0000000000083577
AUTHOR
Michèle Guilloux-benatier
Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR
International audience; Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a pro…
Sorption of wine volatile phenols by yeast lees
The capacity of Saccharomyces cerevisiae yeast lees to sorb 4-ethylguaiacol and 4-ethylphenol was investigated in a synthetic medium and in wine. Active dried yeast was more effective when volatile phenols were diluted in red wine. Partition coefficients between wine model solution and wine yeast lees were determined and compared with those measured for dried active yeast. They showed a larger affinity of volatile phenols for wine yeast lees than for dried active yeast. The effect of yeast lees on volatile phenol sorption was sensitive to yeast autolysis level and to physicochemical parameters, such as ethanol content, temperature and pH. These results could be applied in the technology of …
Non-Botrytis grape-rotting fungi responsible for earthy and moldy off-flavors and mycotoxins
Abstract The grape microflora is complex and includes filamentous fungi, yeasts and bacteria with different physiological characteristics and effects on wine production. Most studies have focused on the wine microbiota, but a few studies have reported the ecology of grape microorganisms. Some of these organisms — such as non-Botrytis bunch rotting fungi, which greatly influence the safety or sensory quality of wine, due to the production of mycotoxins and off-flavors, respectively — are considered to be spoilage agents. We review here the diversity of filamentous fungi on grapes and the factors influencing their development, such as grape ripening stage, environmental factors (climate, rain…
Yeast autolysis in sparkling wine – a review
Sparkling wine produced by the traditional methode champenoise requires a second in-bottle alcoholic fermentation of a base wine, leading to the sparkling wine. This second fermentation is followed by prolonged ageing in contact with yeast cells (lees). The autolysis of yeast occurs during the ageing of sparkling wines. During this process, the yeast releases different compounds that modify the organoleptic properties of the wine. The ageing period is required to give these wines their roundness and characteristic aroma and flavour. Autolysis products also influence the foaming properties of sparkling wine. Yeast autolysis is characterised by the hydrolysis of intracellular biopolymers by y…
Influence of fatty acids on the growth of wine microorganisms Saccharomyces cerevisiae and Oenococcus oeni
The effects of fatty acids, extracted during prefermentation grape skin-contact on Saccharomyces cerevisiae and Oenococcus oeni, were studied. The influence of skin-contact on total fatty acid content was evaluated both in Chardonnay must and in synthetic medium. Prior to alcoholic fermentation, the skin-contact contributes to a large enrichment of long-chain fatty acids (C 16 to C 18:3 ). These results induced a positive effect on yeast growth and particularly on cell viability. In the skin-contact fermented media, levels of C 12 and especially C 10 are lower and macromolecules content higher than in controls. This production of extracellular mannoproteins and the reduction of medium-chain…
PCR ITS-RFLP for Penicillium Species and Other Genera
Among numerous molecular methodologies developed for highly specific identification of filamentous fungi isolates, here we describe restriction digestion analysis of the ITS products as an easy method to identify isolates of filamentous fungi. This technique is a rapid and reliable method appropriate for routine identification of filamentous fungi. This can be used to screen large numbers of isolates from various environments in a short time. The use of different endonucleases allowed generating individual restriction profiles. The individual profiles obtained were combined into composite restriction patterns characteristic of a species. Eleven different genera can be differentiated and amo…
PCR ITS-RFLP: A useful method for identifying filamentous fungi isolates on grapes.
