Identification and characterization of Saccharomyces cerevisiae and Saccharomyces paradoxus strains isolated from Croatian vineyards

AIMS: The identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential. METHODS AND RESULTS: A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 degrees C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins. CONCLUSION: Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.     

Screening and typing of Patagonian wine yeasts for glycosidase activities

AIMS: The purpose of this study was to select autochthonous glycosidase producer yeasts with potential use in industrial production of Patagonian red wines. METHODS AND RESULTS: The study was carried out in oenological autochthonous yeasts from Comahue region (Argentinean North Patagonia). A set of screenable yeast phenotypic characteristics indicative of their potential usefulness in more aromatic red wine production was defined and tested in both, Saccharomyces and non-Saccharomyces populations. Twelve isolates showing six different glycosidase phenotypes were selected and they were characterized at species and strain levels using molecular methods. A close correlation between molecular and phenotypic characteristics was observed. Five strains belonging to Candida guilliermondii, C. pulcherrima and Kloeckera apiculata with highest constitutive beta-glucosidase activity levels without anthocyanase activity were discriminated. Some of them also showed constitutive beta-xylosidase and inductive alpha-rhamnosidase activities. CONCLUSIONS: The extension of the selection of oenological yeast to non-Saccharomyces species provided strains possessing novel and interesting oenological characteristics which could have significant implications in the production of more aromatic young red wine. SIGNIFICANCE AND IMPACT OF THE STUDY: As these non-Saccharomyces are indigenous to wine, they can be used in mixed starters at the beginning or as pure cultures at the end fermentation to contribute in enhancing the wine nuance that is typical of this specific area.     

Biodiversity of Saccharomyces yeast strains from grape berries of wine-producing areas using starter commercial yeasts

The use of commercial wine yeast strains as starters has grown extensively over the past two decades. In this study, a large-scale sampling plan was devised over a period of 3 years in three different vineyards in the south of France, to evaluate autochthonous wine yeast biodiversity in vineyards around wineries where active dry yeasts have been used as fermentation starters for more than 5 years. Seventy-two spontaneous fermentations were completed from a total of 106 grape samples, and 2160 colonies were isolated. Among these, 608 Saccharomyces strains were identified and 104 different chromosomal patterns found. The large majority of these (91) were found as unique patterns, indicating great biodiversity. There were differences in biodiversity according to the vineyard and year, showing that the biodiversity of Saccharomyces strains is influenced by climatic conditions and specific factors associated with the vineyards, such as age and size. Strains that were terroir yeast candidates were not found. The biodiversity of S. cerevisiae strains after harvest was similar to that in the early campaign; moreover, a temporal succession of S. cerevisiae strains is shown. This fact, together with the differences in biodiversity levels verifies that other factors were more important than commercial yeast utilization in the biodiversity of the vineyard.     

Genetic differentiation of the sherry yeasts Saccharomyces cerevisiae

Molecular genetic study of the yeast Saccharomyces cerevisiae isolated at various stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from the primary winemaking yeasts according to several physiological and molecular markers. All sherry strains independently of the place and time of their isolation carry a 24-bp deletion in the ITS 1 region of ribosomal DNA, whereas the yeasts of the primary winemaking lack this deletion. Molecular karyotypes of the sherry yeast from different populations were found very similar.     

Differentiation of industrial wine yeast strains using microsatellite markers

AIMS: To differentiate nine industrial wine strains of Saccharomyces cerevisiae using microsatellite (simple sequence repeats, SSR) markers. METHODS AND RESULTS: Six of the strains were indigenous yeasts currently used as high-density starter monocultures by the Uruguayan wine industry. Unequivocal differentiation of these six native strains and three commercial S. cerevisiae wine strains was achieved by PCR amplification and polymorphism analysis of loci containing microsatellite markers. CONCLUSION: We recommend the use of this reproducible and simple molecular method to routinely discriminate wine yeast strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Microsatellites are superior to other methods for typing yeasts because the results can be exchanged as quantitative data. Knowledge of the frequencies of the alleles for different SSR markers will eventually lead to an accurate typing method to identify industrial wine yeast strains.     

Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains

AIMS: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. METHODS AND RESULTS: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0.2 and 6.7 g l(-1)) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. CONCLUSIONS: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.     

Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking

AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.     

