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Seit den 80er Jahren wird in der Weinherstellung nach Rotweinen gestrebt, die reich an phenolischen Verbindungen sind. Die Stabilisierung und Konservierung dieser phenolischen Komponenten wird jedoch nicht immer erreicht und viele Weine landen in Flaschen, die nicht der ursprunglich erwunschten Zusammensetzung entsprechen. Wahrend des Weinausbaus fuhrt eine gewisse Unausgewogenheit zwischen den extrahierten Bestandteilen und der Anwesenheit bestimmter Kolloide zu einer schlechten Stabilisierung phenolischer Komponenten. Dies findet oft wahrend der Herstellung vor der Flaschenabfullung nicht genug Beachtung.

International audience; Polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis was the most relevant method to follow the diversity of lactic acid bacteria during winemaking. By targeting the rpoB gene, two types of Oenococcus oeni strains were distinguished resulting from a single mutation in the rpoB region targeted in PCR and generating two different electrophoresis profiles. The first one prevailed during fermentation and the second during ageing. Some strains of each type were isolated during winemaking and were studied using several genetic methods (real-time PCR, PCR-random amplified polymorphic DNA, multiple locus sequence typing and the presence of gene markers). Physiological characters related to environmental conditions were examined. The results confirmed the relevance of the rpoB mutation for characterising the two O. oeni subgroups. The relationship between the physiological response to stress and the rpoB genetic groups raised the question of O. oeni intraspecies grouping. A possible division within this species, of great technological interest to the wine industry, was also raised.

International audience; Brettanomyces bruxellensis spoilage is a serious problem for the wine industry. Mainly, by producing 4-ethylphenol and 4-ethylguaiacol, it confers off-odors to the wine and changes its aromatic quality. The presence of B. bruxellensis cells on the berry was speculated but it had never been clearly demonstrated. On grape berries, the microbial ecosystem is highly diverse and the population of B. bruxellensis can be very small. The aim of our study was to reveal and confirm the presence of B. bruxellensis on the surface of grape berries. We developed an enrichment medium for B. bruxellensis in order to overcome the detection limit of the molecular methods (species-specific PCR, ITS-RFLP PCR, PCR-DGGE). This medium, named EBB medium, made it possible to detect B. bruxellensis after 10 days of culture. For the first time, the presence of B. bruxellensis has been clearly established in several vineyards and at different stages of the grape development after the veraison. This work led to the conclusion that the grape berry is the primary source of B. bruxellensis. Grape growers and winemakers should take these results into account when deciding on the treatment to apply in the vineyards and the must. With the information provided here, B. bruxellensis prevention could start in the vineyard.

We found recently that, in yeast cells, the heterologous expression of Bax induces a loss of plating efficiency different from that induced by acute stress because it is associated with the maintenance of plasma membrane integrity (Camougrand, N., Grelaud-Coq, A., Marza, E., Priault, M., Bessoule, J. J., and Manon, S. (2003) Mol. Microbiol. 47, 495-506). Bax effects were neither dependent on the presence of the yeast metacaspase Yca1p and the apoptosis-inducing factor homolog nor associated with the appearance of typical apoptotic markers such as metacaspase activation, annexin V binding, and DNA cleavage. Yeast cells expressing Bax instead displayed autophagic features, including increased accumulation of Atg8p, activation of vacuolar alkaline phosphatase, and the presence of autophagosomes and autophagic bodies. However, the inactivation of autophagy did not prevent and actually slightly accelerated Bax-induced loss of plating efficiency. On the other hand, Bax expression induced a fragmentation of the mitochondrial network, which retained, however, some level of organization in wild-type cells. However, when expressed in cells inactivated for the gene UTH1, previously shown to be involved in mitophagy, Bax induced a complete disorganization of the mitochondrial network. Interestingly, although mitochondrially targeted green fluorescent protein was slowly degraded in the wild-type strain, it remained unaffected in the mutant. Furthermore, the slow loss of plating efficiency in the mutant strain correlated with a loss of plasma membrane integrity. These data suggest that Bax-induced loss of growth capacity is associated with maintenance of plasma membrane integrity dependent on UTH1, suggesting that selective degradation of altered mitochondria is required for a regulated loss of growth capacity.

International audience; AIMS: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. METHODS AND RESULTS: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. CONCLUSIONS: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.

