皖北地区白酒大曲中酵母菌的筛选与鉴定

为了分离筛选功能优良的大曲酵母菌株,采用传统分离培养方法,从皖北某白酒企业春季中温大曲中分离得到89株酵母菌,选择6株优势菌株进行复筛实验。通过菌落特征、细胞形态、生理生化特性等常规分析方法,筛选出生长优势明显,产酒精能力较强的2株酵母菌株(N4、N6)。结合18S-ITS r DNA序列测定和系统发育分析,确定N4为大隐球酵母(Cryptococcus magnus),N6为近玫色锁掷孢酵母(Sporidiobolus pararoseus),属于锁掷酵母属(Sporidiobolus)。筛选的2株酵母菌作为皖北地区白酒酵母菌的重要菌种资源,在白酒生产中具有一定的应用潜力。     

甘蔗内生酵母菌的分离鉴定及发酵性能

为研究甘蔗内生酵母菌的种类并筛选出发酵性能良好的酵母菌株,该文采用组织分离法对甘蔗中的内生酵母菌进行分离,通过形态学、生理生化以及26S rDNA D1/D2区序列分析法进行鉴定。利用杜氏管发酵法初筛产气性能较好的菌株,确定筛选菌株的最适生长条件,然后采用摇瓶发酵法对其进行耐糖、耐盐以及耐酒精等耐受性试验,复筛出发酵性能优良的菌株。结果表明,从甘蔗中共分离得到内生酵母菌63株,鉴定为8种：葡萄有孢汉逊酵母(Hanseniaspora uvarum)、季也蒙毕赤酵母(Meyerozyma guiliermondii)、汉逊德巴利酵母(Debaryomyces hansenii)、嗜油假丝酵母(Candida oleophila)、类筒假丝酵母(Candida zeylanoides)、异常威克汉姆酵母(Wickerhamomyces anomalus)、Cystobasidium minutum以及Papiliotrema terrestris。从63株酵母菌中,筛选一株生长性能和发酵性能优良的菌株J-6,在温度26℃、pH5.0的条件下,24 h时生长速率达到最大值,其最大值为1.99...     

树莓酒自然发酵过程中酵母菌的分离与鉴定

目的:从树莓酒自然发酵过程中分离和鉴定酵母菌,目的是筛选适宜树莓酒发酵特点的专用酵母菌株。方法:采用传统微生物分离纯化技术与和26S r DNA D1/D2区序列分析相结合的方法,从树莓酒自然发酵的不同时期分离纯化酵母菌,结合菌落和细胞显微形态特征进行区分,随机选择代表菌株进行26S r DNA D1/D2区序列分析。结果:从树莓酒自然发酵过程中共分离出323株酵母菌,形态学初步鉴定为12类,分子鉴定为分属于5个属的7种酵母菌,分别为葡萄汁有孢汉逊酵母(Hanseniaspora uvarum)、东方伊萨酵母(Issatchenkia terricola)、酿酒酵母(Saccharomyces cerevisiae)、覆膜孢酵母属亚种(Saccharomycopsis crataegensis)、叉开假丝酵母(Candida diversa)、东方拟无枝酸菌(Candida sorboxylosa)和假丝酵母属亚种(Candida stellimalicola)。结论:传统的微生物培养形态区分与分子鉴定技术结合,对筛选树莓酒自然发酵过程中的酵母菌准确有效。     

天山一号冰川产脂肪酶酵母菌的筛选及其系统发育分析

通过对天山一号冰川底部沉积层耐低温酵母菌的分离和其中产脂肪酶酵母菌株的筛选,了解冰川微生物生理多样性和系统发育多样性,为低温脂肪酶生物技术的研发奠定基础。采用RB琼脂平板涂布分离可培养酵母菌,通过橄榄油选择培养基和吐温80选择培养基筛选产脂肪酶的耐低温菌株。对分离菌株表型特征、最适生长温度进行比较,结合26S r DNA基因序列同源性分析确定产脂肪酶菌株的多样性和系统进化地位。从57株分离物中筛选到12株产脂肪酶的耐低温酵母菌株,其中7株属于隐球菌属(Cryptococcus),2株属于掷孢酵母属(Sporobolomyces),2株属于红酵母属,1株属于Mrakiellaspp。天山一号冰川冻土中产脂肪酶的耐低温酵母菌多样性较丰富,依据其生长温度的范围,均属于耐冷菌。     

