Influence of SO<Subscript>2</Subscript> on the evolution of volatile compounds through alcoholic fermentation of must stabilized by pulsed electric fields

The aim of this work was to study the influence of sulphur dioxide (SO₂) on the formation of volatile compounds by yeast through wine alcoholic fermentation. Thus Parellada must was microbiologically stabilized using a pulsed electric field (PEF) treatment and inoculated with Saccharomyces cerevisiae Na33 strain. Fermentation was carried out with or without SO₂ and the results showed that the evolution of the volatile compounds profile throughout the process was similar. The content of volatile acids in wine obtained by using sulphur dioxide was not significantly different from that fermented without adding the compound. However, the final content of total alcohols and esters was significantly different even thought the differences were small. Consequently, when grape must is treated by PEF the sulphur dioxide concentration usually used in winemaking could be reduced to safer levels or even eliminated without an important effect on the volatile compounds content of the final product. Therefore, the absence of sulphur dioxide should not have a negative impact on the sensory characteristics of wine.     

Fermentation of sulphite-free white musts with added lysozyme and oenological tannins: Nitrogen consumption and biogenic amines composition of final wines

The influence on amino acid consumption and biogenic amines composition of white wines obtained by replacing SO₂ during fermentation with lysozyme and tannins was studied. At the same time, the fermentative performance of two low SO₂ producing selected yeasts strains was evaluated. For this purpose, a series of laboratory-scale fermentations of fresh white must and a HPLC-DAD method for the analysis of amino acids, biogenic amines and ammonium ion were undertaken. The presence of SO₂ or lysozyme affected the consumption of nitrogen as a function of the yeast strain, while oenological tannin had no substantial influence. Strain 1042 increased the consumption of total YAN in the presence of SO₂, as a consequence of the enhanced utilization of ammonium ion and a number of amino acids. By contrast, strain 333 tended not to change the total YAN uptake, whatever the juice treatment, and reduced the consumption of aspartic and glutamic acids, GABA and other compounds in the case of samples added with SO₂. When compared with lysozyme addition, for both strains, SO₂ increased the consumption of alanine and glutamine, the latter being a major contributor to the assimilable nitrogen of the must. No influence of must treatments was found on the content of biogenic amines in the final wines.     

Effectiveness of dimethyldicarbonate to stop alcoholic fermentation in wine

The alcoholic fermentation of Botrytis-affected wines is stopped by the addition of high concentrations of sulfur dioxide (SO₂). The natural microbial unstability of these wines and the binding phenomena forces winemakers to periodically add sulfur dioxide during maturation, leading to a high concentration of a maximum of 400 mg/l in the bottled wine. Dimethyldicarbonate (DMDC) is now considered as a reliable fungicide which could be partially used instead of SO₂, especially just before bottling. This study investigated the use of DMDC to stop alcoholic fermentation. The experiment was carried out on pure cultures of three yeast species present in this type of wine (Saccharomyces cerevisiae, Candida stellata and Zygosaccharomyces bailii). The results were very promising and suggested that DMDC was more effective than SO₂. The yeast cells died after the addition of DMDC whereas they partially entered into a viable but non-culturable (VBNC) state with SO₂. However, the same experiment carried out on botrytized must, whose fermentation was carried out using indigenous microflora, was less conclusive. It pointed out that DMDC, used in a concentration of 200 mg/l, was more effective than SO₂ but leading to the same results: the entering of a part of the cells into a VBNC state. DMDC could be used to stop alcoholic fermentation, but could not replace SO₂. Nevertheless, the concentrations of SO₂ added in this type of wine could be reduced in this way.     

