活率在75%以上的菌株有8 株,管家基因rpoA序列同源性分析结果表明高耐酸性的两株菌株为植物乳杆菌（L. plantarum）,为开发功能性乳酸菌食品提供了一定理论支持。     

植物源益生乳酸菌的筛选及其特性

为筛选具有益生特性的植物源乳酸菌,以传统发酵蔬菜中分离的1 000 株乳酸菌为出发菌株,进行了耐酸性、耐胆盐能力、抑菌性、体外抗氧化能力、药敏性、溶血性和氨基酸脱羧酶活性等特性研究,并对筛选菌株进行了16S rDNA 鉴定。经pH 3.0 MRS培养得乳酸菌82 株,再经pH 2.5 MRS培养得乳酸菌49 株;49 株菌经0.3%胆盐测试,均具有耐胆盐能力;根据镜检形态结合发酵植物源的不同从中挑选19 株乳酸菌进行药敏性、溶血性、抑菌性、氨基酸脱羧酶活性和1,1-二苯基-2-三硝基苯肼自由基清除实验。结果表明,19 株菌对所选20 种抗生素多数表现敏感,其中4 株菌对20 种抗生素都较敏感;19 株菌对供试致病菌都有不同程度抑制能力且都无溶血性;经氨基酸脱羧酶活性试剂盒结合聚合酶链式反应扩增检测表明,19 株菌无产生物胺的潜在威胁;有5 株菌体外抗氧化能力高于40%。可见19 株菌均具有益生菌的基本特性。经16S rDNA鉴定,7 株为发酵乳杆菌、6 株为植物乳杆菌,嫩江杆菌、戊糖片球菌、利莫西杆菌、戊糖乳杆菌、屎肠球菌、短乳杆菌分别各1 株。     

一株产细菌素乳杆菌的鉴定及其细菌素编码基因的获得

应用双层平板打孔法,从自制樱桃酒里分离的乳杆菌中筛选到一株对革兰氏阳性细菌和革兰氏阴性细菌均有明显抑制作用的菌株,命名为LD 1.0008。通过API 50 CHL糖发酵产酸实验、16S rDNA基因序列分析及其特异性recA基因多重聚合酶链式反应（polymerase chain reaction,PCR）,鉴定LD 1.0008为植物乳杆菌。排除有机酸的干扰,用多种蛋白酶处理后,其抑菌活性均有明显降低,而用过氧化氢酶处理后其抑菌活性基本不变,从而确定LD 1.0008所产生的抑菌物质为细菌素。使用已报道的10 对植物乳杆菌细菌素基因片段设计的引物对菌株LD 1.0008进行PCR扩增,发现其至少含有4 个植物乳杆菌素相关编码基因,即plnD、plnO、plnV和plnW。     

植物乳杆菌活的非可培养状态的初步研究

利用16S rDNA序列分析法测得实验室保藏的乳酸菌的16S rDNA序列,并与基因库中基因序列进行同源性比较,经鉴定此菌为植物乳杆菌.并研究了植物乳杆菌进入活的非可培养状态(Viable but Non-cuhurable,VBNC)的诱导条件和复苏条件.结果显示,在诱导条件为MRS液体培养基、pH5.5～6.2、温度-20℃和有氧环境时,植物乳杆菌在12d之后进入了VBNC状态;复苏条件为添加有6％吐温-80的MRS液体培养基和普通MRS液体培养基分别在48h和96h之内可以使VBNC的植物乳杆菌复苏.     

婴儿粪便长双歧杆菌的分离与多样性分析

本研究旨在分析婴儿粪便长双歧杆菌的多样性,采用改良MRS培养基从哈尔滨地区健康婴儿粪便中分离双歧杆菌,采用生理生化实验结合16S rDNA和热应激蛋白60基因(heat-shock protein 60,hsp60)同源性分析鉴定分离株,利用同源性分析及随机扩增多态性DNA(random amplified polymorphism DNA,RAPD)、多位点序列分型(multilocus sequence typing,MLST)技术进一步分析长双歧杆菌多态性。实验从11个婴儿体内分离得到18株厌氧的细菌菌株,经形态学分析、生理生化实验、16S rDNA测序及hsp60测序实验发现,其中7株为长双歧杆菌长亚种,3株为长双歧杆菌婴儿亚种。RAPD和MLST结果表明:7株长双歧杆菌长亚种为6个基因型,3株长双歧杆菌婴儿亚种为2个基因型,上述结果说明不同人源婴儿粪便长双歧杆菌基因型差异较大。     

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.     

