Phenotypic and genotypic identification of yeasts from cheese

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.     

Yeast species associated with spontaneous wine fermentation of Cabernet Sauvignon from Ningxia,China

The eastern base of the Helan Mountains in Ningxia is a fast developing wine production area in China. Of urgent necessity to the Ningxia wine industry is to be able to produce wines with typical regional characteristics. It is well known that autochthonous yeast species and strains play an important role in introducing local character or terroir into the winemaking practice. The aim of this study was to investigate indigenous yeast species diversity and preselect desirable S. cerevisiae strains in the Ningxia region. Four hundred wine-related yeast colonies were isolated from Cabernet Sauvignon musts in three vineyards at the beginning, middle and final stages of spontaneous fermentations. Yeast species were first classified according to colony morphologies on Wallerstein Laboratory Nutrient Agar (WL) and confirmed by sequencing of the D1/D2 domain of the 26S rRNA gene. Nine unique colony morphology types were profiled on WL agar and three new types were found. After sequence analysis of representative colonies from each WL group, nine yeast species were identified, namely H. uvarum, H. occidentalis, M. pulcherrima, C. zemplinina, H. vineae, I. orientalis, Z. bailii, P. kluyveri and S. cerevisiae. The non-Saccharomyces yeasts appeared mainly in the early stage of the fermentation while H. uvarum, I. orientalis and P. kluyveri were able to remain in the final stage. Fourty-six S. cerevisiae isolates were preselected according to physiological characteristics and twelve strains with valuable fermentation properties were obtained.     

Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees

Three endophytic yeast, one isolated from stems of wild cottonwood (Populus trichocarpa), two from stems of hybrid poplar (P. trichocarpa × Populus deltoides), were characterized by analyzing three ribosomal genes, the small subunit (18S), internal transcribed spacer (ITS), and D1/D2 region of the large subunit (26S). Phenotypic characteristics of the yeast isolates were also obtained using a commercial yeast identification kit and used for assisting the species identification. The isolate from wild cottonwood was identified to be closest to species Rhodotorula graminis. The two isolates from hybrid poplar were identified to be species Rhodotorula mucilaginosa. In addition, the three yeast isolates were observed to be able to produce indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. To our knowledge, the yeast strains presented in this study were the first endophytic yeast strains isolated from species of Populus.     

Distribution of Ochratoxin A producing strains in the A. niger aggregate

Ochratoxin A (OA) production of 92 isolates belonging to the A. niger aggregate was tested. All these isolates were grouped into the two proposed species A. niger and A. tubingensis, according to their ITS-5.8S rDNA RFLP patterns. The distribution of the isolates into the two species was very similar since 52.2% were classified as pattern T (corresponding to A. tubingensis), and 47.8% were classified as pattern N (corresponding to A. niger). Six out of the 92 isolates studied produced OA. All the OA producing strains were classified as pattern N while none of the isolates classified as pattern T produced OA.     

Yeast species associated with orange juice: evaluation of different identification methods

Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.     

Molecular characterization of carotenogenic yeasts from aquatic environments in Patagonia, Argentina

Fifteen aquatic environments (lakes, lagoons and rivers) of glacial origin in the northern Andean Patagonia (Argentina) were surveyed for the occurrence of red yeasts. Subsurface water samples were filtered and used for colony counting and yeast isolation. A preliminary quantitative analysis indicated that total yeast counts ranged between 0 and 250 cells l⁻¹. A polyphasic approach including physiological and molecular methods was used for the identification of 64 carotenogenic yeast strains. The molecular characterisation of the isolates was based on the mini/microsatellite-primed PCR technique (MSP-PCR) employing the (GTG)₅ and the M13 primers. Comparison of representative fingerprints of each group with those of the type strains of pigmented yeasts allowed the expeditious identification of 87.5% isolates. The sequence analysis of the D1/D2 domains of the 26S rDNA was employed to confirm identifications and in the characterization of the unidentified MSP-PCR groups. Teleomorphic yeast species were detected by performing sexual compatibility assays. The isolates corresponded to 6 genera and 15 yeast species, including four new yeast species of the genera Cryptococcus (1), Rhodotorula (1) and Sporobolomyces (2). Rhodotorula mucilaginosa was found in the majority of the samples and represented ca. 50% of the total number of isolates. However, this yeast was not detected in aquatic environments with very low anthropic influence. Other frequent yeast isolates were teleomorphic yeast species of Rhodosporidium babjevae, R. kratochvilovae and Sporidiobolus salmonicolor. This study represents the first report on red yeast occurrence and biodiversity in northwestern Patagonia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular assessment of indigenous yeast population from traditional balsamic vinegar

AIMS: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. METHODS AND RESULTS: Using the restriction analysis of the ribosomal region 5.8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5.8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. CONCLUSIONS: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.     

