葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株,利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究,探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布.共鉴定出13属26种,其中优势属为汉逊酵母属Hanseniaspora(5种),假丝酵母属Candida(4种),毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种).对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较,以探讨不同地理起源地酵母种内序列稳定性及其变异.     

葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株, 利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究, 探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布。共鉴定出13属26种, 其中优势属为汉逊酵母属Hanseniaspora(5种), 假丝酵母属Candida(4种), 毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种)。对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较, 以探讨不同地理起源地酵母种内序列稳定性及其变异。     

田间施药对自然发酵葡萄酒酵母菌群落结构的影响

【背景】酵母菌是葡萄酒发酵过程中一类非常重要的微生物,其多样性及群体组成对葡萄酒的质量有重要贡献。影响葡萄酒中酵母菌组成的因素有很多,但目前尚未见葡萄园田管理对葡萄酒酵母菌群落结构影响方面的报道。【目的】探索田间施药对自然发酵葡萄酒酵母菌群落结构的影响。【方法】采用分离培养、常规分子生物学鉴定和Illumina MiSeq宏基因组测序结合的方法分析不同样品中的酵母菌群落结构情况。【结果】从不使用内吸收型化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Saccharomyces、Zygoascus、Issatchenkia等7个属8个种的酵母菌,宏基因组测序结果表明有Pichia(29.42%)、Saccharomyces(21.91%)、Issatchenkia(17.99%)、 Hanseniaspora(12.10%)、 Candida(7.47%)、 Zygosaccharomyces(5.32%)、Schizosaccharomyces (3.07%)、Aureobasidium (0.29%)等属的酵母菌参与发酵;使用常规化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Cryptococcus等5个属6个种的酵母菌,宏基因组测序结果表明有Pichia (41.66%)、Hanseniaspora (21.54%)、Candida(19.11%)、 Zygosaccharomyces(7.78%)、 Schizosaccharomyces(4.04%)、 Cryptococcus(3.21%)、Saccharomyces (1.12%)、Aureobasidium (0.49%)等属的酵母菌参与发酵。【结论】两样品中酵母菌比例有显著差异,表明在酿酒葡萄的园田管理中化学农药的使用对自然发酵葡萄酒的酵母菌群落结构有较大影响。     

Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains

Mixed inoculation of Saccharomyces cerevisiae strains is used in winemaking for achieving high sensory quality of the wine. However, information on the diversity and population of yeasts during inoculated fermentation is very limited. In this study, we evaluated the effect of mixed inocula with different inoculation timing on the yeast community during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inoculation of both strains. Wallersterin Laboratory Nutrient (WLN) medium and sequence of the 26S rDNA D1/D2 domain were used to compare the diversity of yeast species. Five species, including Candida diversa, Hanseniaspora opuntiae, H. uvarum, Issatchenkia orientalis and I. terricola, were identified in the grape must, with Issatchenkia sp. being predominant (67.5 %). Three to four species were involved in each fermentation treatment. The fermentations by mixed inocula presented more yeast species than by pure inocula. Interdelta sequence typing was used to identify S. cerevisiae strains. Ten genotypes were identified among 322 isolated S. cerevisiae strains. Their distribution varied among different stages of fermentations and different inoculation treatments. The inoculated strains were not predominant, while indigenous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided information on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.     

Identification of indigenous yeast flora isolated from the five winegrape varieties harvested in Xiangning,China

Inoculated fermentation by selected indigenous yeast strains from a specific location could provide the wine with unique regional sensory characteristics. The identification and differentiation of local yeasts are the first step to understand the function of yeasts and develop a better strain-selection program for winemaking. The indigenous yeasts in five grape varieties, Chardonnay, Cabernet Franc, Cabernet Sauvignon, Marselan, and Merlot cultivated in Xiangning, Shanxi, China were investigated. Eight species of seven genera including Aureobasidium pullulans, Candida zemplinina, Hanseniaspora uvarum, Hanseniaspora occidentalis, Issatchenkia terricola, Metschnikowia pulcherrima, Pichia kluyveri, and Saccharomyces cerevisiae were identified using Wallerstein Laboratory Nutrient medium with sequencing of the 26S rDNA D1/D2 domain. H. uvarum and S. cerevisiae were the predominant species, while most non-Saccharomyces species were present in the whole fermentation process at different levels among the grape varieties. The genotypes of S. cerevisiae from each microvinification were determined by using interdelta sequence analysis. The 102 isolates showed eight different genotypes, and genotype III was the predominant genotype found. The distribution of S. cerevisiae strains during the fermentation of Marselan was also studied. Six genotypes were observed among the 92 strains with different genotypes of competitiveness at different sampling stages. Genotype V demonstrated the potential for organizing starter strains and avoiding inefficient fermentation. In general, this study explored the yeast species in the grapes grown in Xiangning County and provided important information of relationship of local yeast diversity and its regional wine sensory characteristics.     

