Limit SO2 content of wines by applying High Hydrostatic Pressure

Sulphur dioxide (SO₂) has an important role in wine industry as an antioxidant, antioxidasic and antiseptic additive. However, since SO₂ is also responsible for allergic reactions, it is of great interest its replacement with alternative additives or technologies. The potential use of High Pressure (HHP) as an alternative antimicrobial technique to preserve wine with reduced amounts of SO₂ was explored in this study. Red wine samples containing 0, 30, 60 and 100 mg/L total sulphur dioxide (SO₂) were subjected to pressure treatment at 350 MPa for 10 min at 8 °C. Another group of non-treated samples containing similar amounts of SO₂ was used as control. Color parameters, total flavanols and antioxidant activity were determined by spectrophotometry while for individual anthocyanin content and tannin structural characteristics HPLC and HPLC/MS were employed respectively. Analyses were initially performed every two months (up to 6 months of storage) and thereafter at 12 months. Sensory analysis was also performed at the end of storage period by a trained panel. Pressurised wines with <60 mg/L SO₂ showed reduced total flavanols, anthocyanins and antioxidant activity values and higher hue after six months of storage when compared to the non-treated samples. These results were in line with the sensory evaluation of the treated samples, perceived as less aromatic, more oxidized and with higher intensity of dried fruit odor. However, after twelve months of storage these differences between the non-pressurised and pressurised wines, which contained over 60 mg/L SO_2, were not significant indicating that HHP could potentially preserve wine when combined with addition of reduced contents of SO_2.Industrial relevanceSince SO₂ is a cheap and effective wine preservative; its complete replacement seems not feasible at the moment. However, a more realistic approach is the reduction of the quantities required for wine protection and preservation by combining the antimicrobial action of the HHP process and the antioxidant effect of Sulphur Dioxide. This work shows that HHP can potentially be employed in winemaking to complement the action of SO₂ making thus possible to reduce the required doses in wine.     

Chemical and physical methodologies for the replacement/reduction of sulfur dioxide use during winemaking: review of their potentialities and limitations

Sulfur dioxide (SO₂) is probably one of the most versatile and efficient additives used in winemaking due to its antiseptic and antioxidant properties. This compound is also important for minimizing phenolic polymerization rate and color loss during wine aging. However, allergies caused by SO₂-derived compounds, namely the sulfites, are becoming more frequent, causing symptoms such as headaches, nausea, gastric irritation, and breathing difficulties in asthma patients. Consequently, the legislated maximum concentration of SO₂ allowed in wines has been gradually reduced. For this reason, it is crucial in a competitive global winemaking market strategy, to reduce or even eliminate the use of SO₂ as a preservative and to search for new healthier and safe strategies. This work gives an overview of the main methodologies that have been proposed so far and that have potential to be used in winemaking as an alternative to SO₂. The addition of compounds such as dimethyl dicarbonate, bacteriocins, phenolic compounds, and lysozyme, and the use of physical methods, namely pulsed electric fields, ultrasound, ultraviolet radiation, and high pressure are discussed and critically evaluated.     

Some new findings on the potential use of biocompatible silver nanoparticles in winemaking

There is currently an increasing commercial demand for silver nanoparticles (Ag NPs) due to their wide applicability in various markets. Because of their powerful antimicrobial properties, these nanoparticles are frequently used for food-associated consumer products. In this paper, the effect of two Ag NPs coated with biocompatible materials – PEG-Ag NPs 20 (polyethylene glycol) and GSH-Ag NPs (reduced glutathione) – to control microbial growth in wines was assessed. Both Ag NPs were subjected to an in vitro three-step digestion, and changes in their morphology and an assessment of their cytotoxicity against Caco-2 cells were determined. Both Ag NPs were effective against the different microbial population present in tested wines. Regarding their in vitro digestion, the size and shape of the nanoparticles were almost unaltered in the case of GSH-Ag NPs, while in PEG-Ag NPs 20 some particle agglomeration was observed. Overall, these results suggest that Ag NPs may reach the intestine in a nano-scaled form. Finally, Caco-2 cell experiments seemed to exclude toxicity of Ag NPs at the intestinal epithelium.Industrial relevanceTraditionally, sulphur dioxide (SO₂) has been used by oenologists to control the microbial population in wine. As a result of increasing evidence of possible health risks associated with this additive in wine, there has been growing interest in finding new alternatives to replace it. Silver nanoparticles display a broad spectrum of antimicrobial activity, so they could constitute a very promising approach to reducing SO₂ in winemaking.     

