随机扩增多态性法分析西藏牦牛奶酪中乳酸菌的遗传多样性

目的：利用随机扩增多态性（randomly amplified polymorphic DNA,RAPD）法对西藏地区牦牛奶酪中27 株乳酸菌基因型进行同源性分析。方法：利用5 个随机引物对Mg2+浓度、dNTP用量、退火温度、模版用量/引物用量（ng/pmol）4 个条件做单因素梯度试验,建立最佳反应条件,筛选最佳引物,然后对27 株乳酸菌和4 株乳酸菌标准菌株进行随机扩增,用NTsys 2.10e软件对扩增条带进行聚类和遗传相似性系数分析,分析结果与16S rRNA测序得到的菌种鉴定结果进行对比。结果：31 株菌遗传相似系数在0.72～1.00之间,当相似性系数在0.82时,菌株被分成了8 组,菌株按照不同种属聚类,聚类结果同16S rRNA测序结果基本一致,同时成功将Lactobacillus casei和Lactobacillus paracasei两个亚种区分开。结论：RAPD技术可以较好地应用于西藏地区牦牛奶酪中乳酸菌亲缘性关系分析。     

PCR技术结合RAPD图谱鉴定食品中双歧杆菌的研究

通过筛选16S rDNA序列库,设计了一对双歧杆菌属特异性引物P175和P874在试验确定的反应条件下,该引物对能正确区分双歧杆菌和非双歧杆菌。RAPD技术用于种及菌株的鉴定。选用10种引物对9株标准菌株基因组DNA进行扩增,对其指纹图谱分析表明S 256引物扩增的图谱可于双歧杆菌菌种及菌株鉴定的依据。通过对图谱相似性进行聚类分析,揭示了双歧杆菌属基因组的多态性及其遗传发育关系。     

不同来源酿酒酵母菌株的随机扩增多态DNA分析

研究中试用了20个随机引物对16株不同来源的酿酒酵母菌株全基因组进行了随机扩增多态DNA分析,其中OPG06,OPG11和OPG20三条适宜引物具有鉴别作用,每一引物均可扩增1～10条DNA片段,大多数片段分子量大小在100～2000bp之间,共扩增出34条RAPD谱带,多态性为85.3%,获得了稳定清晰的菌株RAPD指纹图谱。RAPD分析结果表明,不同来源的酿酒酵母菌株之间的遗传相似系数在37.5%～94.1%之间,反映出较高的遗传差异性,并可通过聚类分析将16株不同来源的酿酒酵母菌株按亲缘关系的远近分为6个类群。结果表明,利用RAPD标记技术在基因水平上对酿酒酵母菌株进行分子鉴定和分型是可行的。     

川西高原牦牛酸奶子乳酸菌遗传多样性及系统发育研究

采用16S rDNA基因的限制性酶切片段长度多态性分析(16S rDNA-PCR-RFLP)和16S～23S rDNA基因间区(ISR)的酶切多态性分析(ISR-PCR-RFLP)技术,研究分离自川西高原牦牛酸奶子的81株乳酸菌的遗传多样性,并结合16S rDNA序列分析技术研究16S rDNA-PCR-RFLP的各类群代表菌株的系统发育关系。结果表明：供试菌株共有23种16S rDNA遗传型,供试菌株按61%相似系数可分为4个类群,按81%相似系数,可分为16个类群,结合序列分析结果,乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)细菌为主要类群;16S～23S rDNA基因的ISR扩增产物的酶切分析表明,供试菌株共有19种ISR遗传型,按60%相似系数,供试菌株可分为4个类群,按80%相似系数,供试菌株可划分为14个类群。     

牦牛奶酪中乳酸菌对抗生素的药敏性

分析牦牛奶酪中乳酸菌对常用抗生素的药敏性。采用纸片扩散法测定分离自牦牛奶酪的39 株乳酸菌对8 种常用抗生素的药敏性。结果表明：39 株乳酸菌都有耐药性,主要对万古霉素、新霉素、链霉素和萘啶酮酸有抗性,抗性菌株分别达到了36 株（92.3%）、32 株（82.1%）、38 株（97.4%）和39 株（100%）,菌株的耐药性有一定的种间差异,部分菌株有多重耐药性。     