Restriction digestion analysis of the ITS products was tested as an easy method to identify isolates of filamentous fungi on grapes. Endonucleases SduI, HinfI, MseI, HaeIII were used. Endonucleases BfmI, Cfr9I, Hpy188I, MaeII or PspGI were used as necessary to complete discrimination. The 43 species studied generated 42 different composite profiles. Only the species P. thomii and P. glabrum gave the same composite profile. 96.3% strains tested could be differentiated to the species level with only four enzymes. Hundred ninety nine strains of filamentous fungi were isolated from various vineyards in Burgundy and identified by this method. Penicillium (58.5%) was the genus the most frequently…
Lysis of yeast cells by Oenococcus oeni enzymes
exhibited extracellular β (1→3) glucanase activity. This activity increased when cells were cultivated with glycosidic cell-wall macromolecules. In addition, the culture supernatant of the organism effectively lysed viable or dead cells of Saccharomyces cerevisiae. This lytic activity appeared in the early stationary phase of bacterial growth. Yeast cells at the end of the log phase of growth were the most sensitive. The optimum temperature for lysis of viable yeast cells was 40°C, which is very different from the temperatures observed in enological conditions (15–20°C). Moreover, the rate of the lytic activity was significantly lower in comparison with yeast cell wall-degrading activities …
Origines des levures Saccharomyces et non-Saccharomyces présentes dans le moût : Vignoble ou cuverie ?
International audience; Les levures de l’espèce Saccharomyces cerevisiae ont longtemps été considérées comme les seules responsables du processus de fermentation. On sait aujourd’hui que différentes espèces de levures Saccharomyces et non-Saccharomyces (NS) sont présentes dans les moûts et qu’un certain nombre d’entre elles participe à la fermentation alcoolique (FA). Bien qu’il soit aujourd’hui largement admis que les principales sources de levures dans le moût de raisin sont les baies de raisin et le matériel de vinification (Fleet et Heard, 1993 ; Mortimer et Polsinelli, 1999) (figure 1), la part apportée par chacune de ces deux sources est encore mal définie. De plus, d’autres sources p…
Oligopeptide assimilation and transport by Oenococcus oeni
International audience; Aims: Oenococcus oeni is a slow-growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino-acid medium. We aimed to characterize the oligopeptide use of this micro-organism. Methods and Results: Several peptides of two to eight amino-acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino-acid residues tested and to hydrolys…
Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions
Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25 degrees C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermenta…
Effect of nitrogen limitation and nature of the feed upon Oenococcus oeni metabolism and extracellular protein production
Aims: The aim of the study was to characterize the effect of various nitrogen sources on Oenococcus oeni growth, carbon source utilization, extracellular protease activity and extracellular proteins. More generally, the goal is to understand how nitrogen-based additives might act to enhance malolactic fermentation in wine. Methods and Results: Five yeast extracts were used. As the amino acid and nitrogen analyses revealed, they were similar in global amino acid composition, except for arginine level. Nevertheless the ratio of amino acids between free/bound, and low/high molecular weight fractions were highly different. One of the yeast extracts led to a significant protease activity in th…
Les grands types de sols viticoles de Pomerol et Saint-Emilion
<p style="text-align: justify;">Les vins les plus réputés de la région viticole de Pomerol et Saint-Emilion sont produits sur des sols foncièrement différents, dérivant de roches mères présentant une grande diversité.</p><p style="text-align: justify;">Les variations que l'on peut noter d'un cru à l'autre sont liées, non pas aux caractéristiques chimiques de ces sols, mais surtout à leurs propriétés physiques (texture, structure, porosité, perméabilité) avec leurs conséquences sur le mode et la profondeur de l'enracinement.</p><p style="text-align: justify;">+++</p><p style="text-align: justify;">The most famous wines from the wine-growing region of…
MOOC : "Open Wine University 2"
Plan du cours Semaine 1 : Introduction Entre sol et climat Micro-organismes du vin Premiers pas en dégustation + support sur la vinification des vins blancs et des vins rouges Semaine 2 : Le vin en danger ? Climat et viticulture Principales maladies et ravageurs de la vigne Economie mondiale du vin après la crise du phylloxéra Semaine 3 : Façonner le vin Géologie et sols des terroirs bourguignons Vin et diversité microbienne Marchés du vin Semaine 4 : Maîtriser son terroir Construction du terroir de la vigne Vinification des vins spéciaux : l'exemple de la production de vins jaunes du Jura Défauts du vin Semaine 5 : Mettre le vin à table Mise en bouteille du vin Vieillissement du vin Le vin…
Diversity of yeast strains of the genus Hanseniaspora in the winery environment: What is their involvement in grape must fermentation?
