Diversity of Saccharomyces and non-Saccharomyces yeasts in three red grape varieties cultured in the Serranía de Ronda (Spain) vine-growing region

For the first time, an ecological survey of wine yeasts present in grapes growing in two vineyards located in the region of “Serranía de Ronda” (Málaga, southern Spain) has been carried out. During the 2006 and 2007 vintages, grapes from different varieties were aseptically collected and allowed to ferment spontaneously in the laboratory. From a total of 1586 colonies isolated from microvinifications, 1281 were identified according to ITS polymorphisms and their identity confirmed by sequencing of the D1/D2 region of 26S rDNA. Most of the isolates (84%) corresponded to thirteen different non-Saccharomyces species with Kluyveromyces thermotolerans, Hanseniaspora guilliermondii, Hanseniaspora uvarum and Issatchenkia orientalis accounting for 42.7% of the total. Mitochondrial DNA restriction analysis from the Saccharomyces cerevisiae isolates revealed a low diversity since only eleven different profiles were found, nine of them corresponding to local strains and two to commercial ones that had been used in different campaigns and that very likely were disseminated from the winery to the adjacent vineyard. A different distribution of strains was found in the three grape varieties studied.     

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.     

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol.     

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.     

Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus

This study investigated the formation and utilization of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Time-course papaya juice fermentations were carried out using pure cultures of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 and a mixed culture of the two yeasts at a ratio of 1:1000 (R2:NCYC2251). Changes in S. cerevisiae cell population, Brix, sugar consumption and pH were similar in the mixed culture and in the S. cerevisiae monoculture. There was an early growth arrest of W. saturnus in the mixed culture fermentation. A range of volatile compounds were produced during fermentation including fatty acids, alcohols, aldehydes and esters and some volatile compounds including those initially present in the juice were utilized. The mixed culture fermentation of S. cerevisiae and W. saturnus benefited from the presence of both yeasts, with more esters being produced than the S. cerevisiae monoculture and more alcohols being formed than the W. saturnus monoculture. The study suggests that papaya juice fermentation with a mixed culture of S. cerevisiae and W. saturnus may be able to result in the formation of more complex aroma compounds and higher ethanol level than those using single yeasts.     

Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae

Discovery, characterisation and use of novel yeast strains for winemaking is increasingly regarded as a way for improving quality and to provide variation, including subtle characteristic differences in fine wines. The objective of this work was to evaluate the use of a native apiculate strain, selected from grapes, Hanseniaspora vineae (H. vineae) 02/5A. Fermentations were done in triplicate, working with 225 L oak barrels, using a Chardonnay grape must. Three yeast fermentation strategies were compared: conventional inoculation with a commercial Saccharomyces cerevisiae strain, ALG 804, sequential inoculation with H. vineae and then strain ALG 804 and spontaneous fermentation. Yeast strain identification was performed during fermentation, in which the apiculate strain was found to be active, until 9% of alcohol in volume, for the co-fermentation and the spontaneous fermentation was completed by three native S. cerevisiae strains. Basic winemaking parameters and some key chemical analysis, such as concentration of glycerol, biogenic amines, organic acids, and aroma compounds were analysed. Sensory analysis was done using a trained panel and further evaluated with professional winemakers. Sequential inoculation with H. vineae followed by S. cerevisiae resulted in relatively dry wines, with increased aroma and flavour diversity compared with wines resulting from inoculation with S. cerevisiae alone. Wines produced from sequential inoculations were considered, by a winemaker’s panel, to have an increased palate length and body. Characteristics of wines derived from sequential inoculation could be explained due to significant increases in glycerol and acetyl and ethyl ester flavour compounds and relative decreases in alcohols and fatty acids. Aroma sensory analysis of wine character and flavour, attributed to winemaking using H. vineae, indicated a significant increase in fruit intensity described as banana, pear, apple, citric fruits and guava. GC analysis of the relative accumulation of 23 compounds to significantly different concentrations for the three fermentation strategies is discussed in relation to aroma compound composition.     