白平菇不同菌株菌丝体的RAPD分析及系统进化关系的研究

本研究采用RAPD技术分析白平菇四个不同株系的DNA序列多态性,用15个随机引物对菌株基因组DNA进行PCR扩增,其中3个引物可以扩增出较好的多态性条带图谱,共产生66条清晰稳定的带。通过对供试菌株遗传相似系数的计算和系统聚类分析,构建了树状聚类图谱,在分子水平上分析了白平菇的种质遗传学差异,为白平菇菌株鉴定提供快速有效的技术和方法。     

新疆伊犁牧区传统手工奶酪中微生物多样性及其功能分析

为探究新疆伊犁牧区传统手工奶酪中的可培养乳酸菌和细菌多样性,首先采用Illumina MiSeq高通量测序技术对细菌16S rRNA基因的V1～V3可变区进行测序分析,结果显示：14 份奶酪中共存在493 种细菌属和780 种细菌种,其中奶酪中的优势细菌属和优势乳酸菌属为雷尔氏菌属和乳杆菌属,优势乳酸菌种为瑞士乳杆菌,且奶酪中还存在致病菌。在此基础上,利用传统培养法从MRS培养基中筛选出68 株可培养疑似乳酸菌,经生理生化实验及16S rDNA序列同源性分析技术对其进行属种鉴定。结果表明：68 株疑似乳酸菌分别为短乳杆菌、融合魏斯氏菌、屎肠球菌、粪肠球菌、耐久肠球菌、鸟肠球菌、棉籽糖肠球菌、植物乳杆菌、瑞士乳杆菌,其中融合魏斯氏菌为奶酪中的优势乳酸菌种。在乳酸菌的属种鉴定上,传统培养法能够更准确地将乳酸菌鉴定到种水平。奶酪微生物基因COG功能预测结果显示：奶酪中的微生物富含与氨基酸转运和代谢有关的基因。将各个样品的微生物群落组成与功能组成进行比较,发现各个样品的细菌群落组成虽存在明显差异,但其功能组成却具有高度相似性。     

青稞酒酒曲与奶酪中乳酸菌的筛选与应用

通过传统的分离、培养方法从西藏青稞酒酒曲和西藏、新疆奶酪中分别得到1株和3株乳酸菌,采用16S rRNA基因序列同源性分析的方法对菌株进行鉴定.研究进行了这4株乳酸菌发酵制备酸奶的比较实验,发现这4株乳酸菌均具备乳酸发酵制成酸奶的能力,而且制成的酸奶品质良好.表明从酒曲和奶酪中分离乳酸菌是筛选生产酸奶菌株的良好途径.丰富乳酸菌多样性,提供优良菌株,为酸奶行业的可持续发展提供保证.     

55株芽孢杆菌16S rRNA基因序列测定与系统发育学分析

采用16S rRNA基因序列分析法对中国工业微生物菌种保藏管理中心(CCIC)保藏的55株枯草芽孢杆菌(Bacillus subtilis)进行复核鉴定。菌株经纯化培养,以改良CTAB法提取总DNA,采用细菌16S rRNA通用引物、TD-PCR方法(touchdown-PCR)进行16S rRNA基因序列扩增,PCR产物纯化后直接进行序列测定,序列经人工校对后用Clustal X进行比对分析,最后用MEGA3.1软件构建系统发育树。系统发育分析结果表明:55株枯草芽孢杆菌中有52株菌种与原鉴定结果一致,有3株菌种与原鉴定结果存在差异,其中2株鉴定结果为巨大芽孢杆菌(B.megaterium),另1株鉴定结果为地衣芽孢杆菌(B.licheniformis)。     

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。     

Strain-level characterization of nonstarter lactic acid bacteria in Norvegia cheese by high-resolution melt analysis

The nonstarter lactic acid bacteria (NSLAB) constitute an important microbial group found during cheese ripening and they are thought to be fundamental to the quality of cheese. Rapid and accurate diagnostic tests for NSLAB are important for cheese quality control and in understanding the cheese ripening process. Here, we present a novel rapid approach for strain-level characterization through combined 16S rRNA gene and repetitive sequence-based high-resolution melt analysis (HRM). The approach was demonstrated through the characterization of 94 isolates from Norvegia, a Gouda-type cheese. The HRM profiles of the V1 and V3 variable regions of the 16S rRNA gene of the isolates were compared with the HRM profiles of 13 reference strains. The HRM profile comparison of the V1 and V3 regions of the 16S rRNA gene allowed discrimination of isolates and reference strains. Among the cheese isolates, Lactobacillus casei/paracasei (62 isolates) and Lactobacillus plantarum/Lactobacillus pentosus (27 isolates) were the dominant species, whereas Lactobacillus curvatus/Lactobacillus sakei were found occasionally (5 isolates). The HRM profiling of repetitive sequence-based PCR using the (GTG)(5) primer was developed for strain-level characterization. The clustering analysis of the HRM profiles showed high discriminatory power, similar to that of cluster analysis based on the gel method. In conclusion, the HRM approach in this study may be applied as a fast, accurate, and reproducible method for characterization of the NSLAB microflora in cheese and may be applicable to other microbial environments following selective plate culturing.     