云南程海湖酵母菌多样性及应用

【目的】针对云南丽江永胜县境内程海湖环境的特殊性,研究高原湖泊环境中酵母菌的多样性,初步探索程海湖环境中酵母菌的利用价值。【方法】对程海湖的湖水和其周边土壤样品中的酵母菌进行分离;应用26S rDNA的D1/D2区域序列分析,并结合形态及生理生化指标对分离获得的酵母菌进行鉴定;采用筛选培养基对已鉴定酵母菌进行产酶定性实验,分析高原湖泊中酵母菌的多样性及可应用性。【结果】分离得到酵母菌64株,对其中63株进行鉴定,归属于9个属22个种(包括4个疑似新种或新变种);地霉属Geotrichum和隐球酵母属Cryptococcus是2种环境中的共有属;在产酶活性筛选中发现有9株产胞外酶活性的菌株,其中YM24373既产蛋白酶又可产淀粉酶。【结论】研究结果显示程海湖中酵母菌组成具有较为丰富的多样性,其应用价值值得进一步研究。     

白木香内生真菌的分离鉴定及其抑菌活性

从白木香Aquilaria sinensis（Lour．）Gilg木质部的树脂形成部位和健康部位中共分离获得42株内生真菌,经初步鉴定,产孢的33株分属于3目4科7属,其余未产孢的9株暂归为无孢菌群。采用杯碟法和MTT法分别测定了各菌株的发酵上清液对3种病原菌的体外抑菌活性和2种肿瘤细胞的体外细胞毒活性。结果表明,白木香木质部健康部位内生真菌以枝顶孢霉属为优势属,而树脂形成部位的内生真菌种类比健康部位要多,且以青霉属为优势属。其中26株至少能抑制一种指示菌,占总数的61．9％;7株对指示瘤株具有细胞毒活性,占总数的16．7％。抑菌活性菌株主要分布在枝顶孢霉属和青霉属。枝顶孢霉属菌株抑菌活性较强,其抗菌活性成分值得进一步研究。     

内蒙古西部区酸粥中酵母菌的分离鉴定及优势菌分析

从内蒙古地区采集28份酸粥样品,从中分离出40株酵母菌,并对其进行了分子生物学鉴定和生物多样性分析。26S rDNA D1/D2区域 (600bp左右)碱基序列分析结果表明,酸粥中的酵母菌有Issatchenkia orientalis、Saccharomyces cerevisiae、Geotrichum sp.、Candida pararugosa、 Candida parapsilosis、Trichosporon asahii、Trichosporon coremiiforme、Clavispora lusitaniae和Candida tropicalis。经过分析,Issatchenkia orientalis(75%,Frequency percentage)为酸粥中的优势菌。     

Thermotolerant varieties of Bacillus licheniformis isolated from desert environments

One hundred and nineteen thermotolerant and thermophilic Bacillus strains isolated from solar-heated and non-heated environments in Jordan were classified by numerical techniques. Some strains were classified into thermophilic taxa which did not equate with established species. However, most of the isolates were identified phenotypically as Bacillus licheniformis, a conclusion supported by the high DNA hybridization which was detected between these strains and a reference strain of this species (gt64% at optimal renaturation temperature). Several of the B. licheniformis isolates had a higher ratio of iso-C₁₅ and iso-C₁₇ fatty acids to the anteiso equivalents in their membranes than the reference strain of B. licheniformis and they grew more strongly at high temperature than the reference strain. This suggests that the B. licheniformis isolates represent thermotolerant variants of this species.     

Isoenzyme patterns: a valuable molecular tool for the differentiation of Zygosaccharomyces species and detection of misidentified isolates

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species.     