Isolation and selection of yeasts from wine grape ecosystem secreting cold-active pectinolytic activity

The present study was undertaken with the purpose of selecting yeasts from wine grapes that are able to produce extracellular cold-active pectinases. After two consecutive selections yeast isolates were identified by pheno- and genotyping, and pectinolytic activity was preliminarily characterised at proximate winemaking conditions. Out of 1023 indigenous microorganisms isolated from grape skins of D.O. San Rafael (Mendoza, Argentina) viticulture region, 565 (55%) showed pectinolytic activity on plates and, among them, 96 (17%) were chosen in a primary selection. Ten isolates were finally selected for exhibiting the greatest activity at low temperature (12 °C) and identified as Aureobasidium pullulans. GM-R-22 strain demonstrated the highest pectinolytic activity (0.751 U/mL) at pH 3.5 and 12 °C. Yeast pectinases were constitutively produced. This study is the first report about strains of A. pullulans producing pectinases which are able to show good activity at low temperature. These pectinolytic strains could be of interest in wine production.     

Acidification of grape marc for alcoholic beverage production: effects on indigenous microflora and aroma profile after distillation

Grappa is an Italian alcoholic beverage obtained from distillation of grape marc, the raw material derived from separation of must during the winemaking process. Marc is stored for a period lasting from few days to several weeks, when fermentation of residual sugars occurs mainly by yeast activity. Many distilleries have adopted different solutions to manage this critical phase in order to avoid spoilage microorganisms: marc acidification is the most widely diffused. In this work, Prosecco grape pomace was acidified with sulphuric acid (to pH 2.9) and stored, whereas non-acidified grape marc was used as control (pH 3.9). Samples for microbiological analysis were collected at the beginning of the storage period, after 15 and 43 days. At the beginning of the ensilage (time T0) the indigenous microflora was represented both by yeasts and bacteria at a concentration of about 10⁶ cfu/g. During the first 15 days, when the fermentation generally takes place, yeast population grew considerably (up to 10⁷ cfu/g) in acidified grape marc, where bacterial population was maintained at low levels. Moreover, yeast populations recovered at the three sampling times in both treated and untreated marc were genetically characterised. This analysis showed that the species succession lead to non-Saccharomyces species dominance (in particular Issatchenkia and Pichia genera) in both conditions although acidified marc showed a lower percentage of Saccharomyces at any sampling time analysed, this meaning that non-Saccharomyces species were favoured in this environment. Gas chromatographic analysis showed a remarkable change in the aromatic profile of distilled grape marcs at the end of the storage, thus evidencing that concentration of monitored volatile compounds usually produced by microflora was generally lowered by the acidification treatment.This work demonstrates for the first time the strong effect of a persistent acidification treatment both on the microbiota of grape pomace and on the aromatic profile of the distillate. Indeed, the lowering of the pH caused significant changes in yeast-bacteria populations ratio and in yeast species turnover. These microbiological changes determine an improvement of the aromatic profile of the distillate, due to the reduction of the main volatile products associated with potential off-flavours.     

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: A strategy to enhance acidity and improve the overall quality of wine

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine.     

Formation of vinylphenolic pyranoanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies

The strains of two species of yeast, Saccharomyces cerevisiae and Pichia guillermondii, both with high hydroxycinnamate decarboxylase (HCDC) activity (56% and 90% respectively), were used in the fermentation of musts enriched with grape anthocyanins, to favour the formation of highly stable vinylphenolic pyranoanthocyanin pigments. The different strains were used to ferment the must separately, simultaneously, or sequentially, the latter involving an initial period using the yeast with the greatest HCDC activity (P. guillermondii). The must was made from concentrated grape juice diluted to 220 g/l of sugar, and enriched with grape anthocyanins to 100 mg/l and with p-coumaric acid to 120 mg/l. The pH was fixed to 3.5. All 50 ml micro-fermentations were done in triplicate. The development of anthocyanin-3-O-glucoside precursors, the decarboxylation of p-coumaric acid, and the formation of 4-vinylphenol and vinylphenolic pyranoanthocyanin derivatives were studied during the fermentation. The fermentation strategy used and the yeast HCDC activity significantly influenced the formation of vinylphenolic pyranoanthocyanins. The latter molecules were separated, identified, and quantified using high performance liquid chromatograph with diode array detection and electrospray-mass spectrometry (HPLC-DAD-ESI/MS). The volatile compounds profile was screened during fermentation using gas cromatogrphy-flame ionisation detection (GC-FID), in order to detect and quantify the main molecules. The best results were reached with the sequential fermentation (3.74 mg/l of malvidin-3-O-glucoside-4-vinylphenol). This work shows that during mixed or sequential fermentations carried out with non-Saccharomyces or highly fermentative Saccharomyces strains, with high HCDC activity, the content of stable pigments can be increased without sensorial modifications.     

Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes: Potential use of Hanseniaspora uvarum as a starter culture

Several yeasts were isolated from Campbell Early grapes (Vitis labrusca cultivar Campbell Early), the major grape cultivar in Korea, grown in two different regions. PCR-RFLP analysis of the ITS I-5.8S-ITS II region showed that 34 isolates out of a total of 40 were in the same group. Phylogenetic analysis revealed that the major strain belonged to one species, Hanseniaspora uvarum, although they displayed some nucleotide mismatches between them. During spontaneous alcohol fermentation at 20 °C, the two grape musts containing 24 °Brix sugar exhibited similar fermentation patterns with differences in final alcohol production and yeast viable counts. PCR analysis of the yeasts randomly isolated during the fermentation using an intron splice site primer showed changes in yeast flora between 8 and 10 days of fermentation. We found that the dominant yeasts displaying various PCR patterns using the primer remained the same throughout the early stages of fermentation, as determined by molecular typing of their ITS regions using PCR-RFLP, and these yeasts were identical to those isolated from grape berries. Among the isolates, the strain designated SS6 was selected based on its potassium metabisulfite resistance, alcohol production (distillation method), and flavor (by sniffing test) of grape juice. When Campbell Early grape must was inoculated with H. uvarum SS6 cells, no differences in fermentation pattern were observed compared with that inoculated with cells of Saccharomyces cerevisiae W-3, an industrial wine yeast strain. However, SS6 wine showed higher levels of organic acid (especially lactic acid), aldehydes, and minor alcohols (except n-propyl alcohol), as well as a higher score in sensory evaluation, compared to those of W-3 wine.     

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).     

Quantifying the individual effects of ethanol and temperature on the fitness advantage of Saccharomyces cerevisiae

The presence of Saccharomyces cerevisiae in grape berries and fresh musts is usually very low. However, as fermentation progresses, the population levels of this species considerably increase. In this study, we use the concept of fitness advantage to measure how increasing ethanol concentrations (0-25%) and temperature values (4-46 °C) in wine fermentations affects competition between S. cerevisiae and several non-Saccharomyces yeasts (Hanseniaspora uvarum, Torulaspora delbrueckii, Candida zemplinina, Pichia fermentans and Kluyveromyces marxianus). We used a mathematical approach to model the hypothetical time needed for S. cerevisiae to impose itself on a mixed population of the non-Saccharomyces species described above. This approach also took into consideration the influence of environmental factors and the initial population levels of S. cerevisiae (0.1, 1.0 and 10.0%). Our results suggest that Saccharomyces niche construction via ethanol production does not provide a clear ecological advantage (at least not until the ethanol concentration exceeds 9%), whereas a temperature rise (above 15 °C) does give S. cerevisiae a considerable advantage. The initial frequency of S. cerevisiae considerably influences the time it needs to impose itself (until it reaches a final frequency of 99% in the mixed culture), the lowest time values being found at the highest initial frequency. In light of these results, the application of low temperatures in the wine industry could favor the growth and survival of non-Saccharomyces species for a longer period of time.     