广谱抑菌乳酸菌的筛选及其细菌素相关基因分析

采用琼脂扩散法,从分离自新疆传统酸奶的8株乳酸菌中筛选出1株抑菌效果较好并具有广谱抑菌性的菌株B6,通过革兰氏染色、生理生化及16S rDNA序列比对,鉴定该菌为植物乳杆菌。植物乳杆菌B6发酵液经排除酸、过氧化氢实验后,仍有抑菌效果,且对蛋白酶敏感,判定该菌产细菌素。通过设计10对编码合成细菌素相关基因的特异性引物,以菌株B6的DNA为模板,进行聚合酶链式反应快速鉴定。结果表明,菌株B6含有编码IIb类细菌素结构基因plnE/F/J/K,plnJ与植物乳杆菌C11相比,序列仅在前导肽区出现一处氨基酸突变,相似度为99%,plnE/F/K序列与植物乳杆菌C11的这3个基因序列完全相同。因此鉴定植物乳杆菌B6产生的是IIb类细菌素。     

Isolation of bacteriocinogenic Lactobacillus plantarum strains from ben saalga, a traditional fermented gruel from Burkina Faso

A collection of lactic acid bacteria isolated from ben saalga, a traditional fermented gruel from Burkina Faso, was screened for bacteriocin production. Seven isolates were selected for their broad antimicrobial spectra, which overall included strains of Bacillus cereus, Bacillus licheniformis, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella enterica. Cluster analysis of RAPD-PCR patterns revealed that six of the isolates represent different strains. The six selected strains were identified as Lactobacillus plantarum by 16S rDNA sequencing, species-specific PCR and multiplex PCR of the recA gene. PCR amplification revealed the presence of genes of the plantaricin cluster described in L. plantarum C11. Among them, strain 5.2.2 carried the largest number of genes from this cluster.     

Lactobacillus casei, dominant species in naturally fermented Sicilian green olives

This study investigated the phenotypic and genotypic characteristics of lactic acid bacteria in naturally fermented green olives, collected from different areas of Sicily. Both classical biochemical tests and PCR/Restriction Fragments Length Polymorphism (RFLP) of 16S rDNA were used to characterize the isolates. The identity of the isolates was obtained by the partial sequencing analysis of the 16S rDNA. The BioMerieux software assigned the 13 heterofermentative strains to the Lactobacillus brevis species; 24 homofermentative strains were classified as Lactobacillus casei and the remaining 11 homofermentative lactobacilli were identified as Lactobacillus plantarum. The rapid ID 32 STREP test identified coccal-shaped strains as Enterococcus faecium species. The PCR/RFLP analysis showed a remarkable bacterial heterogeneity within the isolates. The 16S rDNA partial sequencing did not confirm biochemical identification, revealing a strong dominance of isolates belonging to the L. casei species. It is noteworthy that this species has never been reported as dominant species in fermented vegetables.A combination of molecular and biochemical analysis allowed the identification of species involved in natural food fermentations.     

Efficacy of recA gene sequence analysis in the identification and discrimination of Lactobacillus hilgardii strains isolated from stuck wine fermentations

Conventional phenotypic methods sometimes lead to misidentification of some heterofermentative wine lactobacilli such as Lactobacillus hilgardii, Lactobacillus buchneri, and Lactobacillus brevis. We establish the specificity of 16S rDNA sequencing in the differentiation of these species and in the rejection of the Lactobacillus vermiforme species name. Moreover, we succeeded in differentiating these heterofermentative species by means of recA gene sequence comparison. Short homologous regions were amplified by PCR with degenerate consensus primers, sequenced, and 280 bp were analysed and considered for the inference of phylogenetic trees. The phylogram obtained was coherent and clearly separated the three species. The recA gene sequence was a reliable and useful method that allowed a good discrimination among closely related species. The validity of the recA gene sequence, restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR), and random amplified polymorphic DNA (RAPD) to study the L. hilgardii intraspecies heterogeneity was tested in five strains isolated from stuck wine fermentations at the same winery in the same vintage. The results indicated that L. hilgardii is a heterogeneous species. Since L. hilgardii is a malolactic species that can influence the final quality of the wine, the presence of oenological relevant genes, such as those involved in ethyl carbamate or biogenic amine production, was investigated.     

Tannase activity by lactic acid bacteria isolated from grape must and wine

We examined a range of oenological lactic acid bacteria species and reference strains for their potential to degrade tannins. Bacterial tannase activity was checked by a spectrophotometric and a visual reading method. None of the strains belonging to the oenological species of the genus Lactobacillus, Leuconostoc, Oenococcus or Pediococcus were tannase producers, with the exception of Lactobacillus plantarum. All the L. plantarum strains analyzed were positive for tannase activity and their identities were reconfirmed by L. plantarum PCR-specific assay or by sequencing the 16S rDNA. Tannase activity could be considered an important criterion for the selection of malolactic starter cultures since it might confer advantages in the winemaking process by reducing astringency and haze in wine.     

Phenotypic and genotypic identification of lactic acid bacteria isolated from ethnic fermented bamboo tender shoots of North East India

Mesu, soidon, soibum and soijim are ethnic fermented bamboo tender shoot products prepared by the people in North East India. Microbiological analysis of mesu, soidon, soibum and soijim showed the population dominated by lactic acid bacteria (LAB) ranging up to 10(8) cfu g(-1). The phenotypic characterisation of predominant LAB isolated from the fermented bamboo shoot products was based on general morphology, physiological tests, API and Biolog systems. The genotypic characterisation of LAB was based on RAPD-PCR, rep PCR, species-specific PCR techniques, 16S rRNA gene sequencing and DNA-DNA hybridisation. Predominant functional LAB strains associated with the fermented bamboo shoot products were identified as Lactobacillus brevis, Lb. plantarum, Lb. curvatus, Pediococcus pentosaceus, Leuconostoc mesenteroides subsp. mesenteroides, Leuc. fallax, Leuc. lactis, Leuc. citreum and Enterococcus durans.     