Genetic diversity and population structure of Saccharomyces cerevisiae strains isolated from different grape varieties and winemaking regions

We herein evaluate intraspecific genetic diversity of fermentative vineyard-associated S. cerevisiae strains and evaluate relationships between grape varieties and geographical location on populational structures. From the musts obtained from 288 grape samples, collected from two wine regions (16 vineyards, nine grape varieties), 94 spontaneous fermentations were concluded and 2820 yeast isolates were obtained that belonged mainly (92%) to the species S. cerevisiae. Isolates were classified in 321 strains by the use of ten microsatellite markers. A high strain diversity (8-43 strains per fermentation) was associated with high percentage (60-100%) of fermenting samples per vineyard, whereas a lower percentage of spontaneous fermentations (0-40%) corresponded to a rather low strain diversity (1-10 strains per fermentation).For the majority of the populations, observed heterozygosity (Ho) was about two to five times lower than the expected heterozygosity (He). The inferred ancestry showed a very high degree of admixture and divergence was observed between both grape variety and geographical region. Analysis of molecular variance showed that 81-93% of the total genetic variation existed within populations, while significant differentiation within the groups could be detected. Results from AMOVA analysis and clustering of allelic frequencies agree in the distinction of genetically more dispersed populations from the larger wine region compared to the less extended region. Our data show that grape variety is a driver of populational structures, because vineyards with distinct varieties harbor genetically more differentiated S. cerevisiae populations. Conversely, S. cerevisiae strains from vineyards in close proximity (5-10 km) that contain the same grape variety tend to be less divergent. Populational similarities did not correlate with the distance between vineyards of the two wine regions. Globally, our results show that populations of S. cerevisiae in vineyards may occur locally due to multi-factorial influences, one of them being the grape variety.     

Phenotypic landscape of Saccharomyces cerevisiae during wine fermentation: evidence for origin-dependent metabolic traits

The species Saccharomyces cerevisiae includes natural strains, clinical isolates, and a large number of strains used in human activities. The aim of this work was to investigate how the adaptation to a broad range of ecological niches may have selectively shaped the yeast metabolic network to generate specific phenotypes. Using 72 S. cerevisiae strains collected from various sources, we provide, for the first time, a population-scale picture of the fermentative metabolic traits found in the S. cerevisiae species under wine making conditions. Considerable phenotypic variation was found suggesting that this yeast employs diverse metabolic strategies to face environmental constraints. Several groups of strains can be distinguished from the entire population on the basis of specific traits. Strains accustomed to growing in the presence of high sugar concentrations, such as wine yeasts and strains obtained from fruits, were able to achieve fermentation, whereas natural yeasts isolated from "poor-sugar" environments, such as oak trees or plants, were not. Commercial wine yeasts clearly appeared as a subset of vineyard isolates, and were mainly differentiated by their fermentative performances as well as their low acetate production. Overall, the emergence of the origin-dependent properties of the strains provides evidence for a phenotypic evolution driven by environmental constraints and/or human selection within S. cerevisiae.     

Field-flow fractionation as analytical technique for the characterization of dry yeast: correlation with wine fermentation activity

Important oenological properties of wine depend on the winemaking yeast used in the fermentation process. There is considerable controversy about the quality of yeast, and a simple and cheap analytical methodology for quality control of yeast is needed. Gravitational field flow fractionation (GFFF) was used to characterize several commercial active dry wine yeasts from Saccharomyces cerevisiae and Saccharomyces bayanus and to assess the quality of the raw material before use. Laboratory-scale fermentations were performed using two different S. cerevisiae strains as inocula, and GFFF was used to follow the behavior of yeast cells during alcoholic fermentation. The viable/nonviable cell ratio was obtained by flow cytometry (FC) using propidium iodide as fluorescent dye. In each experiment, the amount of dry wine yeast to be used was calculated in order to provide the same quantity of viable cells. Kinetic studies of the fermentation process were performed controlling the density of the must, from 1.071 to 0.989 (20/20 density), and the total residual sugars, from 170 to 3 g/L. During the wine fermentation process, differences in the peak profiles obtained by GFFF between the two types of commercial yeasts that can be related with the unlike cell growth were observed. Moreover, the strains showed different fermentation kinetic profiles that could be correlated with the corresponding fractograms monitored by GFFF. These results allow optimism that sedimentation FFF techniques could be successfully used for quality assessment of the raw material and to predict yeast behavior during yeast-based bioprocesses such as wine production.     

Authentication and identification of Saccharomyces cerevisiae‘flor’ yeast races involved in sherry ageing

Yeasts involved in velum formation during biological ageing of sherry wine have to date been classified into four races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, rouxii) according to their abilities to ferment different sugars. It has been proposed that race succession during biological ageing is essential for the development of the organoleptical properties of sherry wines. In this work we studied the physiological characteristics, the molecular differentiation and the phylogenetic relationships of the four races employing type and reference strains from culture collections and natural environments. Using restriction analysis of the ribosomal region that includes the 5.8S rRNA gene and internal transcribed regions (5.8S-ITS) we were able to differentiate 'flor' and non-'flor' S. cerevisiae yeast strains. However, no correlation between fermentation profile, mitochondrial DNA restriction analysis or chromosomal profiles and these races was found. Moreover, sequences of the D1/D2 domain of the 26S rRNA gene and the 5.8S-ITS region from these strains were analysed and no genetic differences were noted suggesting that 'flor' yeast cannot be grouped into four different races and the four races are identified as S. cerevisiae. Since the yeasts isolated from velum in sherry wine present a unique 5.8S rRNA pattern different from the rest of the Saccharomyces cerevisiae strains we propose that they should be included as a single race or variety inside the S. cerevisiae taxon.     