橙汁腐败酵母菌的分子鉴定及其形态特征分析

目的：结合多种酵母菌鉴定方法,分析腐败橙汁中酵母菌的种类,为快速检测和控制商品橙汁中酵母菌污染奠定基础,也为酵母菌种类的快速鉴定提供参考。方法：以分离自腐败橙汁的8株酵母菌株为材料,观察菌落和细胞形态。用引物ITS1/ITS4扩增菌株5.8S rDNA 及其ITS间隔区(5.8S-ITS),对扩增产物用HhaⅠ、Hae Ⅲ 和 HinfⅠ进行限制性酶切片段多态性(RFLP)分析和测序;用引物NL1/NL4扩增26S rDNA D1/D2区并测序。结果：菌落特征观察将菌株初分为6组,细胞学观察粗分为3组,分子方法都分成4组。用限制性内切酶HhaⅠ、Hae Ⅲ和HinfⅠ对5.8S-ITS区产物进行 RFLP分析,观察到4种不同的图谱类型。5.8S-ITS 区和26S rDNA D1/D2区的序列分析结果相似,8株分离菌与GenBank中的4种酵母菌参考菌株序列一致性达99%以上。分离株与参考菌株以两区序列构建的NJ系统树都分成4枝：Y1与克鲁维毕赤酵母(Pichia kluyveri),Y12-3、Y18-1与发酵毕赤酵母(Pichia fermentans),Y22、Y23、Y26和Y56与Meyerozyma guilliermondii,Y47与Wickerhamomyces anomalus分别聚为一枝。结论：结合形态分析和核酸分析,将8株腐败橙汁分离菌鉴定为P. kluyveri var. kluyveri、P. fermentans、M. guilliermondii和W. anomalus 4个种,其中M. guilliermondii在腐败橙汁中首见报道;ITS- RFLP分析、26S rDNA D1/D2区测序与菌株形态特征结合能有效鉴定酵母菌,核糖体DNA分析可鉴定酵母到生物学种,菌落形态特征可反映种以下的遗传差异,因此,采用分子方法鉴定时不能忽视形态特征分析的重要性。     

果汁饮料腐败酵母菌——出芽短梗霉的分离与鉴定

目的:从腐败果汁饮料中分离得到一株黑酵母,对其进行特性研究,为果汁饮料中腐败酵母菌的控制提供参考。方法:以NL1和NL4为引物,对分离到的酵母菌菌株3814B基因组进行26S rDNA D1/D2区域序列扩增。结论:菌株3814B具有耐高渗性,能够在50%葡萄糖琼脂上生长;酵母菌的菌落形态随培养时间延长发生变化,菌落发育形态和菌体细胞特征对菌株鉴定具有直观、简洁的优点;PCR扩增后的基因碱基序列经Gene Bank公布的同源基因碱基序列比对,把分离到的菌株3814B鉴定为出芽短梗霉(Aureobasidium pullulans)。结论:分子鉴定和形态学鉴定相结合对于果汁中腐败酵母的鉴定更为准确有效。     

宁夏御马葡萄酒厂野生酵母菌株的分离筛选及分子鉴定

利用26S rDNA D1/D2区序列分析法,对分离自宁夏御马葡萄园的22株野生酵母菌进行了鉴定,同时构建了22株供试菌株与相关模式菌株的系统发育树,分析了供试菌株与已知酵母菌的亲缘关系及其分类地位.结果表明:供试菌株被鉴定为5属7种,分别是酿酒酵母、巴氏酵母、葡萄汁有孢汉生酵母、克鲁维毕赤酵母、美极梅奇酵母和核果梅奇酵母.     