Replacement of sulfur dioxide by lysozyme and oenological tannins during fermentation: influence on volatile composition of white wines

BACKGROUND: In recent years the use of sulfur dioxide, a commonly used additive in winemaking, has been questioned because of its toxic effects on human health. Studies have been conducted to find alternatives that can effectively substitute for this additive in all its various technological functions. In previous work, lysozyme and oenological tannins were found as possible substitutes in controlling bacterial undesirable fermentations and phenolic oxidation. However, data on the volatile composition of wines obtained by that protocol are lacking. In this work, the effects on volatile composition of white wines by the substitution of SO₂ during fermentation with lysozyme and tannin were studied. At the same time, the technological performance of two strains of yeast that produce low amounts of SO₂ were evaluated. RESULTS: The results showed that both SO₂ and lysozyme prevented the development of undesirable bacterial fermentations. The study of volatile compounds shows differences in the alcohol, acid and ester contents among the final products: wines fermented with strain 1042 and lysozyme had higher total alcohol concentration, while the addition of SO₂ promoted higher production of 3‐methyl‐1‐butanol, 3‐methylthio‐1‐propanol, phenylethyl alcohol and 4‐hydroxy‐benzenethanol. Esters, as a total, were influenced by the different strain and tannins addition, while amounts of medium‐chain fatty acid ethyl esters and their corresponding fatty acids were found in higher amounts in wines coming from fermentations with lysozyme. The sensory analysis revealed a preference for wines to which lysozyme and tannins had been added. CONCLUSION: The data suggest that the addition of lysozyme and oenological tannins during alcoholic fermentation could represent a promising alternative to the use of SO₂ and for the production of wines with reduced content of SO₂. The composition of the volatiles in the final wines was affected by the different vinification protocols (mainly with regards to alcohols and ethyl esters). Copyright © 2009 Society of Chemical Industry     

Pulsed Electric Field treatment after malolactic fermentation of Tempranillo Rioja wines: Influence on microbial,physicochemical and sensorial quality

During wine stabilization, control of the microbial population is required in order to ensure a quality wine. Pulsed Electric Field (PEF) was applied to commercial wines in a continuous flow to reduce the amount of SO₂ added after the end of malolactic fermentation to manage microbial populations. Samples of wine from three commercial wineries of the Rioja Qualified Designation of Origin were treated with 30 mg/L SO₂, with 15 mg/L SO₂ and PEF, and only with PEF. The inactivation of the microbiota with the PEF treatment was greater than the inactivation achieved with SO₂ four days after treatment. Six months later, the lactic acid bacteria population was still viable in some of the wines. Regarding the physicochemical characterization of the wines, the PEF treatment was positive for quality because the wines had greater color intensity and lower volatile acidity. Moreover, organoleptic analysis indicated different scoring depending mainly on the characteristics of the original wine. Despite these positive results, further studies are advisable to optimize the control of lactic acid bacteria.Industrial relevanceThe application of PEF during wine stabilization after MLF can be a viable alternative for the oenological industry to reduce the SO₂ dosage used in winemaking.     

Effects of different vinification technologies on physico-chemical properties and antioxidant activity of ‘Falanghina’ and ‘Bombino bianco’ wines

The present research was aimed to compare the traditional white vinification with a combined cryomaceration–vinification in reductive conditions, by evaluating their effects on chemical and physical indices and on antioxidant compounds of ‘Falanghina’ and ‘Bombino bianco’, two wine grape cultivars of southern Italy. According to the results, the two cultivars showed similar behaviour in response to the same vinification protocol. The processing conditions mainly affected physico-chemical characteristics such as titratable acidity, pH, SO₂, tartaric and malic acids, phenolic concentrations, and antioxidant activity. Titratable acidity decreased with cryomaceration due to the tartrate precipitation, whereas the malic acid was greatly extracted from pulps and skins. In the reductive conditions applied, the SO₂ was protected by the presence of ascorbic acid; thus, the wines exhibited higher free/total SO₂ ratio. Cryomaceration combined with vinification in reductive conditions allowed a greater extraction of phenolic compounds from skins and, consequently, a greater antioxidant activity compared to the traditional winemaking. This behaviour was due to both the protection exerted by the low temperatures and the solubilisation of proanthocyanidins and flavans reactive with vanillin favoured by the high free SO₂ content.     