Effect of storage conditions on the volatile composition of wines obtained from must stabilized by PEF during ageing without SO2

The aim of this work was to study the effect of the storage conditions on the evolution of volatile composition of white wines aged in bottles without the addition of SO₂. Therefore Parellada must was stabilized by pulsed electric fields (PEF) and fermented without the employment of SO₂ and later on the wine was aged in bottles, without addition of this preservative, at low and controlled temperature and at room temperature. The obtained results showed that the concentration of some important compounds for wine aroma such as isoamyl acetate, and ethyl esters of fatty acids was higher in the wines stored at low and controlled temperature than in those aged at room temperature. However, the temperature favoured the formation of total alcohols during the aging of wines in the bottles. Consequently, from the point of view of the aromatic quality, the conservation of white wines obtained from fermentation of must processed by PEF and aged in bottles without the addition of SO₂ was improved under controlled storage conditions than at room temperature.

Industrial relevance

SO₂ is used as a preservative agent in wine due to its multi-action in the wine conservation. Although neither carcinogenicity nor mutagenic effects have been found in SO₂, this compound has an influence on human health. For that reason, several competent international organizations (WHO, FAO, OIV) have set down maximum limits for wines as well as promote a reduction of its concentration in wines. Therefore, potential industrial applications of this work include the possibility to produce and store wines without SO₂. This has been achieved by stabilizing the musts with PEF. The wines produced under these conditions can be conserved without this additive when used under controlled conditions of storage.     

Co-inoculation of different Saccharomyces cerevisiae strains and influence on volatile composition of wines

Wine is the result of the performance of different yeast strains throughout the fermentation in both spontaneous and inoculated processes. 22 Saccharomyces cerevisiae strains were characterized by microsatellite fingerprinting, selecting 6 of them to formulate S. cerevisiae mixed cultures. The aim of this study was to ascertain a potential benefit to use mixed cultures to improve wine quality. For this purpose yeasts behavior was studied during co-inoculated fermentations. Aromatic composition of the wines obtained was analyzed, and despite the fact that only one strain dominated at the end of the process, co-cultures released different concentrations of major volatile compounds than single strains, especially higher alcohols and acetaldehydes. Nevertheless, no significant differences were found in the type and quantity of the amino acids assimilated. This study demonstrates that the final wine composition may be modulated and enhanced by using suitable combinations of yeast strains.     

Effect of the addition of different quantities of amino acids to nitrogen-deficient must on the formation of esters, alcohols, and acids during wine alcoholic fermentation

In this work the effect of the addition of different quantities of amino acids to a nitrogen-deficient must on the formation of volatile compounds during the wine alcoholic fermentation was studied. To do so, fermentations of Mazuelo must were carried out, to which were added ammonium and 45, 120, and 250 mg/l of amino acids. The results showed that the formation of total esters, isoamyl acetate, and 2-phenylethyl acetate was directly proportional to the quantity of amino acids added to the must while the synthesis of diethyl succinate, and ethyl 3-hydroxybutyrate was inversely proportional to this addition. The alcohols, with the exception of tyrosol and 2-phenylethanol, did not show any direct correlation between their formation and the addition of amino acids. The quantity of amino acids added to the must favoured the formation of total acids but it had scant influence on the individual synthesis of the acids. So, it may be stated that, in general, the addition of amino acids in the concentrations under study to a nitrogen-deficient must, favoured the formation of volatile compounds in the wine.     

Influence of yeast strain on binding of sulphur dioxide in wines,and on its formation during fermentation

The amounts of sulphur dioxide bound by acetaldehyde, pyruvic acid and α-ketoglutaric acid during fermentation of three grape juices by eight wine yeasts (Saccharomyces sp.) are reported. These constituents accounted for 49–83 % (mean 69) of the measured bound SO₂, depending on the yeast strain and juice. the maximum range of concentrations of the binding components for individual wines were 10–48 ppm for acetaldehyde, 9–77 ppm for pyruvic acid and 5–63 ppm for α-ketoglutaric acid, depending on yeast strain and grape juice. the validity of the calculations was verified by an experiment with SO₂ and the three binding compounds in a multicomponent model system. The acetaldehyde content was related to the total SO₂ present, which itself was determined by the strain of yeast. SOz bound in the wines after a further SO₂ addition was correlated significantly with pyruvic and α-ketoglutaric acids, but not with acetaldehyde. Certain yeasts produced SO₂ during fermentation in grape juice and in synthetic media with defined sulphur sources. More SO₂ was produced at pH 3.6 than 3.0 in the absence of added sulphate in grape juice. Sulphate was the best sulphur source for SO₂ production in synthetic media, although some yeasts were able to produce smaller amounts of SO₂ from l-cysteine and reduced glutathione.     

