Changes in sour rotten grape berry microbiota during ripening and wine fermentation

This study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes. Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health status.     

新疆慕萨莱思自然发酵过程中酵母菌表型多样性及优势菌分析

目的：研究新疆慕萨莱思自然发酵过程中酵母菌种群表型多样性与其优势菌,探讨慕萨莱思传统工艺对其主要菌群结构的影响。方法：来自于新疆阿瓦提一古作坊的慕萨莱思酿制原料、原料处理液及发酵液(自然发酵过程中)共19份样品被用于酵母菌分离,分离株利用WL培养基培养归类,筛选代表株,对代表株进行形态观察、生理生化特征检测与类平均连算法聚类分析,探讨新疆慕萨莱思自然发酵过程中酵母菌表型多样性及优势菌群。结果：分离得到217株酵母菌,13种WL培养类型,8个表观群。13株代表菌株经初步鉴定为7个属,疑似为13个种,表明慕萨莱思酵母菌具有丰富的多样性。Hanseniaspora spp.为葡萄果皮、果汁及皮渣中的优势菌,S.cerevisiae为慕萨莱思自然发酵过程中起发者及唯一一种优势菌,非酿酒酵母偶尔在发酵液中发现。结论：慕萨莱思酿制过程中,葡萄原料原有的酵母菌经熬煮工序几乎被全部杀死,自然发酵中唯一优势菌S.cerevisiae可能来自酿制场所和设备,并具有较高适应能力。     

Bacterial species associated with sound and Botrytis-infected grapes from a Greek vineyard

Grape bacterial microbiota plays central roles in the quality of grapes and wine, yet its diversity remains poorly described. In the present study, bacterial species associated with sound and Botrytis-infected grapes of two cultivars originating from the same vineyard were assessed. Isolates were identified by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and sequence analysis of partial 16S rRNA gene. Comparable counts were recorded between Botrytis-infected and sound grape samples. In all cases, the majority of isolates belonged to different species of Enterobacteriaceae. The dominant species in the vineyard was Klebsiella oxytoca that was found in different combinations with Citrobacter freundii, Enterobacter spp., Erwinia sp., Pantoea dispersa, Tatumella ptyseos or other species. In fermenting musts, those populations declined while other species evolved, like Lactobacillus plantarum and Enterobacter ludwigii. Populations in botrytised samples persisted longer during spontaneous fermentations. Present study suggests that bacterial diversity on grapes may be wider than previously described.     

Application of fluorescence in situ hybridisation (FISH) to the analysis of yeast population dynamics in winery and laboratory grape must fermentations

To analyse the yeast population diversity during wine fermentations, specific fluorescein-labelled oligonucleotide probes targeted to the D1/D2 region of the 26S rRNA of different yeast species known to occur frequently in this environment were designed and tested with reference strains. The probes were then used to identify wine must isolates and to follow, in combination with plate counts, the evolution of yeast populations in two winery fermentations of white and red grape musts. In both cases, a high diversity of non-Saccharomyces yeast species was detected, including Candida stellata, Hanseniaspora uvarum, H. guilliermondii, Kluyveromyces marxianus, K. thermotolerans and Torulaspora delbrueckii. Some of these species (e.g., K. marxianus, K. thermotolerans and T. delbrueckii) were present in significant amounts during the tumultuous fermentation stage, despite the predominance of Saccharomyces cerevisiae cells following the inoculation of the wine musts with a starter strain. To further clarify the yeast population dynamics at the late phase of the fermentations, and because winery conditions do not allow a reliable control of experimental variables, strains isolated from the industrial musts were used to conduct two laboratory microvinifications in synthetic grape juice, using different ratios of S. cerevisiae/non-Saccharomyces in the inocula. Under these conditions, the results were similar to those obtained in the winery, showing a yeast profile with mixed species throughout the first fermentation stage, i.e. until about 40-50% of the total sugar was consumed. Non-Saccharomyces yeasts were outgrown by S. cerevisiae only after ethanol reached concentrations around 4-5% (v/v), which argues in favour of a potential important role of non-Saccharomyces in the final organoleptic characteristics of the wine.     