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.     

Effects of reduced levels of sulfite in wine production using mixtures with lysozyme and dimethyl dicarbonate on levels of volatile and biogenic amines

Sulphur dioxide (SO₂) is an important preservative for wine, but its presence in foods can cause allergies and this has given impetus to the research for alternatives. The aim of this study was to reduce levels of sulfite in wine production using mixtures with lysozyme and dimethyl dicarbonate and examine the influence on levels of volatile and biogenic amines. To do so, vinifications were carried out using lysozyme, dimethyl dicarbonate (DMDC) and mixtures of these with SO₂ in different concentrations (25 and 50 mg l^?1). Results were compared with a control vinification with only SO₂ (50 mg l^?1). Mixing low concentrations of SO₂ with lysozyme and DMDC reduced the concentration of biogenic amines (histamine, tyramine, putrescine, cadaverine, phenylethylamine + spermidine and spermine). In general, the total concentration of volatile amines (dimethylamine, isopropylamine, isobutylamine, pyrrolidine, ethylamine, diethylamine, amylamine and hexylamine) was higher in the sample fermented only with SO₂. The concentrations of amines with secondary amino groups (dimethylamine, diethylamine, pyrrolidine) were higher in the sample only fermented with SO₂ than those fermented with DMDC and lysozyme or with a mixture of preservatives. When SO₂ was the only preservative in wine, total amine concentration (biogenic and volatile amines) was higher than for the rest of the treatments. Lysozyme by itself, and lysozyme mixed with SO₂, both reduced the formation of biogenic amines but given the antioxidant activity of SO₂ the use of the preservative mixture seems more advisable.     

Influence of SO2 on the consumption of nitrogen compounds through alcoholic fermentation of must sterilized by pulsed electric fields

The aim of this work was to study the influence of SO₂ on the use of nitrogenous compounds by yeast during wine alcoholic fermentation. Thus Parellada must was sterilized by a pulsed electric field treatment and inoculated with Saccharomyces cerevisiae Na33 strain. The fermentations were carried out with SO₂ (20 mg/l) and without SO₂. Results showed that yeast better consumed the amino acids in the first half of fermentation in the presence of SO₂. The final concentration of amino acids in the obtained wine was greater when the must was fermented without SO₂ than when the latter compound was present. Therefore, the presence of SO₂ facilitated the consumption of amino acids and, hence, such wine should have more complex flavour and better microbiological stability than that obtained from fermentation without SO₂.     

Effect of high pressure treatments on the physicochemical properties of a sulphur dioxide-free red wine

The application of high hydrostatic pressure (HHP) in winemaking for substitution of the use of sulphur dioxide is still at a very early stage of development, since knowledge about the effect on physicochemical and sensorial characteristics of the wine during storage is very scarce. In this work, the evolution of colour, antioxidant activity and total phenolic compounds of SO₂-free red wines treated by HHP and aged in bottles was followed for 12 months. The pressurised wines were compared with wine samples prepared with addition of 40 ppm of SO₂ and without any of these two treatments. After 12 months, the pressurised wines presented higher values of CIELab parameters (a^∗, b^∗, and L^∗) and a lower monomeric anthocyanin content (45–61%) when compared to the unpressurised ones. The pressurised wines showed also a better global sensorial assessment, with the pressure treatments imparting aged-like characteristics to the wines. The wine deposits of pressurised wines had higher total phenolic content, namely proanthocyanidins (3- to 10-fold). The results demonstrate that HHP can influence long term red wine physicochemical and sensorial characteristics, hypothesised to be due to an increase of condensation reactions of phenolic compounds, forming compounds with higher degree of polymerisation that became insoluble in wine along storage.     

