Monitoring the influence of high-gravity brewing and fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast

During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active substances, which are vital for the complex flavour of beer. In order to obtain insight into the influence of high-gravity brewing and fermentation temperature on flavour formation, we analysed flavour production and the expression level of ten genes (ADH1, BAP2, BAT1, BAT2, ILV5, ATF1, ATF2, IAH1, EHT1 and EEB1) during fermentation of a lager and an ale yeast. Higher initial wort gravity increased acetate ester production, while the influence of higher fermentation temperature on aroma compound production was rather limited. In addition, there is a good correlation between flavour production and the expression level of specific genes involved in the biosynthesis of aroma compounds. We conclude that yeasts with desired amounts of esters and higher alcohols, in accordance with specific consumer preferences, may be identified based on the expression level of flavour biosynthesis genes. Moreover, these results demonstrate that the initial wort density can determine the final concentration of important volatile aroma compounds, thereby allowing beneficial adaptation of the flavour of beer.     

Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.     

Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines

Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast.     

Metabolic profiling as a tool for revealing Saccharomyces interactions during wine fermentation

The multi-yeast strain composition of wine fermentations has been well established. However, the effect of multiple strains of Saccharomyces spp. on wine flavour is unknown. Here, we demonstrate that multiple strains of Saccharomyces grown together in grape juice can affect the profile of aroma compounds that accumulate during fermentation. A metabolic footprint of each yeast in monoculture, mixed cultures or blended wines was derived by gas chromatography - mass spectrometry measurement of volatiles accumulated during fermentation. The resultant ion spectrograms were transformed and compared by principal-component analysis. The principal-component analysis showed that the profiles of compounds present in wines made by mixed-culture fermentation were different from those where yeasts were grown in monoculture fermentation, and these differences could not be produced by blending wines. Blending of monoculture wines to mimic the population composition of mixed-culture wines showed that yeast metabolic interactions could account for these differences. Additionally, the yeast strain contribution of volatiles to a mixed fermentation cannot be predicted by the population of that yeast. This study provides a novel way to measure the population status of wine fermentations by metabolic footprinting.     

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast

Low‐temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low‐temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:776–783, 2014     

Influence of wine fermentation temperature on the synthesis of yeast-derived volatile aroma compounds

The yeast Saccharomyces cerevisiae synthesises a variety of volatile aroma compounds during wine fermentation. In this study, the influence of fermentation temperature on (1) the production of yeast-derived aroma compounds and (2) the expression of genes involved in aroma compounds’ metabolism (ADH1, PDC1, BAT1, BAT2, LEU2, ILV2, ATF1, ATF2, EHT1 and IAH1) was assessed, during the fermentation of a defined must at 15 and 28°C. Higher concentrations of compounds related to fresh and fruity aromas were found at 15°C, while higher concentrations of flowery related aroma compounds were found at 28°C. The formation rates of volatile aroma compounds varied according to growth stage. In addition, linear correlations between the increases in concentration of higher alcohol and their corresponding acetates were obtained. Genes presented different expression profiles at both temperatures, except ILV2, and those involved in common pathways were co-expressed (ADH1, PDC1 and BAT2; and ATF1, EHT1 and IAH1). These results demonstrate that the fermentation temperature plays an important role in the wine final aroma profile, and is therefore an important control parameter to fine-tune wine quality during winemaking.     

Characterization of volatile fraction of typical Irpinian wines fermented with a new starter yeast

Non-Saccharomyces yeasts are microorganisms that play an important role in the fermentation dynamics, compositions and flavour of wine. The aromatic compounds responsible for varietal aroma in wine are mainly terpenes, of which the most important group are the monoterpenes because of their volatility and odour if present in a free form. In fact, some terpenyl-glycosides do not contribute to the aroma unless they are hydrolysed. The glycosylated form of terpenes can be converted by hydrolysis with β-glycosidases produced by yeasts during the winemaking process, into aromatic compounds. In this study we utilized a non-Saccharomyces yeast, with a high extra-cellular glycosidase activity, isolated from grapes of cultivars typical of Irpinia region. This strain, identified as a Rhodotorula mucillaginosa (strain WLR12), was used to carry out an experimental winemaking process and the results were compared with those obtained with a commercial yeast starter. Chemical and sensorial analysis demonstrated that the wines produced with WLR12 strain had a more floral aroma and some sweet and ripened fruit notes compared to those obtained with commercial yeast. The data also showed an increasing of the free terpenes fraction that, however, did not significatively modify the bouquet of the wines.     