荞麦红曲酒的酿造

采用红曲霉固态培养荞麦制曲,液态发酵产洒的方法酿造荞麦红曲酒。固态培养5d的荞麦红曲加水转入液态发酵,添加少量酿酒活性干酵母以及大米根霉糖化液有助于发酵过程,并且使成品酒风味更好。     

Partial 26S rDNA restriction analysis as a tool to characterise non-Saccharomyces yeasts present during red wine fermentations

Restriction patterns of amplified regions of ribosomal large subunit RNA encoding genes (26S rDNA) were evaluated as a routine methodology to examine yeast species diversity during red wine fermentation. The results were confirmed by sequencing of D1/D2 region of 26S rDNA. Red wine production was carried out using a yeast starter culture together with different commercial products, namely enzymes, fermentation activators and tannins and their influence on the non-Saccharomyces yeast population was studied. Yeast strains were isolated using lysine agar as a selective medium for non-Saccharomyces yeasts, after morphological characterisation of colonies. Amplification of 26S rDNA followed by digestion with three restriction enzymes applied to the 121 isolates, generated 19 profiles and a very high correlation with sequencing results was achieved. Although a starter yeast culture was added, results showed that several yeast species were present during all stages of fermentation, independent of the conditions tested, emphasizing the diversity of microorganisms associated with winemaking. On the other hand commercial additives did not significantly influence the diversity of yeast population during the fermentation process. For non-Saccharomyces strains, restriction patterns of a PCR amplified 26S rDNA region proved to be an adequate tool for clustering strains at species level and enabled the monitoring of yeast population dynamics during red wine fermentation.     

Yeast associated with spontaneous fermentations of white wines from the "Txakoli de Bizkaia" region (Basque Country,North Spain)

The microbiota of eight spontaneous fermentation of white wine from different grape varieties and different wineries from the "Txakoli de Bizkaia" region (Basque country, North Spain), in 1996 and 1997 campaigns was studied. The yeast population was higher in grapes harvested in 1997, in which late summer and early autumn was warmer and drier. Eight species belonging to five genera were identified in total. The most frequent genera in grapes were Rhodotorula in 1996 and Kloeckera in 1997. Saccharomyces bayanus was the most frequent species during vigorous and final fermentation, and it was occasionally isolated from grapes and must. Only another Saccharomyces spp., i.e., S. kluyvery, was identified in some samples from 1997.     

Characterization of the yeast ecosystem in grape must and wine using real-time PCR

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.     

Identification of lactic acid bacteria isolated from South African brandy base wines

In brandy base wines, no sulphur dioxide is used and it therefore is ideal for the proliferation of lactic acid bacteria. As part of an extensive taxonomic survey within the ecological framework of South African vineyards and wineries, and the influence of naturally occurring lactic acid bacteria on the quality of wine and brandy, a total of 54 strains were isolated from grape juice and at different stages of brandy base wine production. The strains were identified using numerical analysis of total soluble cell protein patterns, 16S rRNA sequence analyses and polymerase chain reaction (PCR) using species-specific primers. The predominant species was Oenococcus oeni (22 strains), but Lactobacillus brevis (8 strains), Lactobacillus paracasei (8 strains) and Lactobacillus plantarum (6 strains) were also isolated frequently. Many of the O. oeni strains were isolated from brandy base wines after completion of spontaneous malolactic fermentation (MLF). The Lactobacillus spp. were isolated from all the different stages of brandy base wine production. Lb. plantarum was the dominant species in the juice, but disappeared during the later stages of production. However, Lactobacillus hilgardii, Lb. brevis and Lb. paracasei were also isolated from base wine after spontaneous MLF. Strains identified as Lactobacillus vermiforme were isolated during the alcoholic fermentation and after MLF have been completed. Total soluble cell protein patterns grouped O. oeni strains into two phenotypic groups. Two phenotypic clusters have also been identified for the Lb. brevis isolates. The Lb. paracasei isolates all grouped in one cluster. This is the first report of the presence of Lb. paracasei and Lb. vermiforme in brandy base wines. The presence of the Lactobacillus spp. could be correlated to the decrease in quality of the base wine and distillate, while O. oeni strains were found to have a more favourable influence on the quality of base wine and distillates. These results shed some light on the ecology and oenological influence of lactic acid bacteria (LAB) on the quality of South African brandy.     

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.     

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.     

红曲黄酒酿造用曲及传统酿造过程中酵母菌的多样性研究

目的:探讨红曲黄酒酿造用曲中酵母菌多样性以及传统酿造过程中酵母菌菌群结构变化,为我国传统红曲黄酒中酵母菌资源的利用和对传统酿酒的有效控制及现代化酿酒新工艺的建立提供基础数据。方法:收集福建各地区的红曲黄酒酿造用曲20份,从中分离纯化出300株酵母菌,通过ITS1-5.8S-ITS2的PCR-RFLP指纹图谱对酵母菌进行分型,从各个分型类群中随机选取代表菌株,利用26S rDNA基因D1/D2区域序列分析进行分类鉴定;并采用PCR-RFLP快速分型鉴定技术分析红曲黄酒传统酿造过程中酵母菌菌群结构的变化。结果:从红曲黄酒酿造用酒曲中总共分离鉴定出12种类型酵母菌,其中扣囊复膜孢酵母(Saccharomycop-sis fibuligera)、酿酒酵母(Saccharomyces cerevisiae)和弗比恩毕赤酵母(Pichia fabianii)是酒曲中3种主要的酵母菌类型。红曲黄酒传统酿造过程酵母菌群的跟踪分析共鉴定出4种酵母菌,即酿酒酵母、扣囊复膜孢酵母、季也蒙毕赤酵母(Pichia guilliermondii)、粘性红圆酵母(Rhodotorula mucilaginosa)。在酿造前期扣囊复膜孢酵母是优势酵母菌,而在酿造的后期,酿酒酵母完全取代之成为优势菌。结论:红曲黄酒酿造用酒曲中的酵母菌具有丰富的生物多样性,红曲黄酒传统酿造过程酵母菌菌群结构处于动态变化,最终酿酒酵母成为酿造体系的优势酵母菌。     