Isolated yeast populations of Chardonnay grape must during spontaneous fermentation were compared to those isolated on grape berries and in a winery environment before the arrival of the harvest (air, floor, winery equipment) and in the air through time. Two genera of yeast, Hanseniaspora and Saccharomyces, were isolated in grape must and in the winery environment before the arrival of the harvest but not on grape berries. The genus Hanseniaspora represented 27% of isolates in the must and 35% of isolates in the winery environment. The isolates of these two species were discriminated at the strain level by Fourier transform infrared spectroscopy. The diversity of these strains observed in t…
Persistence of Two Non-Saccharomyces Yeasts (Hanseniaspora and Starmerella) in the Cellar
International audience; Different genera and/or species of yeasts present on grape berries, in musts and wines are widely described. Nevertheless, the community of non-Saccharomyces yeasts present in the cellar is still given little attention. Thus it is not known if the cellar is a real ecological niche for these yeasts or if it is merely a transient habitat for populations brought in by grape berries during the winemaking period. This study focused on three species of non-Saccharomyces yeasts commonly encountered during vinification: Starmerella bacillaris (synonymy with Candida zemplinina), Hanseniaspora guilliermondii and Hanseniaspora uvarum. More than 1200 isolates were identified at …
Influence des facteurs naturels sur la maturation du raisin, en 1979, à Pomerol et Saint-Emilion
<p style="text-align: justify;">Les vignobles de Pomerol et Saint-Emilion sont implantés sur des sols dérivant de roches-mères variées; ils produisent des vins de haute qualité mais différents suivant leur origine géologique.</p><p style="text-align: justify;">Dans les meilleurs crus, nous avons pu vérifier que les variations mésoclimatiques sont très faibles et ont peu d'influence sur les caractères des vins. Par contre, en 1979, une relation a été mise en évidence entre les conditions de l'alimentation en eau de la vigne (étroitement liées aux propriétés physiques des sols) et la constitution des raisins à maturité (<em>V. vinifera</em> var. Merlot noir).<…
Predominant mycotoxins, mycotoxigenic fungi and climate change related to wine
Wine is a significant contributor to the economies of many countries. However, the commodity can become contaminated with mycotoxins produced by certain fungi. Most information on mycotoxins in wine is from Spain, Italy and France. Grapes can be infected by mycotoxigenic fungi, of which Aspergillus carbonarius producing ochratoxin A (OTA) is of highest concern. Climate is the most important factor in determining contamination once the fungi are established, with high temperatures being a major factor for OTA contamination: OTA in wine is at higher concentrations in warmer southern Europe than northern. Contamination by fumonisins is a particular concern, related to Aspergillus niger produci…
Development of a qPCR assay for specific quantification of Botrytis cinerea on grapes
The aim of this study was to develop a system for rapid and accurate real-time quantitative PCR (qPCR) identification and quantification of Botrytis cinerea , one of the major pathogens present on grapes. The intergenic spacer (IGS) region of the nuclear ribosomal DNA was used to specifically detect and quantify B. cinerea . A standard curve was established to quantify this fungus. The qPCR reaction was based on the simultaneous detection of a specific IGS sequence and also contained an internal amplification control to compensate for variations in DNA extraction and the various compounds from grapes that inhibit PCR. In these conditions, the assay had high efficiency (97%), and the limit o…
FT-IR spectroscopy : A powerful tool for studying the inter- and intraspecific biodiversity of cultivable non-Saccharomyces yeasts isolated from grape must