International audience; Aims: To study the impact of soil-type, grapevine variety and rootstock on grape yield and wine quality in prestigious estates located in the Bordeaux area (France). Methods and results: High-resolution soil maps (scale: 1/3000(th)) were created for seven prestigious red wine-producing estates in Bordeaux. covering a total area of approximately 400 ha. Soil-type, rootstocks and grapevine varieties were recorded for each vineyard block. A Quality Index was created by considering the destination of the grapes produced in each block, whether they were integrated in the first, the second or the third quality wine produced by the estate. Quality Index was averaged over five vintages. Yield was also measured for each vineyard block and averaged over live vintages. PEYROSOL (gravelly soil) was the most frequent soil-type in these estates (45% of the total mapped area). Soils with temporary waterlogging (REDOXISOL). heavy clay soils (PLANOSOL) and sandy-gravelly soils (BRUNISOL) covered around 10% of the mapped area each. Highest quality was obtained on PLANOSOLS, ARENOSOLS (sandy soils), BRUNISOLS and PEYROSOLS. Quality was low on COLLUVIOSOLS (deep soils on colluvium). LUVISOLS (leached acidic soils) and REDUCTISOLS (soils with permanent waterlogging). Cabernet-Sauvignon was the dominant grapevine variety (59% of the mapped area). followed by Merlot (32%), Cabernet franc (8%) and Petit Verdot (1%). On average. the Quality Index was higher for Cabernet-Sauvignon and Merlot compared with Cabernet franc and Petit Verdot. Riparia Gloire de Montpellier (RGM) was by far the most used rootstock. It covered 45% of the mapped area. Including 3309C and 420A. these three rootstocks covered 75% of the total acreage planted in these estates. Highest quality wine was produced with 420A. RCM, 3309C and Gravesac. Highest yields were obtained with 161-49C. 101-14MG, RGM, SO4 and 420A. Conclusions: Soil, grapevine variety and rootstock have a major impact on yield and wine quality in prestigious Bordeaux wine producing estates. Significance and impact of the study: Assessment of a Quality Index by soil-type, cultivar and rootstock can indicate which combinations of soil-type, cultivar and rootstock would best optimise quality performance in Bordeaux vineyards.; Objectifs : Étudier l’effet du sol, du cépage et du porte-greffe sur le rendement et la qualité du vin produit dans des domaines prestigieux du vignoble de Bordeaux. Méthodes et résultats : Des cartes de sol à haute résolution (échelle : 1/ 3000e) ont été réalisées pour sept domaines produisant des vins rouges réputés à Bordeaux sur une superficie totale d'environ 400 ha. Le type de sol, le cépage et le porte-greffe ont été identifiés pour chaque parcelle de vigne. Un indice de qualité a été créé en tenant compte de la destination des raisins produits sur chaque parcelle. Une note différente a été attribuée suivant qu'ils furent intégrés dans la première, la deuxième ou la troisième qualité du vin produit sur chaque domaine. L’indice de qualité a été pondéré sur cinq millésimes. Le rendement a également été évalué pour chaque parcelle de vigne sur cinq millésimes. Le PEYROSOL (sol graveleux) est le sol dominant dans ces domaines (45 % de la superficie totale cartographiée). Les sols présentant une hydromorphie temporaire (REDOXISOLS), les sols argileux (PLANOSOLS) et les sols sablograveleux (BRUNISOLS) couvrent chacun environ 10%de la superficie cartographiée. La plus haute qualité a été obtenue sur les PLANOSOLS, les ARENOSOLS (sols sableux), les BRUNISOLS (sols sablo-graveleux) et les PEYROSOLS. La qualité a été faible sur les COLLUVIOSOLS (sols profonds de colluvions), les LUVISOLS (sols lessivés acides) et les REDUCTISOLS (sols à hydromorphie permanente). Le Cabernet-Sauvignon est le cépage dominant (59 % de la superficie cartographiée), suivi par le Merlot (32 %), le Cabernet franc (8 %) et le Petit Verdot (1 %). En moyenne, l'indice de qualité était plus élevé pour le Cabernet-Sauvignon et le Merlot, comparativement au Cabernet franc et au Petit Verdot. Le Riparia Gloire de Montpellier (RGM) est de loin le porte-greffe le plus utilisé. Il couvre 45%de la surface cartographiée. Les trois porte-greffe les plus plantés (RGM, 3309C et 420A) couvrent 75 % de la superficie totale. La meilleure qualité de vin a été produite avec 420A, RGM, 3309C et Gravesac (indice de qualité > 2,5). Les rendements les plus élevés ont été obtenus avec 161-49C, 101-14 mg, RGM, SO4 et 420A. Conclusion : Le sol, le cépage et le porte-greffe ont un effet majeur sur le rendement et la qualité du vin produit dans des domaines prestigieux du vignoble de Bordeaux. Signification et l'impact de l'étude : Ces données quantitatives et qualitatives permettent d’évaluer les performances de différents types de sol et du matériel végétal associé dans le vignoble de Bordeaux. Elles permettent de formuler des recommandations concernant les associations entre le type de sol, le porte-greffe et le cépage qui permettent de valoriser le mieux quelques-uns des principaux types de sol du vignoble de Bordeaux.

International audience; This study reports on monitoring Oenococcus oeni intraspecific diversity evolution during winemaking. Three different wines were monitored. The proportion of O. oeni species was determined by species-specific PCR and O. oeni strains were distinguished by multiplex PCR-RAPD. Each strain was tested by PCR for 16 significant markers revealed by a previous genetic comparison between a strong oenological potential strain and one with poor oenological potential. Population levels and diversity changed according to winemaking stages, oenological practices and the chemical properties of the wine. In all situations, O. oeni was the best-adapted species. Within the O. oeni group, intraspecific strain diversity decreased and the malolactic fermentation was the result of the most resistant strains with the highest number of markers.