新疆石河子地区酵母菌的生物多样性及系统发育研究

通过对石河子地区酵母菌的筛选,了解石河子地区酵母菌的生物多样性。采用YPD培养基分离纯化菌株,通过26SrDNA基因D1／D2区序列分析确定菌种的系统进化地位。分离筛选出6个属天然酵母菌,分别为：假丝酵母属（Candida）、Meyerozyma属、红酵母属（Rhodotorula）、孢汉逊属（Hanseniaspora）、酿酒酵母（Saccharomycescerevisiae）和毕赤酵母属（Pichia）,共10个种,其中包括6个疑似种。     

乳扇中高产胞外多糖酵母菌的筛选及其抗氧化活性

为获得高产胞外多糖的酵母菌株,以云南地区发酵食品乳扇为原料,采用平板分离结合硫酸-苯酚测定法筛选目标酵母菌,并通过形态学、生理生化及分子生物学方法进行菌株鉴定,最终从乳扇中初步分离筛选出9 株产胞外多糖酵母菌,包括3 株胶红酵母（Rhodotorula mucilaginosa）、1 株白假丝酵母（Candida albicans）、4 株涎沫假丝酵母（Candida zeylanoides）及1 株金黄蝶形担孢酵母（Papiliotrema aurea）。其中筛选得到的金黄蝶形担孢酵母DF-12能较好利用葡萄糖、蔗糖及糖蜜等碳源高效合成胞外多糖,在发酵168 h后胞外多糖产量可达3 510 mg/L,具有较好的工业生产潜力。体外抗氧化能力实验表明DF-12菌株所产胞外多糖有一定清除1,1-二苯基-2-三硝基苯肼自由基和超氧阴离子自由基的能力。本研究为酵母菌胞外多糖在食品、药品领域的应用研究提供了一定的基础。     

酒曲中酵母菌的分离鉴定及果酒发酵的初步研究

酵母菌是决定果酒品质的关键因素。采用稀释涂布平板法分离纯化酒曲中的菌株,以传统形态学及分子系统发育分析对酵母菌进行初步鉴定,从6种市售酒曲样品中分离出10株酵母菌,经 26S rDNA 序列分析,有3株为库德里亚夫毕赤酵母(Pichia kudriavzevii),1株为异常毕赤酵母(Pichia anomala),2株克鲁维酵母(Kluyveromyces marxianus),3株酿酒酵母(Saccharomyces cerevisiae),1株葡萄牙棒孢酵母(Clavispora lusitaniae)。通过初筛将筛选出的酵母菌CK-03用于猕猴桃汁发酵,发酵液经顶空固相微萃取-气质联用技术(solid phase microextractiongas chromatography/mass spectrometry,SPME-GC/MS)测定分析,结果显示共检测出25种挥发性成分,主要为酯类和醇类物质。     

仰韶陶融型白酒大曲可培养微生物多样性研究

研究陶融型白酒大曲可培养微生物的多样性。采用组织培养方法分离陶融型白酒大曲的微生物,提取分离菌株DNA,用酵母通用引物NL1/NL4、细菌通用引物799F/1492R和真菌通用引物ITS1/ITS4对酵母26S rRNA、细菌16S rRNA、霉菌28S rRNA序列扩增,提交测序分析,构建系统发育树。仰韶陶融型白酒大曲酒曲中共分离到酵母213株、细菌386株、霉菌73株,酵母归分为72株形态种,细菌归分为77株形态种,霉菌归分为7株形态种,根据测序结果不同,陶融型白酒大曲微生物被分为21个分类单元。仰韶陶融型白酒大曲可培养微生物具有多样性特征,为其发酵及开发利用提供理论参考。     