Yeast Diversity Isolated from Grape Musts During Spontaneous Fermentation from a Brazilian Winery

Saccharomyces and non-Saccharomyces yeast species from a winery located in Brazil were identified by ribosomal gene-sequencing analysis. A total of 130 yeast strains were isolated from grape surfaces and musts during alcoholic fermentation from Isabel, Bordeaux, and Cabernet Sauvignon varieties. Samples were submitted to PCR–RFLP analysis and genomic sequencing. Thirteen species were identified: Candida quercitrusa, Candida stellata, Cryptococcus flavescens, Cryptococcus laurentii, Hanseniaspora uvarum, Issatchenkia occidentalis, Issatchenkia orientalis, Issatchenkia terricola, Pichia kluyveri, Pichia guilliermondii, Pichia sp., Saccharomyces cerevisiae, and Sporidiobolus pararoseus. A sequential substitution of species during the different stages of fermentation, with a dominance of non-Saccharomyces yeasts at the beginning, and a successive replacement of species by S. cerevisiae strains at the final steps were observed. This is the first report about the yeast distribution present throughout the alcoholic fermentation in a Brazilian winery, providing supportive information for future studies on their contribution to wine quality.     

秦岭地区子囊菌酵母物种多样性研究

从陕西秦岭地区的果实和叶等不同基物上分离得到子囊菌酵母262株,利用26SrDNA的D1/D2区域序列分析并结合形态学特征对这些菌株进行了分类学研究,探讨了该地区子囊菌酵母的物种多样性及其分布。共鉴定出10属31种,其中优势属为假丝酵母属Candida,12种,(其中新种2个,另文发表)、酿酒酵母属Saccharomyces,5种、毕赤酵母属Pichia,5种和有孢汉逊酵母属Hanseniaspora,3种。对分离自陕西秦岭不同地区与海拔的同一个种的不同菌株进行了D1/D2序列分析比较,以探讨子囊菌酵母种内序列稳定性和变异幅度。     

Yeast diversity of Ghanaian cocoa bean heap fermentations

The fermentation of the Theobroma cacao beans, involving yeasts, lactic acid bacteria, and acetic acid bacteria, has a major influence on the quality of the resulting cocoa. An assessment of the microbial community of cocoa bean heap fermentations in Ghana resulted in 91 yeast isolates. These were grouped by PCR-fingerprinting with the primer M13. Representative isolates were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences and partial actin gene sequences leading to the detection of 15 species. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for selected isolates. Pichia kudriavzevii ( Issatchenkia orientalis ), Saccharomyces cerevisiae , and Hanseniaspora opuntiae formed the major components of the yeast community. Hanseniaspora opuntiae was identified conclusively for the first time from cocoa fermentations. Among the less frequently encountered species, Candida carpophila, Candida orthopsilosis, Kodamaea ohmeri, Meyerozyma ( Pichia ) caribbica, Pichia manshurica, Saccharomycodes ludwigii , and Yamadazyma ( Pichia ) mexicana were not yet documented from this substrate. Hanseniaspora opuntiae was preferably growing during the earlier phase of fermentation, reflecting its tolerance to low pH and its citrate-negative phenotype, while no specific temporal distribution was recognized for P. kudriavzevii and S. cerevisiae .     

Molecular profiling of yeasts isolated during spontaneous fermentations of Austrian wines

The aim of the present study was to evaluate the autochthonous yeast population during spontaneous fermentations of grape musts in Austrian wine-producing areas. Investigation of genomic and genetic variations among wine yeasts was a first step towards a long-term goal of selecting strains with valuable enological properties typical for this geographical region. An approach, combining sequences of the D1/D2 domain of the 26S rRNA gene and random amplified polymorphic DNA fingerprinting, was used to characterize yeasts at the species level, whereas the differentiation of Saccharomyces strains was accomplished by amplified fragment length polymorphism fingerprinting. At the beginning of fermentation, representatives of nine genera were identified, with Hanseniaspora and Metschnikowia species characterized most frequently. Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum strains, which were identified throughout the entire fermentation process, showed a high level of genetic diversity. A number of S. cerevisiae strains were common at multiple wineries, but a wide range of strains with characteristic profiles were characterized at individual locations. This biodiversity survey represents a contribution to the investigation and preservation of genetic diversity of biotechnologically relevant yeasts in Austrian wine-making areas.     

Yeast diversity and persistence in botrytis-affected wine fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected ("botrytized") wine fermentations carried out at high ( approximately 30 degrees C) and ambient ( approximately 20 degrees C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by SACCHAROMYCES: In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10(6) cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of CANDIDA: Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

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.     