Selenium biotransformation by Saccharomyces cerevisiae and Saccharomyces bayanus during white wine manufacture: Laboratory-scale experiments

The main purpose of this laboratory-scale study was to evaluate the transformation of inorganic selenium, as sodium selenite, when added to white grape juice as part of the fermentation process of white wine. The participation of yeast, added in the fermentation of the must, is necessary to convert inorganic selenium into organoseleno species. Two different yeasts, Saccharomyces cerevisiae and Saccharomyces bayanus were used for fermentation. The effects of different Se concentrations on cells and their viability during fermentation were evaluated. The alcoholic fermentation that produced wine was not affected by the presence of selenium, regardless of the type of Saccharomyces used. After 21 days of fermentation, the white wine and residual yeast were separated and analysed by ICP-MS and LC–ICP-MS for determination of total selenium and speciation. Selenomethionine was found to be the main Se-species in the selenised white wine. The results obtained are preliminary but they could be considered for future studies using both pilot and full-scale vinification processes.     

The effect of nitrogen source on yeast metabolism and H2S formation

The impacts of aspartic acid and glutamine used as nitrogen supplements for alcoholic fermentations conducted by Saccharomyces were studied. Synthetic grape juice media and commercially prepared grape juices were supplemented with diammonium phosphate, aspartic acid, or glutamine to increase yeast-assimilable nitrogen concentrations to 250 mg N/L prior to fermentation. Two yeast strains, UCD522 and EC1118, were inoculated at 10⁵ CFU/mL and fermentations were monitored for soluble solids, hydrogen sulfide (H₂S), and residual amino acids. In general, unsupplemented media/juices fermented slower than supplemented ones, produced more H₂S, and contained lower concentrations of amino acids after fermentation. Among the supplemented treatments, fermentation rates, H₂S production, and amino acid utilization varied depending on the nitrogen source and yeast strain. Those fermentations supplemented with aspartic acid were generally slower and sometimes did not achieve dryness. In contrast, glutamine additions yielded fermentation rates and H₂S production equivalent or better than other supplemented treatments. Based on these results, the use of glutamine appears promising as an alternative nitrogen supplement for wine production.     

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.     

Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d'Abruzzo red wine quality

The growth of selected, indigenous Saccharomyces cerevisiae added as starters (SRS1, MS72 and RT73) was monitored during Montepulciano d'Abruzzo wine production. In all the fermentations the addition of the starter, caused a decrease of the non-Saccharomyces yeasts. When strains MS72 and RT73 were used as starters they were detected in the first phases of fermentations, while strain SRS1 competed successfully with native yeasts during all the process. Wines obtained by fermentation with the indigenous starters showed some different characteristics, according to the chemical and sensory analyses. This study highlighted that among selected starters with high fermentative capacity, some are able to dominate better than other natural wine yeast biota, whereas some strains can interact and survive besides native yeast populations during the fermentation. As a consequence, the dominance character can have a positive or negative effect on wine quality and has to be considered in the frame of yeast selection in order to improve or characterize traditional wines. Winemakers could choose among different degrees of yeast dominance to modulate the interaction among starter and native wine yeast population.     

Taqman real-time PCR for the detection and enumeration of Saccharomyces cerevisiae in wine

Saccharomyces cerevisiae is the main yeast species responsible for wine fermentation; however, its presence during maturing or barrel-ageing can sometimes result in a reduction in the quality of wine by refermentation. In this work, we developed a quantitative real-time PCR (QPCR) for the rapid detection and quantification of S. cerevisiae in wine. The primers and the hydrolysis probe (TaqMan^®) were designed from the sequence of a DNA fragment present only in S. cerevisiae and absent in other wine yeasts obtained from an RAPD-PCR analysis. The QPCR developed was highly reproducible, allowing the specific detection and quantification of this yeast in artificially contaminated wines, with a detection limit of 78 CFU/mL. Furthermore, the usefulness of the QPCR developed was evaluated through the quantification of the yeast in wine samples obtained from vineyards, confirming the quantitative capacity of the method. The methodology developed was specific, fast and a sensitive tool for the detection and enumeration of S. cerevisiae cells in wine.     