贵州传统小曲酵母菌分子指纹图谱分析

采用26S rRNA基因D1/D2区域序列分析方法将59株分离自贵州5个不同地区传统小曲的酵母菌鉴定为6个酵母菌种：1株阿萨丝孢酵母（Trichosporon asahii）、32株扣囊覆膜孢酵母（Saccharomycopsis fibuligera）、8株Saccharomycopsismalanga、4株伯顿丝孢毕赤酵母（Hyphopichia burtonii）、6株异常威克汉姆酵母（Wickerhamomyces anomalus）和8株酿酒酵母（Saccharomyces cerevisiae）。本研究展示了6个酵母菌种的WL培养基菌落特征和5.8S rRNA基因ITS区限制性片段长度多态性酶切图谱特征，为这6个菌种的快速鉴定提供依据。在菌种鉴定的基础上，本研究进一步采用串联重复-tRNA指纹图谱法分析酵母菌的种内差异，共展示了17个基因型，H. burtonii、W. anomalus与S. fibuligera分别鉴定出4、3种和8种基因型，其中S. fibuligera基因型10、11、12、15、16与17之间的遗传关系较近，而其余菌种内部基因型之间的遗传关系相对较...     

橙汁中两株条件致病酵母的形态及分子鉴定

目的：为建立更加完善的橙汁酵母菌检测体系,进一步精确监控市售橙汁质量,对市售橙汁中分离的两株酵母菌Y25、Y32进行菌落动态发育和细胞特征的观察。方法：以NL1和NL4为引物,对分离到的两株酵母菌基因组进行26S rDNA D1/D2区域序列扩增。结果：酵母菌的菌落形态随培养时间延长发生变化,菌落发育形态和菌体细胞特征对菌株鉴定具有直观、简洁的优点;PCR扩增后的基因碱基序列经GeneBank公布的同源基因碱基序列比对后,相似度在99%以上。结论：把分离到的Y25、Y32鉴定为汉逊德巴利酵母(Debaryomyces hansenii)和近平滑假丝酵母(Candida parapsilosis)。形态学与分子技术的结合使鉴定结果更为准确可靠,将为橙汁中其他腐败微生物的鉴定与监控提供有价值的借鉴。     

冬季甜瓜腐烂病原菌的分离与鉴定

从冬季贮存甜瓜的腐烂组织中分离病原菌,并对其类型进行检测分析。利用马铃薯葡萄糖琼脂(PDA)培养基和溶菌肉汤(LB)培养基培养,依据微生物形态特性,真菌的26S rRNA 序列和细菌的16S rRNA 序列比对以及系统发育进化分析等分子生物学方法,共分离到22 株菌种,其中真菌13 株：包括青霉2 株、链格孢4 株、白地霉4 株、酵母3 株(梅奇氏酵母2 株、毕赤酵母1 株);细菌9 株：包括沙雷氏菌5 株,丁香菌1 株,克雷伯菌、肠杆菌和芽孢杆菌各1 株。根据26S rRNA D1/D2 区序列比对和系统进化树结果显示,两株酵母菌121 和122 可能是梅奇氏酵母属潜在的新种。青霉和链铬孢是甜瓜腐烂致病菌,白地霉和沙雷氏菌是人体致病菌,表明目前甜瓜贮存方法需要进一步改进和完善。     

Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis

Samples of Coffea arabica were collected during the different stages of the fermentation from two production sites in Tanzania. The yeasts community was identified by genotyping using ITS-PCR and sequence analysis of the D1/D2 domain of the 26S rRNA gene. For confirmation, denaturating gradient gel electrophoresis (DGGE) of PCR-amplified 26S rRNA gene was performed to detect yeast directly from coffee samples without cultivation. Yeast counts were in the range 4.0 x 10(4) - 5.0 x 10(7) CFU/g with an increase during fermentation. Three yeasts species were dominant. The predominant yeast found during fermentation and drying was Pichia kluyveri. Pichia anomala was found in high numbers during drying of coffee beans. Hanseniaspora uvarum was the predominant yeast during fermentation but decreased during drying. Kluyveromyces marxianus, Candida pseudointermedia, Issatchenkia orientalis, Pichia ohmeri and Torulaspora delbrueckii occurred in concentrations of 10(3) CFU/g or below in coffee samples. Saccharomyces cerevisiae and Candida xestobii were not isolated by cultivation, but by the DGGE technique. A good agreement was found between the sequence analysis of the D1/D2 domain of the 26S rRNA gene and sequencing of the DGGE bands.     