Inactivation of yeasts in grape juice using a continuous dense phase carbon dioxide processing system

The effects of dense phase CO₂ processing parameters, including temperature (25 and 35 °C), CO₂ concentration (0, 85 and 170 g kg⁻¹) and pressure (6.9, 27.6 and 48.3 MPa), on yeast survival and sensory properties of grape juice were investigated. The dense phase CO₂ process resulted in more than a 6 log reduction in yeast population. As the CO₂ to juice concentration, temperature and pressure increased, the inactivation rate increased. CO₂ in the supercritical state was more effective in inactivating yeast than in the subcritical state. The process did not cause detectable flavor degradation. Dense phase CO₂ processing can be an effective non‐thermal alternative process for pasteurization of grape juice. Copyright © 2005 Society of Chemical Industry     

Diversity of Saccharomyces strains on grapes and winery surfaces: analysis of their contribution to fermentative flora of Malbec wine from Mendoza (Argentina) during two consecutive years

Spontaneous fermentations are still conducted by several wineries in different regions of Argentina as a common practice. Native Saccharomyces strains associated with winery equipment, grape and spontaneous fermentations of Malbec musts from "Zona Alta del Río Mendoza" region (Argentina) were investigated during 2001 and 2002 in the same cellar. Low occurrence of Saccharomyces on grapes and their limited participation during fermentation were confirmed. Strain sequential substitution during fermentation was observed. Between 30% and 60% of yeast population at the end of fermentation was coming from yeasts already present in the winery. A stable and resident Saccharomyces micro-flora in the winery was confirmed. It exhibited a dynamic behaviour during season and between years. Commercial strains were found during fermentation in different percentages, but their presence on winery equipment was low. The present work represents a first approach to winery yeast and spontaneous fermentation Saccharomyces population dynamics in an important viticultural region from Argentina that has never been characterized before. The results obtained have an important significance for the local industry, showing for the first time the real situation of the microbial ecology of alcoholic fermentation in an industrial winery from Mendoza, Argentina.     

Genetic and phenotypic diversity of autochthonous cider yeasts in a cellar from Asturias

This paper analyses yeast diversity and dynamics during the production of Asturian cider. Yeasts were isolated from apple juice and at different stages of fermentation in a cellar in Villaviciosa during two Asturian cider-apple harvests. The species identified by ITS-RFLP corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia guilliermondii, Candida parapsilosis, Saccharomyces cerevisiae and Saccharomyces bayanus/Saccharomyces pastorianus/Saccharomyces kudriavzevii/Saccharomyces mikatae. The species C. parapsilosis is reported here for the first time in cider. The analysis of Saccharomyces mtDNA patterns showed great diversity, sequential substitution and the presence of a small number of yeast patterns (up to 8), present in both harvests. Killer (patterns nos. 22′ and 47), sensitive (patterns nos. 12, 15, 33 and 61) and neutral phenotypes were found among the S. cerevisiae isolates. The detection of β-glucosidase activity, with arbutin as the sole carbon source, allowed two S. cerevisiae strains (patterns nos. 3′ and 19′) to be differentiated by means of this enzymatic activity. Yeast strains producing the killer toxin or with β-glucosidase activity are reported for the first time in autochthonous cider yeasts.     

Combined pH and high hydrostatic pressure effects on Lactococcus starter cultures and Candida spoilage yeasts in a fermented milk test system during cold storage