西藏曲拉和云南乳饼中酵母菌的鉴定及其生物多样性

【目的】探讨西藏曲拉和云南乳饼中酵母菌的生物多样性及其分布特征,为我国传统乳制品中酵母菌资源的利用提供基础数据。【方法】从西藏和云南分别采集的5份曲拉样品和8份乳饼样品中分离出41株酵母菌,利用26SrDNAD1/D2区域序列分析对这些菌株进行了分类鉴定。【结果】曲拉和乳饼样品中酵母菌的总数分别在106-107cfu/g和102-106cfu/g之间,曲拉样品的酵母菌平均数比乳饼样品中的高34倍。共鉴定出10属12种,其中西藏曲拉的优势菌株为发酵毕赤氏酵母(Pichia fermentans)和酿酒酵母(Saccharomyces cerevisiae);云南乳饼的优势菌株为类筒假丝酵母(Candida zeylanoides)和喜仙人掌毕赤氏酵母(Pichia cactophila)。毕赤氏酵母属(Pichia)是曲拉和乳饼的共同优势属。【结论】西藏曲拉和云南乳饼中的酵母菌都具有丰富的生物多样性,但其差异性很大。     

A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.     

红富士苹果园酵母多样性研究

从北京顺义和山东泰安红富士苹果园采集果实、叶片、树皮和土壤等不同基物,分离酵母菌,利用26S rDNA的D1/D2区域序列分析并结合形态学特征和SSCP分析对这些菌株进行了分类学研究,探讨了苹果园酵母的物种多样性及其分布。北京苹果园共分离酵母菌129株,鉴定为13属21种,优势属为Pichia（4个种）,Cryptococcus（3个种）,Pseudozyma（3个种）,子囊菌占较大优势,分布于8属12种,占总种数的57.1%。山东苹果园共分离酵母291株,鉴定为13属26种,优势属为假丝酵母Candida（6个种）,毕赤酵母Pichia（4个种）和隐球酵母Cryptococcus（3个种）,并且子囊菌占较大优势,分布于7属17种,占总种数的65.4%。     

Taxonomic and phenotypic characterization of yeasts isolated from worldwide cold rock-associated habitats

Yeast strains isolated from rock samples collected from worldwide cold regions were identified by sequence analysis of the D1/D2 domains of the 26S rDNA gene and the ITS region followed by molecular phylogeny. Over 77 % of yeasts isolates were Basidiomycota. Cryptococcus (orders Filobasidiales and Tremellales) and Rhodotorula (order Cystobasidiales) were the most frequent genera. About 40 % of yeast isolates belonged to undescribed species.     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.     

Ascomycetous yeasts from tropical intertidal dark mud of southeast Brazilian estuaries

Four different intertidal estuarine sediments had distinct yeast communities. One-hundred-ninety-three yeast isolates were classified in 47 species, with 34 of these in the genus Candida. Candida tropicalis was the only ascomycetous species isolated from all four sites. Other opportunistic pathogens including Candida glabrata, Candida guilliermondii, Candida parapsilosis and Candida krusei were present, especially at the more polluted sites. Pichia species were also frequent isolates with Pichia membranaefaciens, and its anamorph, Candida valida, and other phenotypically similar low assimilation profile species the most frequent. Kluyveromyces aestuarii was prevalent at the only site with well established mangrove vegetation, but not present at the other sites. The sediment yeast communities were distinct from each other, but more similar to each other than to the yeast communities of other ecosystems in the same geographic region.     

Characterization of the yeast flora present in some Turkish high-sugar products

In this study, 129 Turkish high-sugar products were examined in terms of their yeast flora and 73 representative strains were isolated. Yeast isolates were identified at species level by using Apilab Plus (bioMérieux, France), a specific computer program developed for ID 32C strips (bioMérieux, France). While one of the isolates could be identified at genus level as Aureobasidium, 66 of them were identified as 21 species belonging to 8 different genera. The distribution of these isolates were as follows: Candida (38), Rhodotorula (8), Zygosaccharomyces (7), Cryptococcus (6), Saccharomyces (3), Debaryomyces (2), Pichia (1) and Torulaspora (1). Approximately 70% of the isolates were found to have the ability to grow on media with 50% (w/w) glucose. Hence, they were characterized as xerotolerant strains. Although Zygosaccharomyces rouxii is known as the most xerotolerant yeast species, only two strains of Z. rouxii could be isolated from Turkish high-sugar foods. During identification studies, it was observed that ID 32C test strips should certainly be supported by morphological and physiological tests for obtaining more reliable identification results. If not, closely related yeast species such as anamorph and telemorph forms can not be distinguished.