Influence of addition of yeast autolysate on the formation of amines in wine

Amines, substances which can be toxic or have a negative effect on wine aroma, are formed during alcoholic and malolactic fermentations depending on various factors. In this work the influence of the addition of yeast autolysate to must on the concentration of amines in wine is studied. For this purpose Chardonnay must supplemented with the yeast autolysate was used; a second sample was also used without it (control sample). This autolysate provided amino acids and long‐chain fatty acids (C16 and C18). The enrichment of must by these nutrients did not produce an increase of amines during alcoholic fermentation despite the fact that consumption of amino nitrogen in general, and of some precursor amino acids in particular, was somewhat higher in the supplemented sample. After malolactic fermentation it was observed that the concentration of biogenic amines was higher in the wine from the supplemented must. Copyright © 2006 Society of Chemical Industry     

Influence of addition of ammonium and different amino acid concentrations on nitrogen metabolism in spontaneous must fermentation

The effect of the addition of different amino acid concentrations in must on yeast nitrogen metabolism during alcoholic fermentation was studied. To do this, fermentations of Mazuelo must, poor in nitrogen compounds, were carried out. Ammonium and different concentrations of amino acids (0, 45, 120, 250 and 450 mg/l) were added to the must. Addition of 45, 120 and 250 mg/l of proteic amino acids to the must increased the rate of fermentation. Proline was mainly consumed in fermentations with smaller amounts of amino nitrogen and, at the same time, this amino acid showed the highest residual concentration in the final wines. The consumption of other proteic amino acids was directly proportional to their concentration in the musts, with the exception of leucine and isoleucine that were synthesized. However, a difference in the percentages of the amino acids consumed by the yeasts was observed. The percentages of aspartic acid, alanine and arginine consumed were higher in the fermentations supplemented with amino acids than in the fermentation where only ammonium was added. The percentages of tyrosine and phenylalanine consumed gradually increased with increase of their initial concentration.     

The grape must non-Saccharomyces microbial community: impact on volatile thiol release

Several studies have reported the beneficial influence of non-Saccharomyces yeasts and their potential applications in the wine industry, mainly in mixed-culture fermentation with S. cerevisiae. The potential impact of 15 non-Saccharomyces strains from 7 species on 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) release in model medium and Sauvignon Blanc must was evaluated after partial fermentation. Whereas the impact of non-Saccharomyces on 4MSP release in both media was low, some M. pulcherrima, T. delbrueckii and K. thermotolerans strains had a high capacity to release 3SH, despite their minimal fermentation activity. As previously demonstrated for Saccharomyces yeast, this contribution is strain dependant. Taking into account their dynamic and quantitative presence during the whole process, the real impact of non-Saccharomyces yeast on 4MSP and 3SH release was evaluated using a recreated community simulating the yeast ecosystem. Our results revealed a positive impact on 3SH release in Sauvignon Blanc wines by promoting non-Saccharomyces yeast activity and delaying the growth of S. cerevisiae. Some non-Saccharomyces yeast strains are capable of making a positive contribution to volatile thiol release in wines, essentially during the pre-fermentation stage in winemaking, when this microbiological sub-population is dominant.     