Homofermentative lactic acid bacteria of a traditional cheese, Comlek peyniri from Cappadocia region

Comlek peyniri is a typical artisanal cheese in Central Anatolia. This type of cheese was made by using the indigenous lactic acid bacteria (LAB) flora of cow or ewes' milk. Majority of the samples were taken from fresh cheese because the aim was to isolate homofermentative LAB. Initially 661 microbial isolates were obtained from 17 cheese samples. Only 107 were found to be homofermentative LAB. These isolates were selected and identified by using both phenotypic and molecular methods. Phenotypic identification included curd formation from skim milk, catalase test, Gram staining and light microscopy, growth at different temperatures and salt concentrations, arginine hydrolysis, gas production from glucose, and carbohydrate fermentation. Molecular identification was based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S rRNA gene-ITS (internally transcribed spacer) region. By combining the phenotypic and molecular identification results, isolates belonging to each of the following genera were determined at species or subspecies level: 54 Lactococcus lactis subsp. lactis, 21 Enterococcus faecium, 3 Ec. faecalis, 2 Ec. durans, 10 Ec. sp., 15 Lactobacillus paracasei subsp. paracasei, and 2 Lb. casei strains. Technological characterisation was also performed by culturing each of the strains in UHT skim milk, and by monitoring pH change and lactic acid production at certain time intervals through the 24 h incubation. Results of the technological characterisation indicated that 33% of the isolates (35 strains) were capable of lowering the pH of UHT milk below 5.3 after 6 h incubation at 30 degrees C. Thirty four of these strains were Lc. lactis subsp. lactis, and only one was an Ec. faecium strain.     

Interlaboratory random amplified polymorphic DNA typing of Yersinia enterocolitica and Y. enterocolitica-like bacteria

A random amplified polymorphic DNA (RAPD) protocol was developed for interlaboratory use to discriminate food-borne Yersinia enterocolitica O:3 from other serogroups of Y. enterocolitica and from Y. enterocolitica-like species. Factors that were studied regarding the RAPD performance were choice of primers and concentration of PCR reagents (template DNA, MgCl(2), primer and Taq DNA polymerase). A factorial design experiment was performed to identify the optimal concentrations of the PCR reagents. The experiment showed that the concentration of the PCR reagents tested significantly affected the number of distinct RAPD products. The RAPD protocol developed was evaluated regarding its discrimination ability using 70 different Yersinia strains. Cluster analysis of the RAPD patterns obtained revealed three main groups representing (i) Y. pseudotuberculosis, (ii) Y. enterocolitica and (iii) Y. kristensenii, Y. frederiksenii, Y. intermedia and Y. ruckeri. Within the Y. enterocolitica group, the European serovar (O:3) and the North American serovar (O:8) could be clearly separated from each other. All Y. enterocolitica O:3 strains were found in one cluster which could be further divided into two subclusters, representing the geographical origin of the isolates. Thus, one of the subclusters contained Y. enterocolitica O:3 strains originating from Sweden, Finland and Norway, while Danish and English O:3 strains were found in another subcluster together with O:9 and O:5,27 strains. The repeatability (intralaboratory) and reproducibility (interlaboratory) of the RAPD protocol were tested using 15 Yersinia strains representing different RAPD patterns. The intralaboratory and the interlaboratory studies gave similarity coefficients of the same magnitude (generally >70%) for the individual strains. In the present study, it was shown that interreproducible RAPD results could be achieved by appropriate optimisation of the RAPD protocol. Furthermore, the study reflects the heterogeneous genetic diversity of the Y. enterocolitica species.     