Ozone in wineries and wine processing: A review of the benefits,application, and perspectives

Ozone (O₃) is an emerging eco-friendly technology that has been widely used in the beverage industry due to its broad spectrum of usages, such as fermentation, microbial inactivation, Clean-in-Place (CIP) systems, and postharvest treatment. Wine is among the most financially profitable sectors of the beverage industry. Ozone technology as an alternative approach to conventional methods to inhibit microbes in wine processing and wineries has attracted researchers' attention as this emerging technology will probably play important roles in wineries in the future. This review discusses the prospective applications of ozone in winemaking and wineries and elaborates on ozone's antimicrobial effects on the control of the broad spectrum of microorganisms during wine processing. Also, this paper provides discussions on its effects of O₃ on wine quality and the benefits this emerging technology can bring to wineries. Ozone treatments can improve yeast fermentation by impacting the yeast ecology of postharvested wine grapes, mainly by affecting apiculate yeasts and adjusting the population of undesirable yeasts, such as Brettanomyces spp., during the fermentation process. Furthermore, ozone treatment may enhance wine's anthocyanin concentration, physicochemical properties, color, pH, oxidative stability, and concentration of pleasant volatile compounds and esters. This article presents important information to have a better understanding of the impact of ozone treatment on different stages of wine preparation.     

Mechanisms of oxidative browning of wine

Oxidative browning is a long-standing problem in winemaking and sulphur dioxide is the generally used antioxidant to control wine browning. However, concerns over its ability to induce severe allergic reactions have created a great need for its reduction or replacement in recent years. Furthermore, ascorbic acid seems unsuccessful as an alternative antioxidant in winemaking. So a comprehensive understanding of the chemistry of wine browning is essential to study new antioxidant(s) for the protection of wine from browning. The review systematically outlines the mechanisms of wine browning including enzymic and non-enzymic browning happening in winemaking and discusses the central roles performed by iron and copper in the chemistry of wine oxidation. Iron and copper might play a central role in initiating wine non-enzymic browning, and this has important implications in studying antioxidant substitute for sulphur dioxide in wine.     

Effect of added sulphur dioxide levels on the fermentation characteristics of strawberry wine

The aim of this study to evaluate the effects of sulphur dioxide (SO₂) on strawberry wine fermentation and on the quality of the final wine product. Major aroma compounds, reducing sugars, ethanol, titratable acid and microflora were analyzed during the fermentation of strawberry wine supplemented with 0–100 mg/L SO₂. As the amount of added SO₂ increased, the consumption of reducing sugars and soluble solids and ethanol production decreased during early fermentation, but increased during late fermentation. During the fermentation process, the concentrations of 2‐phenethanol, isobutanol and isopentanol significantly increased and those of n‐propanol, isoamyl acetate and ethyl lactate decreased with increasing amount of added SO₂. The production of n‐butanol, ethyl acetate and ethyl butyrate was slightly dependent on the amount of added SO₂. Yeast cells were the dominant microbe in the fermenting strawberry pulp, and indigenous bacteria and fungi populations decreased rapidly or disappeared because of their sensitivity to SO₂. It was concluded that 60–80 mg/L SO₂ should be added during strawberry wine fermentations to improve wine quality. Copyright © 2016 The Institute of Brewing & Distilling     

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.     

Effect of the Closure Type on the Evolution of the Physical‐Chemical and Sensory Characteristics of a Montepulciano d'Abruzzo Rosé Wine

The present work studied the effect of the kind of closure (a screw cap, a natural cork, and 2 synthetic closures) on the evolution of the oxygen content and on the physical‐chemical and sensory characteristics of a Montepulciano d'Abruzzo rosé wine during the 1st 12 mo of bottle aging. The chemical analyses concerned the parameters more involved in the oxidative reactions (SO₂, acetaldehyde, phenols, wine color), as well as the main fermentative volatile compounds. The kind of closure influenced the oxygen content in wines, free and total SO₂ concentration, and wine color (color intensity and hue). During bottle aging, free and total SO₂ concentration was significantly and negatively correlated with absorbance at 420 nm (A420), whereas the correlations with A520 were weak. Probably, the limited extent of the variations in red color (A520), when varying SO₂ concentration, were due to the low pH of this rosé wine. No effect of the kind of closure on phenols and the main fermentative volatile compounds was observed. The wines bottled with cork closures (N trials), after 12 mo of storage, had higher color intensity and hue, measured by spectrophotometry, and were visually distinguished from the other trials for the more intense pink reflections. On the whole, under the conditions of this work, all the used closures guaranteed a good preservability to the rosè wines during the 1st year of bottle aging, and the changes in composition did not significantly affect wine sensory characteristics. Therefore, these synthetic closures can represent an alternative to the cork closures for a medium to long term bottle aging of these wines.     