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.     

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America

AIMS: The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. METHODS AND RESULTS: Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. CONCLUSIONS: Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.     

Survival of inoculated Saccharomyces cerevisiae strain on wine grapes during two vintages

AIMS: To investigate the influence of a specific ecological niche, the wine grape, on the survival and development of Saccharomyces cerevisiae. METHODS AND RESULTS: A strain with a rare phenotype was sprayed onto the grape surfaces and monitored through two vintages using a specific indicative medium and analysing the internal transcribed spacer regions in the 5.8S rDNA. During the ripening process, there was a progressive colonization of the surface of the undamaged and damaged grapes by epiphytic yeasts, up to the time of harvest. The damaged wine grapes showed a much greater epiphytic yeast population. However, the inoculated S. cerevisiae strain showed a scarce persistence on both undamaged and damaged wine grapes, and the damaged grapes did not appear to improve the grape surface colonization of this strain. CONCLUSIONS: Results indicated that wine grape is not a favourable ecological niche for the development and colonization of S. cerevisiae species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this work are further evidence that S. cerevisiae is not specifically associated with natural environments such as damaged and undamaged wine grapes.     

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.     

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.     

The uses of AFLP for detecting DNA polymorphism, genotype identification and genetic diversity between yeasts isolated from Mexican agave-distilled beverages and from grape musts

AIMS: The objectives were to determine the variability and to compare the genetic diversity obtained using amplified fragment length polymorphism (AFLP) markers in analyses of wine, tequila, mezcal, sotol and raicilla yeasts. METHODS AND RESULTS: A molecular characterization of yeasts isolated from Mexican agave musts, has been performed by AFLP marker analysis, using reference wine strains from Italian and South African regions. CONCLUSIONS: A direct co-relation between genetic profile, origin and fermentation process of strains was found especially in strains isolated from agave must. In addition, unique molecular markers were obtained for all the strains using six combination primers, confirming the discriminatory power of AFLP markers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of molecular characterization between yeasts isolated from different Mexican traditional agave-distilled beverages, which shows high genetic differences with respect to wine strains.     

A survey ofSaccharomyces populations associated with wine fermentations from the Apulia region (South Italy)

The aim of this paper was to investigate the genetic and phenotypic characteristics of yeasts isolated from samples of grape musts collected from four different areas of Apulia region. The 68 yeast isolates were identified asSaccharomyces cerevisiae by PCR-RFLP of 5.8S-ITS region of the rRNA gene. Individual isolates were differentiated by RAPD-PCR and AFLP. The following oenological traits were studied: fermentation power, resistance to cycloheximide, alcohol and SO₂, formation of SO₂ and H₂S, β-glucosidase activity, and production of biogenic amines and secondary compounds. Many phenotypes were common to several yeasts isolated from the four different areas, such as high SO₂ resistance and fermentation power. In addition, someS. cerevisiae isolates showed a β-glucosidase activity and others had a high resistance to cycloheximide. All the strains formed biogenic amines. Solid Phase Microextraction was used to determine secondary compounds produced in wine by the single yeast cultures.     

Application of Multi Locus Sequence Typing to the analysis of the biodiversity of indigenous Saccharomyces cerevisiae wine yeasts from Lebanon

AIMS: To assess suitability of Multi Locus Sequence Typing (MLST) for investigating the biodiversity of wine yeast strains. This method was compared with established ones like microsatellite analysis or amplification of genomic regions flanked by repeated (delta) elements. METHODS AND RESULTS: DNA fragments were amplified and sequenced for 26 loci representing housekeeping genes, open reading frames (ORFs) of unknown functions or intergenic regions. A set of seven loci was tested on 84 Saccharomyces cerevisiae strains, including 65 strains isolated from traditional wineries in Lebanon, commercial wine strains and Asian isolates. An overall sequence diversity of 2.05% was observed, consisting of single nucleotide polymorphisms, 60% of them occurring in a heterozygous state. The number of polymorphic sites per locus varied between 4 and 14. The same set of strains was analysed by microsatellite typing on six polymorphic loci and by interdelta amplification. CONCLUSIONS: Clustering of MLST profiles clearly differentiated the Asian group of strains from Lebanese and European commercial strains that appear closely related. The current MLST scheme appears less discriminatory (92.27%) on closely related wine yeasts than microsatellite or interdelta typing (>99%). SIGNIFICANCE AND IMPACT OF THE STUDY: MLST is a highly reliable method for relatedness inference and promising for wine yeast typing.     

Typing of Saccharomyces cerevisiae and Kloeckera apiculata strains from Aglianico wine

AIMS: Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation. METHODS AND RESULTS: Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae. CONCLUSIONS: The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.