Identification of yeasts associated with milk products using traditional and molecular techniques

An integrated approach including phenotypic (morphological, biochemical and physiological characterization) and genotypic (RAPD-PCR, sequencing of D1/D2 domain of 26S rRNA encoding gene) methods was used for the identification of yeasts isolated from different milk products. There were 513 isolates in all, 460 ascomycetous and 53 basidiomycetous yeasts. The yeast isolates were characterized on the basis of their biochemical and physiological properties, and the D1/D2 domain of 26S rDNA was sequenced in selected strains. Relying on the obtained results from both the data-sets, corresponding type strains were selected and compared with the respective yeast isolates from milk products by RAPD fingerprinting. The strains showing a degree of similarity >80% were considered conspecific. By means of the applied techniques it was possible to identify 92% yeast isolates at species level. Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Yarrowia lipolytica and Candida zeylanoides are the most frequently isolated species. The majority of the yeasts were isolated from fresh and sour curd cheese. A comparison of the results obtained by phenotypic and genotypic investigation revealed that the identification based on classical methods was supported by genotypic characterization in only 54% of examined isolates. The results described in this work show that the applied molecular identification is a reliable approach to the identification of yeasts associated with milk products in contrast to the conventional biochemical and physiological tests. The identification of new yeast species requires additional genetic markers such as sequencing of different genes or DNA:DNA hybridization.     

Isolation of lactic acid bacteria from Lighvan cheese, a semihard cheese made from raw sheep milk in Iran

The lactic acid bacteria contributing to Lighvan cheese ripening during the different stages of production were investigated. Isolated strains from different culture media were identified phenotypically to species and subspecies level. In total, 413 strains were isolated from raw milk, 1-day-old cheese and fully ripened cheese. The most abundant species belonged to Enterococcus faecium (87 isolates), Lactococcus lactis ssp. lactis (68 isolates), Enterococcus faecalis (55 isolates) and Lactobacillus plantarum (48 isolates). E. faecium, Lc. lactis and Lb. plantarum were the predominantly isolated strains from ripened cheese. Therefore, they may contribute considerably to the aroma and flavour development of Lighvan cheese.     

Biodiversity and origin of the microbial populations isolated from Masske,a traditional Iranian dairy product made from fermented Ewe's milk

Masske is a traditional Iranian dairy product containing 50% butterfat made from fermented ewe's milk. Overall, 672 bacterial isolates were collected from ewe's milk, fermented milk (FM) and Masske samples that were produced in households located in southern regions of the Khorasan Province in Iran. To identify lactic acid bacteria in these samples, a total of 79 Gram‐positive and catalase‐negative isolates were analysed. The identification of isolates was achieved by phenotypic and sequential analysis of the 16S rRNA gene. Enterococcus faecium and Aerococcus viridans were the most frequently isolated species in the samples, but the most commonly present bacteria in Masske were Streptococcus thermophilus.     

RAPD-PCR快速鉴定啤酒污染菌的研究

本研究主要评价RAPD-PCR技术在快速鉴定啤酒污染菌中的应用。首先评价了CTAB法和试剂盒提取DNA模板对RAPD指纹稳定性的影响以及乳酸菌专一性BP引物扩增的16SrDNA的5’末端740bp序列用于鉴定菌种的可行性,采用PCR产物直接测序鉴定分离的污染菌,构建标准污染菌库。对分离菌M-13的RAPD指纹聚类分析表明,相同来源的同一种菌能很好地归类在一起。根据建立的快速鉴定流程和初步确定的菌种相似性阈值(SCC),对8株分离菌的鉴定表明,RAPD-PCR技术是一项简单、快捷、可靠性强的鉴定技术,为研究啤酒酿造过程的污染菌提供了一个非常有用的快速鉴定工具。     

Evaluation of the ability of Yarrowia lipolytica to impart strain-dependent characteristics to cheese when used as a ripening adjunct

Four Yarrowia lipolytica strains were tested as cheese-ripening adjuncts with milk culture in cheese production to evaluate their effects on the microbiological and biochemical features of the cheeses. The Y. lipolytica strains were able to overcome the other naturally occurring yeasts and were compatible with lactic acid bacteria (LAB). Fourier transform infrared (FTIR) profiles and analysis of free fatty acids (FFAs) released during ripening showed that the strains induced a marked lipolysis and gave rise to different FFAs accumulation over time with respect to the control. Strain-dependent protein breakdown patterns were identified and these biochemical differences resulted in different cheese organoleptic characteristics.