SO2 reduction in distilled grape spirits by three methods

Sulphur dioxide (SO₂) is the main additive in grape wine production and cannot be eliminated by simple distillation from the base wine, lees or fermented pomace. This research was conducted to confirm the effectiveness of calcium oxide, powdered activated charcoal and hydrogen peroxide on SO₂ reduction in distilled grape spirits using a double distillation method. Three treatments were conducted on the first distillate. Each method was assessed for its effectiveness on SO₂ reduction and its influence on the volatile compounds present, which were analysed both by GC and sensory evaluation. The total residual SO₂ content in the second distillate, which had been treated with calcium oxide, was about 2.00 mg L^?1. The value was below 1.00 mg L^?1 for the activated charcoal and hydrogen peroxide treatments. The calcium oxide treatment was the most convenient method, when compared with the other two treatments, but it also caused the largest decrease in acids and esters. The activated charcoal‐treated distillate had the highest sensory evaluation. However, there were some drawbacks, such as the loss of esters and a higher cost for temperature maintenance (70°C for ~12 h). The hydrogen peroxide treatment resulted in the lowest pH and most of the volatile compounds were retained, but the sensory evaluation was second to the activated charcoal treatment. Copyright © 2013 The Institute of Brewing & Distilling     

Wine bottle closures: physical characteristics and effect on composition and sensory properties of a Semillon wine 1. Performance up to 20 months post-bottling

A Semillon wine was bottled using 14 different closures: a screw‐cap type, two grades of conventional natural cork, two ‘technical cork’ closures (natural cork with a synthetic component), and 9 closures manufactured from synthetic polymer material. Closure performance was evaluated for physical aspects (e.g. extraction force and energy, change in closure diameter, and ease of closure reinsertion), and for wine composition and sensory properties. Wine under the screw cap closure retained the greatest concentration of sulfur dioxide (SO₂) and ascorbic acid and had the slowest rate of browning. For other closures the trend of SO₂ loss relative to the screw cap closure was apparent from an early stage of testing, and was most evident in the group of synthetic closures, intermediate in the conventional corks, and least evident in the technical cork closures. The loss of SO₂ was in general highly correlated with an increase in wine browning (OD₄₂₀) and the concentration of SO₂ in the wine at six months was a strong predictor of future browning in the wine, particularly after eighteen months. Neither the concentration of dissolved oxygen at bottling (0.6–3.1 mg/L), nor the physical closure measures were predictors of future browning. For several closures upright storage tended to accelerate loss of SO₂ from the wine, but in many cases this effect was marginal. The closures differed widely in regard to physical characteristics, and in general synthetic corks appeared least ‘consumer‐friendly’ in terms of extraction forces, energies, and ease of closure re‐insertion, but there was a trend for natural cork closures to exhibit larger variability in physical characteristics than technical cork and synthetic closures. Sensory analysis indicated large differences in wine flavour properties, with closures which tended to result in the best retention of free SO₂ having wine sensory scores for ‘citrus’ that were generally high whilst scores for the attributes ‘developed’/‘oxidised’ were low. The situation was reversed for wine under closures that performed poorly in the retention of free SO₂. It was found that below a critical level of free SO₂ remaining in the wine, closures exhibited substantially higher ‘oxidised’ aroma. Whilst trichloroanisole‐type (TCA) taint was a noticeable problem for some cork and technical cork closures, any plastic‐type taint appeared not to be a problem with most synthetic closures.     

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.     

Use of propidium monoazide for the enumeration of viable Oenococcus oeni in must and wine by quantitative PCR

Malolactic fermentation is an important step in winemaking, but it has to be avoided in some cases. It's carried out by lactic acid bacteria belonging mainly to the genus Oenococcus, which is known to be a slow growing bacterium. Classical microbiological methods to enumerate viable cells of Oenococcus oeni in must and wine take 7–9 days to give results.