加饭酒大罐发酵过程中真菌群落的动力学研究

采用分离培养法和基于核糖体RNA基因间隔序列的RISA图谱分析技术,研究了绍兴加饭黄酒大罐发酵过程中醪液内真菌群落的动力学变化及所含真菌群落情况。研究结果显示,发酵醪液中共涉及9属13种真菌。它们分别是犁头霉属(Absidiasp.),根毛霉属(Rhizomucor variabilis),曲霉属(Aspergillus oryzae,As-pergillus niger,Aspergillussydowii,Emericellanidulans),青霉属(Penicilliumcitrinum),伊萨酵母属(Issatch-enkia orientalis),毕赤氏酵母属(Pichia anomala,Pichia burtonii),酵母属(Saccharomyces cerevisia),红酵母属(Rhodotorula mucilaginosa),念珠菌属(Clavisporalusitaniae)。其中犁头霉、米曲霉属和异常毕赤氏酵母在整个发酵过程中均出现,既是发酵投料时的原始菌群,也是发酵醪液中的主要菌群。酿酒酵母和黏质红酵母在发酵前期含量较少,在中后期数量逐渐增多,并成为发酵醪液中的优势菌群。     

Yeast dynamics during the fermentation of brined green olives treated in the field with kaolin and Bordeaux mixture to control the olive fruit fly

The yeast microbiota associated with naturally fermented and inoculated green table olives, differently treated in the field with non-conventional repellent and antiovipositional products in the control of Bactrocera oleae, was analysed using a combination of culture-dependent and -independent molecular fingerprinting. The routine yeast isolation gave rise to 118 strains, whose identification was performed by PCR-RFLP of the internal transcribed spacer (ITS) regions. Total DNA was extracted directly from the brine throughout fermentation by means of an experimental protocol that included the removal of Taq polymerase inhibitors. Denaturing Gradient Gel Electrophoresis (DGGE) of 26S rRNA gene PCR amplicons highlighted the yeast community. Comparison of both culture-dependent and independent methods indicated that the yeast species Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida diddensiae and Issatchenkia orientalis were dominant during fermentation despite the addition of the Lactobacillus plantarum starter used in brining. The resultant isolated species were unaffected by treatments in field, except for C. diddensiae whose growth was delayed by kaolin.     

Identification of corynebacterium bovis and other coryneforms isolated from bovine mammary glands

Bovine mastitis remains the most economically important disease in dairy cows. Corynebacterium bovis, a lipid-requiring Corynebacterium spp., is frequently isolated from the milk of infected mammary glands of dairy cows and is associated with reduced milk production. A total of 212 coryneform bacteria isolated from the milk of dairy cows were obtained from mastitis reference laboratories in the United States and Canada. All isolates had been presumptively identified as Corynebacterium bovis based on colony morphology and growth in the presence of butterfat. Preliminary identification of the isolates was based on Gram stain, oxidase, catalase, and growth on unsupplemented trypticase soy agar (TSA), TSA supplemented with 5% sheep blood, and TSA supplemented with 1% Tween 80. Of the 212 isolates tested, 183 were identified as Corynebacterium spp. based on preliminary characteristics. Of the strains misidentified, one was identified as a yeast, two as Bacillus spp., 11 as Enterobacteriaceae, 18 as staphylococci, one as a Streptococcus spp., and one as an Enterococcus spp. Eighty-seven coryneforms were selected for identification to the species level by direct sequencing of the 16S rRNA gene, the Biolog system and the API Coryne system. Fifty strains were identified as C. bovis by 16S rRNA gene similarity studies: the Biolog and API Coryne systems correctly identified 54.0 and 88.0% of these strains, respectively. The other coryneforms were identified as other Corynebacterium spp., Rhodococcus spp., or Microbacterium spp. These data indicate that the coryneform bacteria isolated from bovine mammary glands are a heterogeneous group of organisms. Routine identification of C. bovis should include Gram-stain, cell morphology, catalase production, nitrate reduction, stimulated growth on 1% Tween 80 supplemented media, and beta-galactosidase production as the minimum requirements.     