Evaluation of Fermentation Efficiency of Yeast Strains and their Effect on Quality of Young Wines

The yeast has important role in fermentation of wine grapes and wine quality. The fermentation of wine grapes affect by efficiency of particular yeast strain, sugar content, pH, available temperature, etc. To evaluate the efficiency of yeast strains (Premier Cuvee, RS-1, RS-2, RS-3 and natural), present study was conducted on two wine grape varieties viz.; Sauvignon Blanc (White) and Cabernet Sauvignon (Red). Efficiency of yeast strains was evaluated in terms of conversion rate of sugar into alcohol. As per recorded data, strain RS-3 (Pichia kudriavzevii) was found more efficient than other strains in fermentation of Cabernet Sauvignon with efficiency of 84.4 per cent but in case of Sauvignon Blanc, the commercial culture Premier Cuevee was found superior over RS-3. The quality parameters of young wines of both the varieties were also affected by the used strains. Considering the efficiency and impact on various parameters of wines, local strain, i.e., RS-3 was found at par with commercial culture (Premier Cuvee). The RS-3 strain has potential to produce quality wines. However, studies on effects of RS-3 strain on some specific quality parameters of wines like varietal aroma compounds, flavours etc. are needed.     

Genetically different wine yeasts isolated from Austrian vine-growing regions influence wine aroma differently and contain putative hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii

To evaluate the influence of the genomic properties of yeasts on the formation of wine flavour, genotypic diversity among natural Saccharomyces cerevisiae strains originating from grapes collected in four localities of three Austrian vine-growing areas (Thermenregion: locations Perchtoldsdorf and Pfaffst?tten, Neusiedlersee-Hügelland: location Eisenstadt, Neusiedlersee: location Halbturn) was investigated and the aroma compounds produced during fermentation of the grape must of 'Grüner Veltliner' were identified. Amplified fragment length polymorphism analysis (AFLP) showed that the yeast strains cluster in four groups corresponding to their geographical origin. The genotypic analysis and sequencing of the D1/D2 domain of 26S rRNA encoding gene and ITS1/ITS2 regions indicated that the Perchtoldsdorf strains were putative interspecies hybrids between S. cerevisiae and Saccharomyces kudriavzevii. Analysis of the aroma compounds by GS/MS indicated a region-specific influence of the yeasts on the chemical composition of the wines. The aroma compound profiles generated by the Perchtoldsdorf strains were more related to those produced by the Pfaffst?tten strains than by the Eisenstadt and Halbturn strains. Similar to the Pfaffst?tten yeasts, the putative hybrid strains were good ester producers, suggesting that they may influence the wine quality favourably.     

Modulating aroma compounds during wine fermentation by manipulating carnitine acetyltransferases in Saccharomyces cerevisiae

The wine yeast Saccharomyces cerevisiae is central in the production of aroma compounds during fermentation. Some of the most important yeast-derived aroma compounds produced are esters. The esters ethyl acetate and isoamyl acetate are formed from alcohols and acetyl-CoA in a reaction catalysed by alcohol acetyltransferases. The pool of acetyl-CoA available in yeast cells could play a key role in the development of ester aromas. Carnitine acetyltransferases catalyse the reversible reaction between carnitine and acetyl-CoA to form acetylcarnitine and free CoA. This reaction is important in transferring activated acetyl groups to the mitochondria and in regulating the acetyl-CoA/CoA pools within the cell. We investigated the effect of overexpressing CAT2, which encodes the major mitochondrial and peroxisomal carnitine acetyltransferase, on the formation of esters and other flavour compounds during fermentation. We also overexpressed a modified CAT2 that results in a protein that localizes to the cytosol. In general, the overexpression of both forms of CAT2 resulted in a reduction in ester concentrations, especially in ethyl acetate and isoamyl acetate. We hypothesize that overproduction of Cat2p favours the formation of acetylcarnitine and CoA and therefore limits the precursor for ester production. Carnitine acetyltransferase expression could potentially to be used successfully in order to modulate wine flavour.     

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.     

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.     

Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol

Branched-chain amino acids (BCAAs) are key substrates in the formation of fusel alcohols, important flavour components in fermented foods. The first step in the catabolic BCAA degradation is a transaminase step, catalyzed by a branched-chain amino acid transaminase (BCAAT). Saccharomyces cerevisiae possesses a mitochondrial and a cytosolic BCAAT, Bat1p and Bat2p, respectively. In order to study the impact of the BCAATs on fusel alcohol production derived from the BCAA metabolism, S. cerevisiae BCAAT-deletion mutants were constructed. The BCAA l-leucine was exogenously supplied during cultivations with mutants of S. cerevisiae. BAT1 deletion is not essential for fusel alcohol production, neither under glucose nor under ethanol growth conditions. The 3-methyl-1-butanol production rate of bat1Delta-cells on ethanol was decreased in comparison with that of wild-type cells, but the cells were still able to produce 3-methyl-1-butanol. However, drastic effects in fusel alcohol production were obtained in cells lacking BAT2. Although the constructed bat2Delta-single deletion strain and the bat1Deltabat2Delta-double deletion strain were still able to produce 3-methyl-1-butanol when grown on glucose, they were incapable of producing any 3-methyl-1-butanol when ethanol was the sole carbon source available. In the circumstances used, gene expression analysis revealed a strong upregulation of BAT2 gene activity in the wild type, when cells grew on ethanol as carbon source. Apparently, the carbon metabolism is able to influence the expression of BCAATs and interferes with the nitrogen metabolism. Furthermore, analysis of gene expression profiles shows that the expression of genes coding for other transaminases present in S. cerevisiae was influenced by the deletion of one or both BCAATs. Several transaminases were upregulated when a BCAAT was deleted. Strikingly, none of the known transaminases was significantly upregulated when BAT2 was deleted. Therefore we conclude that the expression of BAT2 is essential for 3-methyl-1-butanol formation on the non-fermentable carbon source, ethanol.     

Dynamics of indigenous and inoculated yeast populations and their effect on the sensory character of Riesling and Chardonnay wines

To study the impact of yeast populations on wine flavour and to better understand yeast growth dynamics, wines were produced by the (i) indigenous microflora, (ii) vigorous yeast starter EC1118 and (iii) slowly fermenting yeast Assmannshausen. Sensory analysis revealed that wines differed depending on the fermentation type. However, these yeast-related differences did not exceed the varietal character. Both added starter cultures clearly dominated the Saccharomyces population from the middle of fermentation onwards. The starter cultures differed in their repression of indigenous non- Saccharomyces yeast. EC1118 limited growth of non- Saccharomyces yeasts more strongly than Assmannshausen. Sulphite addition further repressed growth of non- Saccharomyces yeasts. On completion, more than one Saccharomyces strain was present in each fermentation, with the largest variety in the non-inoculated and the smallest in the EC1118-inoculated fermentation. Results from the two genetic assays, karyotyping, and PCR using δ-primers were not fully equivalent, limiting the usefulness of δ-PCR in studies of native Saccharomyces yeasts.     

Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts

Two volatile thiols, 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical varietal aroma compounds in Sauvignon Blanc (SB) wines. These aromatic thiols are not present in the grape juice but are synthesized and released by the yeast during alcoholic fermentation. Single deletion mutants of 67 candidate genes in a laboratory strain of Saccharomyces cerevisiae were screened using gas chromatography mass spectrometry for their thiol production after fermentation of SB grape juice. None of the deletions abolished production of the two volatile thiols. However, deletion of 17 genes caused increases or decreases in production by as much as twofold. These 17 genes, mostly related to sulfur and nitrogen metabolism in yeast, may act by altering the regulation of the pathway(s) of thiol production or altering substrate supply. Deleting subsets of these genes in a wine yeast strain gave similar results to the laboratory strain for sulfur pathway genes but showed strain differences for genes involved in nitrogen metabolism. The addition of two nitrogen sources, urea and di-ammonium phosphate, as well as two sulfur compounds, cysteine and S-ethyl-L-cysteine, increased 3MH and 3MHA concentrations in the final wines. Collectively these results suggest that sulfur and nitrogen metabolism are important in regulating the synthesis of 3MH and 3MHA during yeast fermentation of grape juice.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

Influence of the yeast strain on the changes of the amino acids,peptides and proteins during sparkling wine production by the traditional method

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002