International audience; The efficiency of the FT-IR technique for studying the inter- and intra biodiversity of cultivable non-Saccharomyces yeasts (NS) present in different must samples was examined. In first, the capacity of the technique FF-IR to study the global diversity of a given sample was compared to the pyrosequencing method, used as a reference technique. Seven different genera (Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Issatchenkia, Metschnikowia and Pichia) were identified by FT-IR and also by pyrosequencing. Thirty-eight other genera were identified by pyrosequencing, but together they represented less than 6% of the average total population of 6 musts. Among the sp…
Comparison of Components Released by Fermented or Active Dried Yeasts after Aging on Lees in a Model Wine
Comparison of different components released during autolysis with fermented or active dried yeast, which has never been reported previously, is related in this paper. Three autolysates were elaborated with Saccharomyces cerevisiae in a model wine (pH 3.5) at 30 or 18 degrees C. Composition of the autolysate appears to depend on both the growth medium and the physiological state of the yeast. The autolysate obtained from active dried yeast presents a higher total nitrogen concentration (a factor of 1.5-1.9 for the fermented yeast autolysate), a greater proportion of free amino acids (42 vs 16-25%), the lowest proportion of oligopeptides (25 vs 31-37%) and polypeptides (27 vs 45-34%), and an …
Quantification des Brettanomyces par qPCR
International audience; Brettanomyces bruxellensis est une levure d’altération du vin avec de faibles besoins nutritionnels, résistante à l’éthanol et aux faibles pH, lui permettant une implantation en vin durant ou après la fermentation alcoolique (Conterno et al., 2006). B. bruxellensis est capable de produire des phénols volatils (éthyl-4-phénol, éthyl-4-gaïacol et éthyl-4-catéchol) (Oelofse et al., 2008). Ces molécules volatiles odorantes amènent un caractère phénolé et animal au vin connu sous le nom de « caractère Brett ». D’autres molécules (2-acétyltétrahydropyridine et 2-éthyltétrahydropyridine) produites par B. bruxellensis sont également responsables d’une déviation organoleptiqu…
Influence of yeast autolysis after alcoholic fermentation on the development of <em>Brettanomyces</em>/<em>Dekkera</em> in wine
<p style="text-align: justify;"><em>Brettanomyces</em>, a contaminant yeast, is relatively common in wines and mainly in red wines during barrel aging. The results presented here relate to the effects of yeast lees autolysis on the growth of <em>Brettanomyces</em>. Experiments were realised in a culture medium after alcoholic fermentation, in a hydroalcoholic wine-like solution and in a red wine. <em>Brettanomyces</em> was inoculated at low level : 10<sup>2</sup> cfu/ml and the growth was controlled by counting on agar appropriate medium. Yeast lees from <em>S. cerevisiae</em> were added to these media in the presence or abse…
Saccharomyces cerevisiae-Oenococcus oeni interactions in wine: current knowledge and perspectives.
Winemaking can be summarized as the biotransformation of must into wine, which is performed principally by Saccharomyces cerevisiae strains during the primary or alcoholic fermentation. A secondary fermentation, the so-called malolactic fermentation (MLF) is a biodeacidification that is often encouraged, since it improves wine stability and quality. Malolactic fermentation usually occurs either spontaneously or after inoculation with selected bacteria after alcoholic fermentation. The main organism responsible for MLF, the lactic acid bacterium Oenococcus oeni, develops in physicochemically harsh conditions, which may lead to MLF failure. Furthermore, yeast that ferment must before or toget…
Incidence de la clarification des moûts de raisin sur les fermentescibilltés alcoolique et malolactique
<p style="text-align: justify;">Des essais de débourbage en vinification en blanc, réalisés de 1988 à 1992, mettent en évidence le rôle important joué par la teneur en macromolécules solubles dans le moût de raisin. Les moûts de faible turbidité conduisent en effet à des fermentations alcooliques lentes en relation avec de faibles populations levuriennes. Il est également confirmé que la libération de polysaccharides exocellulaires par les levures au cours de la fermentation alcoolique est d'autant plus élevée que le moût de départ a été fortement appauvri en colloïdes de raisin. Par contre, ce sont dans les lots les plus clarifiés que l'on observe la meilleure fermentescibilité malol…