Natural yeast flora of different varieties of grapes used for wine making in India

The natural Saccharomyces and non-Saccharomyces yeast flora present on the grape berries significantly affect wine production. Six grape varieties, Bangalore blue, Zinfandel, Cabernet, Chenin Blanc, Sauvignon Blanc and Shiraz are being used in India for wine making. The yeast diversity was studied on the basis of morphological, colony, physiological characteristics and 5.8S-ITS sequencing of rDNA of the isolates. Eleven different species belonging to seven genera were identified as: Candida azyma, Candida quercitrusa, Debaryomyces hansenii, Hanseniaspora guilliermondii, Hanseniaspora viniae, Hanseniaspora uvarum, Issatchenkia orientalis, Issatchenkia terricola, Pichia membranifaciens, Saccharomyces cerevisiae and Zygoascus steatolyticus.H. guilliermondii was the predominant species while S. cerevisiae was observed occasionally in the six vine varieties. For the first time, C. azyma was isolated from Bangalore blue and Cabernet varieties grown in different localities. This association may be attributed to the change in cropping pattern from sugarcane to viticulture in the vine growing regions and the known association of C. azyma with sugarcane phylloplane. Further analysis of the indigenous strains and the qualitative and quantitative changes in the flora during fermentation will be useful to understand wine quality and to design preservation strategies to control wine spoilage.     

Lactic acid bacteria and yeasts isolated from the starter doughs for Chinese steamed buns in Thailand

Isolation of lactic acid bacteria (LAB) and yeasts from the starter dough of Chinese steamed buns from four different commercial sources in Thailand was carried out. Thirty-one lactic acid bacteria and eight yeast strains were isolated. Total counts of LAB were from 1.8 × 10⁴ to 10⁸ colonies/g sample, whilst yeasts were very low from 10 to 2.3 × 10² colonies/g sample. The pH values of all starter doughs ranged from 3.36 to 3.52 and the Total Titratable Acidity (TTA) varied from 11.1 to 17.0 ml of 0.1 N NaOH/10 g dough. All LAB isolates were identified as Lactobacillus. The phenotypic characteristics were used to cluster all the LAB isolates into two major groups (Group A and Group B), with the B group subdivided into four groups. Phylogenetic analysis, based upon partial 16S rRNA gene sequences, showed that isolates of Group A, which all contained meso-diaminopimelic acid in their cell wall and produced dl-lactic acid, were closely related to Lactobacillus plantarum, whilst the strains of Group B that produced l-lactic acid were closely related to Lactobacillus casei. For yeasts, eight isolates based on the D1/D2 domain sequences of 26S rRNA were identified as Candida tropicalis, Pichia stipitis, Candida parapsilosis, Issatchenkia orientalis and Saccharomyces cerevisiae.     

Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2-K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.     

本土酿酒酵母的筛选及其低醇葡萄酒特性研究

以天津宝坻产区的三种葡萄为原料,分别从自然发酵的葡萄汁中筛选出340株酵母菌,经生理生化,产酸、酯、尿酶、H2S,发酵能力测定及26SrRNA测序,确定4株为酿酒酵母,用于贵人香低醇白葡萄酒的酿造。以商用酵母为对照,定量分析了不同酵母所酿酒中的香气成分、氨基甲酸乙酯含量,并进行了理化指标和感官测定,发现酵母C508和G611酿造的低醇酒具有更独特的香气特点,氨基甲酸乙酯的含量较低,微生物稳定性较高,更适合低醇酒的酿造。     

Molecular identification and osmotolerant profile of wine yeasts that ferment a high sugar grape must

The objective of this study was to examine the Saccharomyces and non-Saccharomyces yeast populations involved in a spontaneous fermentation of a traditional high sugar must (Vino cotto) produced in central Italy. Molecular identification of a total of 78 isolates was achieved by a combination of PCR-RFLP of the 5.8S ITS rRNA region and sequencing of the D1/D2 domain of the 26S rRNA gene. In addition, the isolates were differentiated by RAPD-PCR. Only a restricted number of osmotolerant yeast species, i.e. Candida apicola, Candida zemplinina and Zygosaccharomyces bailii, were found throughout all the fermentation process, while Saccharomyces cerevisiae prevailed after 15 days of fermentation. A physiological characterization of isolates was performed in relation to the resistance to osmotic stress and ethanol concentration. The osmotolerant features of C. apicola, C. zemplinina and Z. bailii were confirmed, while S. cerevisiae strains showed three patterns of growth in response to different glucose concentrations (2%, 20%, 40% and 60% w/v). The ability of some C. apicola and C. zemplinina strains to grow at 14% v/v ethanol is noteworthy. The finding that some yeast biotypes with higher multiple stress tolerance can persist in the entire winemaking process suggests possible future candidates as starter for Vino cotto production.