The combined effects of high pressure processing (HPP) and pH on the glycolytic and proteolytic activities of Lactococcus lactis subsp. lactis, a commonly used cheese starter culture and the outgrowth of spoilage yeasts of Candida species were investigated in a fermented milk test system. To prepare the test system, L. lactis subsp. lactis C10 was grown in UHT skim milk to a final pH of 4.30 and then additional samples for treatment were prepared by dilution of fermented milk with UHT skim milk to pH levels of 5.20 and 6.50. These milk samples (pH 4.30, 5.20 and 6.50) with or without an added mixture of two yeast cultures, Candida zeylanoides and Candida lipolytica (10⁵ CFU mL⁻¹ of each species), were treated at 300 and 600 MPa (≤20 °C, 5 min) and stored at 4 °C for up to 8 weeks. Continuing acidification by starter cultures, as monitored during storage, was substantially reduced in the milk pressurised at pH 5.20 where the initial titratable acidity (TA) of 0.40% increased by only 0.05% (600 MPa) and 0.10% (300 MPa) at week 8, compared to an increase of 0.30% in untreated controls. No substantial differences were observed in pH or TA between pressure-treated and untreated milk samples at pH 4.30 or 6.50. The rate of proteolysis in milk samples at pH values of 5.20 and 6.50 during storage was significantly reduced by treatment at 600 MPa. Treatment at 600 MPa also reduced the viable counts of both Candida yeast species to below the detection limit (1 CFU mL⁻¹) at all pH levels for the entire storage period. However, samples treated at 300 MPa showed recovery of C. lipolytica from week 3 onwards, reaching 10⁶–10⁷ CFU mL⁻¹ by week 8. In contrast, C. zeylanoides did not show any recovery in any of the pressure-treated samples during storage.     

Inhibitory effect of sour orange (Citrus aurantium) juice on Salmonella Typhimurium and Listeria monocytogenes

The sour orange (Citrus aurantium) juice is commonly used as flavoring and acidifying agent for vegetable salads and appetizers in Turkey. It was aimed to determine the survival and growth pattern of Salmonella Typhimurium and Listeria monocytogenes in sour orange juice. Different concentrations of neutralized and un-neutralized juice samples were inoculated with each of the test microorganisms (∼6 log CFU/mL) separately and then incubated at 4 °C and 37 °C for seven days. It was detected both of the test microorganisms could survive and even grow in neutralized juice samples at 37 °C for two days. However, none of them could survive at the end of seventh day of incubation at 37 °C. Low incubation temperature (+4 °C) increased the survival of the tested microorganisms. Also, it was detected that L. monocytogenes were less resistant to the variable conditions than S. Typhimurium. It was concluded that the antimicrobial effect of sour orange juice mainly depends on the low pH value of the product. However, incubation time and temperature are also effective on the survival of the tested pathogens.     

Effect of aromatic precursor addition to wine fermentations carried out with different Saccharomyces species and their hybrids

This work explores the ability of different yeast strains from different species of the genus Saccharomyces (S. cerevisiae, S. uvarum and S. kudriavzevii) and hybrids between these species to release or form varietal aroma compounds from fractions of grape odourless precursors. The de novo synthesis by the yeasts of some of the varietal aroma compounds was also evaluated. The study has shown that de novo synthesis affects some lipid derivatives, shikimic derivatives and terpenes in all species and hybrids, with some remarkable differences amongst them. The release or formation of aroma compounds from precursors was found to be strongly linked to the yeast or hybrid used, and the triple hybrid S. cerevisiae × S. bayanus × S. kudriavzevii in particular and secondarily the hybrid S. cerevisiae × S. bayanus were highly efficient in the production of most varietal aroma compounds, including γ-lactones, benzenoids, volatile phenols, vanillin derivatives and terpenols. The presence of precursors in the fermenting media caused a surprising levelling effect on the fermentative aroma composition. Altogether, these results suggest that it is possible to modulate wine aroma by employing different yeast species in order to create new wines with different aromatic notes.     

Redox effect on volatile compound formation in wine during fermentation by Saccharomyces cerevisiae

Although redox state is a well-known key process parameter in microbial activity, its impact on wine volatile aroma compounds produced during fermentation has not been studied in detail. In this study we report the effect of reductive and microaerobic conditions on wine aroma compound production using different initial amounts of yeast assimilable nitrogen (YAN: 180 and 400 mg N/l) in a simil grape must defined medium and two S. cerevisiae strains commonly used in wine-making. In batch fermentation culture conditions, reductive conditions were obtained using flasks plugged with Muller valves filled with sulphuric acid; while microaerobic conditions were attained with defined cotton plugs. It was found that significant differences in redox potential were obtained using the different plugs, and with variation of over 100 mV during the main fermentation period.