International audience; The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.

Three different ethanol solutions were sprayed onto Cabernet Sauvignon (Vitis vinifera L.) clusters during the ripening period: 2.5, 5 and 10% by volume in water. Controls were sprayed with water alone. Three different times of spraying were also tested: 8, 10 and 13 weeks post-flowering. One of the observed changes was a lower titratable acidity in grape samples at harvest, when the clusters were sprayed with ethanol at 10 weeks, in comparison with controls. The wines made with grapes treated with ethanol after mid-veraison, had higher ODs at 520 nm than did the controls. This may due to a combined effect of red pigment levels and acidity. In addition, following malolactic fermentation, the acidity levels of wines made with ethanoltreated grapes were slightly higher than those made with the control grapes. Spraying ethanol at 13 weeks post-flowering increased the berry weight by 10% at harvest without decreasing the °Brix value. The corresponding wines had similar degrees of alcohol. This observation was made for the first time in 2001.

Vincent Renouf1,2, Cécile Miot-Sertier2, Marie-Claire Perello2, Gilles de Revel2, Aline Lonvaud-Funel21Laffort, Bordeaux, France; 2UMR Œnologie, INRA-Université Bordeaux, FranceAbstract: The aim of this paper is to study and compare the physiological diversity of different strains of a wine spoilage yeast species: Brettanomyces bruxellensis. The minimum inhibitory concentrations of several drugs on different B. bruxellensis strains were scored on solid nutrient media. This revealed variations in resistance among the B. bruxellensis strains. Their capacity to develop in different wine and must environments: pH, ethanol, and SO2 concentrations, were evaluated by measuring the direct incubation survival rate. The results, compared with those obtained for other wine yeast species, confirmed the remarkable resistance of B. bruxellensis strains to various conditions which inhibit the growth of other species. Nevertheless some differences were observed among the B. bruxellensis strains, thus confirming their physiological diversity. A comparison of their volatile phenol production revealed intraspecific heterogeneity among B. bruxellensis strains. B. bruxellensis is one of the microbial species most resistant to environmental constraints in wine. It is the best adapted to growing in wine and spoiling it by volatile phenol production. However, different B. bruxellensis strains exhibit varying characteristics, particularly their capacity to produce volatile phenols. This implies that certain strains are more prejudicial than others. Further studies are required to determine the molecular causes of this intraspecific diversity.Keywords: Brettanomyces bruxellensis, strain diversity, physiology, volatile phenols

Winemaking is based on complex microbial interactions. They result in alcoholic and malolactic fermentation. In some cases undesirable micro-organisms pass beyond a limit and become prejudicial to wine quality. It is particularly the case of Brettanomyces bruxellensis which produces volatile phenols.Most of wine microbial studies have been focused on only one species and that can lead to incomplete and biased results by neglecting possible interactions between the populations. The aim of this study was to obtain a global survey of wine microflora and its quantitative and qualitative changes during the malolactic fermentation, the last microbial intervention before sulphur dioxide addition. The results were obtained by chemical wine analysis, conventional microbiological methods and molecular tools for microbial identification (PCR-ITS-RFLP, PCR-DGGE). In this study, conducted under cellar scale conditions, several oenological parameters were considered: two different cellars, three grape varieties, MLF in tank or in barrels, use of malolactic starters or indigenous flora.Interactions appeared, mainly between Oenococcus oeni and B. bruxellensis, but also between O. oeni strains. Some explanations are suggested and further investigations are proposed.

Aims: Microorganisms play an important role in winemaking. Most of their metabolisms are beneficial to the wine quality but several species have spoilage effects. Microbiological knowledge are crucial for improving species and strains identification and avoiding contamination by spoilage agents. Our objective was to follow wine microbial consortia at each stage of the process from grapes to bottles, and also on cellar equipment surface: barrels for the yeast analysis and tanks for the bacteria analysis.Methods and results: For a global microbial monitoring, molecular tools are particularly efficient. We chose the PCR-DGGE for direct analyses without cultivation step. We used the rpoB gene as target for the bacteria, and the 26S rRNA gene for the yeast. Species inventories were made during the whole winemaking process, which had never been done before.Conclusion: From the grape surface to storage in bottles, the microbial diversity decreased. Only the most resistant species were able to survive to the ethanol, SO2, low oxygen and low pH constraints. Among them, Pediococcus parvulus for the bacteria and Brettanomyces bruxellensis for the yeast were the most resistant.Significance and impact of study: The use of a molecular method independent of the culture stage of the microorganisms like the PCRDGGE has allowed to have a more exhaustive vision of the diversity of the bacteria and yeasts species and to follow their evolution during the winemaking and in the cellar environment . These follow-ups could be extended to other estates in order to study the causes and the consequences of certain variations. The detection of these two prejudicial species was an argument for the PCR-DGGE approach in wine microbial studies.