纳豆芽孢杆菌和嗜酸乳杆菌融合子的多重PCR鉴定

为建立纳豆芽孢杆菌和嗜酸乳杆菌融合子快速鉴定的多重聚合酶链式反应(polymerase chain reaction,PCR)方法,根据芽孢杆菌的芽孢形成早期因子基因spo0A和13株乳酸菌β-半乳糖苷酶基因的保守序列分别设计引物对P1/P2和P3/P4,以亲本菌株DNA为模板,优化每对引物在适宜退火温度条件下的PCR体系,在最优体系下的特异性分析结果表明P1/P2能特异性扩增出spo0A基因片段(308 bp),而7株乳酸菌全部呈阴性;P3/P4可扩增出所有受试乳酸菌和经表型鉴定确定的阳性融合子中的目标基因片段(576 bp),而对芽孢杆菌无扩增。多重PCR结果表明在P1/P2的最优体系中只有308 bp片段的扩增,而在P3/P4的最优体系中可实现2个片段的共扩增,对融合后获得的50个菌株的鉴定结果与单重PCR和表型鉴定结果100%吻合。该体系中模板DNA 1 ng/μL,每条引物0.4μmol/L,脱氧核糖核苷三磷酸0.25 mmol/L,Taq酶0.06 U/μL;PCR时94℃预变性5 min;每个循环(共30个)94℃30 s、53℃30 s、72℃60 s,产物末端72℃延伸7 min。该方法简便、快速、特异性好,适用于芽孢杆菌、乳酸菌以及二者融合子的快速鉴定。     

A PCR assay for detection of acetic acid-tolerant lactic acid bacteria in acidic food products

A PCR assay for the detection of acetic acid-tolerant lactic acid bacteria in the genera of Lactobacillus and Pediococcus was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 56 different bacterial strains (of 33 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for most tested bacterial members of 16S rRNA gene-based phylogenetic groups (classified in the Lactobacillus casei and Pediococcus group), including Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus plantarum, and Lactobacillus paracasei. For all other bacterial strains and eukaryote tested, results were negative. Bacterial DNA for PCR was prepared with a simple procedure with the use of Chelex 100 resin from culture after growth in deMan Rogosa Sharpe broth (pH 6.0). To test this PCR assay for the monitoring of the acetic acid-tolerant lactic acid bacteria, L. fructivorans was inoculated into several acidic food as an indicator. Before the PCR, the inoculation of 10 to 50 CFU of bacteria per g of food was followed by a 28-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. Including the enrichment culture step, the entire PCR detection process can be completed within 30 h.     

Identification of lactic acid bacteria in traditional fermented yak milk and evaluation of their application in fermented milk products

In this study, 53 strains of lactic acid bacteria (LAB) isolated from Xueo, a traditional fermented yak milk in the western Sichuan Plateau of China, were identified and their use in fermented milk was evaluated. All gram-positive and catalase-negative strains were divided into 6 groups at the level of 87% similarity using amplified ribosomal DNA restriction analysis. These groups were identified as 6 species using 16S rDNA sequence analysis and atpA gene analysis. The dominant LAB strains in Xueo were Enterococcus durans, Lactobacillus fermentum, and Lactobacillus paracasei, accounting for 45.3, 22.6, and 17.0% of isolates, respectively. Milk fermented with most of the representative strains was high in quality, exhibiting relatively high viscosity, moderate acidity, good sensory quality, and high counts of viable LAB. Fermented milk of E. durans SCA16 and L. fermentum SCA52 achieved the highest scores for overall sensory quality. Most strains displayed antimicrobial activity against at least 1 of 9 spoilage microorganisms. Lactic acid was the main factor inhibiting the growth of spoilage bacteria, and H(2)O(2) was also inhibitory to some extent. Excluding the influence of acid and H(2)O(2), strains SCA52 (L. fermentum) and SCA7 (Lactobacillus plantarum) were antagonistic against some of the indicators, suggesting that the 2 strains may produce a bacteriocin-like substance. Therefore, the development of superior strains isolated from Xueo to ferment milk with similar flavor and texture to Xueo is expected.     

葡萄酒中植物乳杆菌的鉴定及其耐受性分析

本论文对4株分离自甘肃、宁夏产区葡萄酒中的乳杆菌进行鉴定并通过调整改良MRS培养基分析植物乳杆菌对酒精浓度、pH和SO2浓度的耐受性.结果表明:16S rDNA鉴定得到3株植物乳杆菌,在单一因素影响下,3株菌在pH 3.0条件下有良好生长,在16％vol酒精浓度可生长,110mg/L SO2浓度下可生长.在综合因素影响...