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.     

Identification of yeasts isolated from raffia wine (<Emphasis Type="Italic">Raphia hookeri</Emphasis>) produced in Côte d’Ivoire and genotyping of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains by PCR inter-delta

Raffia wine is a traditional alcoholic beverage produced in several African countries where it plays a significant role in traditional customs and population diet. Alcoholic fermentation of this beverage is ensured by a complex natural yeast flora which plays a decisive role in the quality of the final product. This present study aims to evaluate the distribution and the diversity of the yeast strains isolated in raffia wine from four sampling areas (Abengourou, Alépé, Grand-Lahou and Adzopé) in Côte d’Ivoire. Based on the D1/D2 domain of the LSU rDNA sequence analysis, nine species belonging to six genera were distinguished. With a percentage of 69.5 % out of 171 yeast isolates, Saccharomyces cerevisiae was the predominant species in the raffia wine, followed by Kodamaea ohmeri (20.4 %). The other species isolated were Candida haemulonii (4.1 %), Candida phangngensis (1.8 %), Pichia kudriavzevii (1.2 %), Hanseniaspora jakobsenii (1.2 %), Candida silvae (0.6 %), Hanseniaspora guilliermondii (0.6 %) and Meyerozyma caribbica (0.6 %). The molecular characterization of S. cerevisiae isolates at the strain level using the PCR-interdelta method revealed the presence of 21 profiles (named I to XXI) within 115 isolates. Only four profiles (I, III, V and XI) were shared by the four areas under study. Phenotypic characterization of K. ohmeri strains showed two subgroups for sugar fermentation and no diversity for the nitrogen compound assimilations and the growth at different temperatures.     

Identification and characterization of yeasts in brem, a traditional Balinese rice wine

Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer (ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae(35 strains), Candida glabrata(six strains), Pichia anomala(three strains) and Issatchenkia orientalis(seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters.     

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。     

Yeasts Associated with Various Amazonian Native Fruits

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Key words: yeast diversity, fruit, Amazonia, PCR-RFLP, 5.8S-ITS     

Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts

AIMS: The objective of this study was to investigate what types of enzymes are being produced by non-Saccharomyces yeasts isolated from grapes in South Africa vineyards and clarified grape juice. These enzyme profiles could pave the way for attributing specific effects in wine to some of these enzymes produced by so-called wild yeasts associated with grape must. METHODS AND RESULTS: In this study 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases beta-glucanases, lichenases, beta-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clarified grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except beta-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species. CONCLUSION: This study clearly revealed the potential of non-Saccharomyces wine yeasts to produce a wide range of useful extracellular enzymes during the initial phase of wine fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Enzymes produced by indigenous yeasts associated with grapes and juice might be harnessed to catalyse desired biotransformations during wine fermentation.     

Analysis of yeast populations during alcoholic fermentation: a six year follow-up study

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.     

The microbiology of Bandji,palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts,lactic acid and acetic acid bacteria

Aim

To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro‐organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa.

Methods and Results

Physicochemical characterization was performed using conventional methods. Identification of micro‐organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro‐organisms were screened respectively by amplification of the ITS1‐5.8S rDNA‐ITS2/16S‐23S rDNA ITS regions and repetitive sequence‐based PCR (rep‐PCR). The physicochemical characteristics of samples were: pH: 3·48–4·12, titratable acidity: 1·67–3·50 mg KOH g^?1, acetic acid: 0·16–0·37%, alcohol content: 0·30–2·73%, sugars (degrees Brix): 2·70–8·50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro‐organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis.

Conclusion

fermentation of palm sap from B. akeassii involved multi‐yeast‐LAB‐AAB cultures at genus, species and intraspecies level.

Significance and Impact of the Study

First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by‐products.     

Monitoring of Saccharomyces and Hanseniaspora populations during alcoholic fermentation by real-time quantitative PCR

Real-time, or quantitative, PCR (QPCR) was developed for the rapid quantification of two of the most important yeast groups in alcoholic fermentation (Saccharomyces spp. and Hanseniaspora spp.). Specific primers were designed from the region spanning the internal transcribed spacer 2 (ITS2) and the 5.8S rRNA gene. To confirm the specificity of these primers, they were tested with different yeast species, acetic acid bacteria and lactic acid bacteria. The designed primers only amplified for the intended group of species and none of the PCR assays was positive for any other wine microorganisms. This technique was performed on reference yeast strains from pure cultures and validated with both artificially contaminated wines and real wine fermentation samples. To determine the effectiveness of the technique, the QPCR results were compared with those obtained by plating. The design of new primers for other important wine yeast species will enable to monitor yeast diversity during industrial wine fermentation and to detect the main spoilage yeasts in wine.