Determination of viable wine yeast using DNA binding dyes and quantitative PCR

The detection and quantification of wine yeast can be misleading due to under or overestimation of these microorganisms. Underestimation may be caused by variable growing rates of different microorganisms in culture media or the presence of viable but non-cultivable microorganisms. Overestimation may be caused by the lack of discrimination between live and dead microorganisms if quantitative PCR is used to quantify with DNA as the template. However, culture-independent methods that use dyes have been described to remove the DNA from dead cells and then quantify the live microorganisms. Two dyes have been studied in this paper: ethidium monoazide bromide (EMA) and propidium monoazide bromide (PMA). The technique was applied to grape must fermentation and ageing wines. Both dyes presented similar results on yeast monitoring. Membrane cell recovery was necessary when yeasts were originated from ethanol-containing media. When applied to grape must fermentation, differences of up to 1 log unit were seen between the QPCR estimation with or without the dye during the stationary phase. In ageing wines, good agreement was found between plating techniques and QPCR. Most of the viable cells were also culturable and no differences were observed with the methods, except for Zygosaccharomyces bailii and Dekkera bruxellensis where much higher counts were occasionally detected by QPCR. The presence of excess dead cells did not interfere with the quantification of live cells with either of the dyes.     

Pre-fermentative replacement of sulphur dioxide by lysozyme and oenological tannins: Effect on the formation and evolution of volatile compounds during the bottle storage of white wines

In this work, the effects on the volatile profile of the pre-fermentative substitution of SO₂ with lysozyme and oenological tannins were studied in white wines. At the same time, in order to understand the changes of volatile compounds in SO₂-free wines, the evolution of volatiles was evaluated over 1 year of storage in bottles. For this purpose, a number of laboratory scale fermentations of SauvignonBlanc musts were carried out and the effects of three variables (SO₂, lysozyme and oenological tannins) were investigated by means of GC–MS analysis. Results showed that the replacement of SO₂ with lysozyme and oenological tannins influenced the volatile composition of wines at the end of the alcoholic fermentation. Wines fermented with SO₂ showed higher total alcohol amounts, while the presence of oenological tannins augmented the level of esters. The presence of SO₂ influenced also the alcohols and esters profiles of wines during bottle storage. Moreover, the presence of oenological tannins displayed a positive role in maintaining the amounts of esters over certain levels in wine stored for 1 year, likely due to their oxygen scavenging ability. By contrast, acids were less affected by the investigated adjuvants both at the end of the alcoholic fermentation and during the storage time.     

Production of white wine by Saccharomyces cerevisiae immobilized on grape pomace

White wine was produced with Saccharomyces cerevisiae cells immobilized on grape pomace, by natural adsorption. The support, the main solid waste from the wine industry, consisted of the skins, seeds and stems. Immobilization was tested using different media, namely complex culture medium, raw grape must and diluted grape must. Grape pomace was revealed to be an appropriate support for yeast cell immobilization. Moreover, grape must was shown to be the most suitable medium as immobilized cells became adapted to the conditions in the subsequent alcoholic fermentation in the wine‐making process. The wines produced, either with immobilized cells or with free cells, were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds); additionally, colour (CIELab) and sensory analysis were performed. The use of immobilized systems to conduct alcoholic fermentation in white wine production proved to be a more rapid and a more efficient process, especially when large amounts of SO₂ were present in the must. Furthermore, the final wines obtained with immobilized cells demonstrated improved sensory properties related to the larger amounts of ethanol and volatile compounds produced. The more intense colour of these wines could be a drawback, which could be hindered by the reutilization of the biocatalyst in successive fermentations. Copyright © 2012 The Institute of Brewing & Distilling     

Effects of Combined Antimicrobial Agents on Fermentation Initiation by Saccharomyces cerevisiae in a Model Broth System

Effects of sodium benzoate, potassium sorbate, sodium metabisulfite (SO₂), butyl p-hydtoxybenzoic acid and their dual combinations were observed against the ability of the wine yeast, Saccharomyces cerevisiae, Montrachet 522, to begin a fermentation in yeast nitrogen base-glucose broth at pH 3.30. Antimicrobial activities of equal molar concentrations of compounds and combinations of compounds were compared. Butylparaben and combinations involving butylparaben most effectively inhibited fermentation initiation. Combinations involving SO₂ had lower antimicrobial activities than either SO₂ or the other individual compound alone indicating antagonistic effects between SO₂ and the other antimicrobial agents tested. Apparent additive antimicrobial effects were observed for butylparaben/sorbate, butylparaben/ benzoate and benzoate/sorbate.