Yeast diversity in crop-growing environments in Cameroon

In the present study, we have investigated the occurrence of yeast flora on several agricultural products coming from crop-growing environments in Cameroon, to provide better knowledge of the biodiversity of yeast flora, and to thus define the impact of this biodiversity on food products. The yeast biodiversity was investigated using traditional culture-dependent methods, along with culture-independent methods. The culture-dependent approach was carried out using both direct and enrichment procedures, to detect the broadest possible presence of yeast species. A total of 151 strains belonging to 26 different yeast species were isolated and identified using restriction pattern analysis of the internal transcribed spacer region 5.8S-ITS and sequence analysis of D1/D2 domain of 26S rRNA gene. The enrichment isolation procedures carried out in high-sugar media allowed the recognition of fermentative species such as Saccharomyces cerevisiae and Torulaspora delbrueckii, which have previously not been detected using direct isolation methodology. The results of culture-independent method using DGGE patterns and sequencing of the DNA bands revealed a lower number of yeast species when compared with the culture-dependent methodology even if the identification of several yeast species not detected by traditional microbiological procedures such as Candida tropicalis and Hanseniaspora uvarum is allowed. Thus, these multiphasic approaches to study yeast biodiversity (culture-dependent and -independent methods) have allowed us to get a more complete picture of the microbial diversity in these natural environments.     

天山一号冰川产果胶酶酵母菌的筛选及其系统发育分析

从前期天山冰川冻土及融水中分离到68株可培养酵母菌中,通过筛选性培养基筛选产低温果胶酶的菌株。选择培养基显示菌株C2、C9、L1和L13可产低温果胶酶。对4株酵母菌最适生长温度进行测定,16 ℃条件下4株酵母菌均能生长,属耐冷菌范畴。通过26S rDNA基因序列分析确定产果胶酶菌种的系统进化地位,在系统发育上,4株产酶菌分别与Cryptococcus macerans DQ377662、Cryptococcus sp. DQ377668、Rhodotorula laryngis JQ768911、Rhodotorula sp. JX124722的同源性最高。     

Molecular Identification of Yeasts Associated with Traditional Egyptian Dairy Products

ABSTRACT:  This study aimed to examine the diversity and ecology of yeasts associated with traditional Egyptian dairy products employing molecular techniques in yeast identification. A total of 120 samples of fresh and stored Domiati cheese, kariesh cheese, and "Matared" cream were collected from local markets and examined. Forty yeast isolates were cultured from these samples and identified using the restriction-fragment length polymorphism (RFLPs) of 5.8S-ITS rDNA region and sequencing of the domains D1 and D2 of the 26S rRNA gene. Yeasts were identified as  Issatchenkia orientalis  (13 isolates),  Candida albicans  (4 isolates),  Clavispora lusitaniae  ( Candida lusitaniae ) (9 isolates),  Kodamaea ohmeri  ( Pichia ohmeri ) (1 isolate),  Kluyveromyces marxianus  (6 isolates), and  Candida catenulata  (7 isolates). With the exception of  C. lusitaniae , the D1/D2 26S rRNA gene sequences were 100% identical for the yeast isolates within the same species. Phylogenetic reconstruction of  C. lusitaniae  isolates grouped them into 3 distinguished clusters. Kariesh cheese was found to be the most diverse in its yeast floras and contained the highest total yeast count compared with other examined dairy products. This was linked to the acidic pH and lower salt content of this cheese, which favor the growth and survival of yeasts in foodstuffs. Stored Domiati cheese also contained diverse yeast species involving isolates of the pathogenic yeast  C. albicans . This raises the possibility of dairy products being vehicles of transmission of pathogenic yeasts.