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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.

Objectif : Les cartes de professionnel de sante permettent notamment l’edition et l’envoi des feuilles de soins electroniques. Ces cartes ne peuvent pas etre pretees, y compris lors des remplacements. Pourtant, les remplacants en medecine generale evoquaient souvent, de facon informelle, un pret et une utilisation des cartes de professionnel de sante des medecins remplaces. Cette etude avait pour objectif principal de determiner la frequence d’utilisation de leurs propres cartes par les medecins generalistes remplacants pour le remboursement des patients en 2013.Methodes : Une enquete retrospective a ete realisee en 2014 aupres des medecins generalistes remplacants en Basse-Normandie par un questionnaire informatise. Les donnees recueillies concernaient leur possession, leur connaissance ainsi que la frequence et les difficultes d’utilisation de leurs propres cartes de professionnel de sante au cours de l’annee 2013.Resultats : Tous les remplacants connaissaient la carte de professionnel de sante mais 71,9 % ne connaissaient pas la carte de professionnel en formation. Une carte etait detenue par 71,9 % des remplacants en 2013, et parmi eux 26,1 % l’avaient utilisee en 2013. 78,1 % des repondants pensaient possible l’utilisation de la carte du medecin remplace. Les principales difficultes d’utilisation identifiees etaient un manque de demande des medecins remplaces, des problemes de configuration des logiciels et d’installation des cartes, une meconnaissance de l’utilite des cartes.Conclusion : La frequence d’utilisation de leurs propres cartes par les remplacants bas-normands etait faible. Une meilleure information des medecins sur l’utilisation et les aides a l’installation de ces cartes parait necessaire.

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; The aim of this study was to investigate the anti-microbial properties of the dimethyldicarbonate (DMDC) towards the wine spoilage yeast Brettanomyces bruxellensis at different winemaking stages. DMDC anti-microbial activity was estimated in red must for diverse wine microorganisms including different strains of B. bruxellensis. DMDC effect before alcoholic fermentation, before malolactic fermentation, and in finished wine were investigated. DMDC was also tested on lees. Microbial monitoring was done by epifluorescence observation and plate numeration. The identification of yeast species and the specific detection of B, bruxellensis were performed with molecular tools. DMDC stopped B. bruxellensis growth at different winemaking stages. But it could also act on fermenting species like Saccharomyces cerevisiae and Oenococcus oeni. Therefore, its use before the end of fermentations should be avoided. On the other hand, the DMDC action was shown to be transitory. Therefore, single addition during ageing could be insufficient. Finally, DMDC could be used just before bottling as an ultimate anti-microbial tool. (c) 2007 Elsevier Ltd. All rights reserved.

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.

Health professional cards are used for electronic production and transmission of medical reimbursement requests. These cards are personal and cannot be lent, including to locums. However, general practice locums often report using the cards of the practitioners they replace. The aim of this study was to assess the use of general practice locum's own professional cards for patient reimbursements in 2013.A retrospective survey by computerized questionnaire was conducted in 2014 in Lower-Normandy among general practice locums. Data were collected on their possession, knowledge,frequency and difficulties using their own cards in 2013.All locums were familiar with health professional cards, but 71.9% did not know about health professional trainee cards. 71.9% of respondents had a card in 2013 and only 26.1% of them had already used the card. 78.1% of respondents thought they could use the card of the practitioner that they replaced' The main difficulties encountered when using health professional cards were the failure of general practitioners to ask locums to use their own cards, problems with software configuration and card installation, and a poor understanding of the purpose of the card.Locums in Lower Normandy in 2013 rarely used their own cards when they have one. Better information concerning use of the cars is required for locums.

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.

Apres les fermentations, il est important de stabiliser microbiologiquement le vin afin d'eviter les alterations microbiennes possibles. Pour cela, toutes les sources de contamination doivent etre envisagees et etudiees. Pour un grand nombre de vins, la fin des fermentations coincide generalement avec l'entonnage. Nous avons etudie la flore microbienne presente a la surface du bois des barriques, en analysant les eaux de rincage de barriques neuves avant leur premiere exposition au vin et lors des soutirages a differentes etapes de l'elaboration du vin. Cinq populations microbiennes differentes: levures totales, levures non-Saccharomyces, bacteries lactiques, bacteries acetiques et bacteries a Gram negatif anaerobiques et anaerobiques facultatives, presentes dans les eaux de rincage des barriques, les vins et les lies correspondantes ont ete denombrees sur des milieux nutritifs selectifs. Les especes et les souches ont ete identifiees par des methodes de biologie moleculaire comme la PCR-DGGE, la PCR-RFLP et l'electrophorese en champ pulse. Puis des essais realises au laboratoire ont permis de simuler et d'evaluer les risques de contamination. Avant leur premiere utilisation, le bois des barriques contient une charge microbienne intrinseque composee de levures appartenant majoritairement a la famille des basidiomycetes (Cryptococcus sp., Bulleromyces sp., Rhodotorula sp) et des bacteries appartenant a la famille des enterobacteries (Serratia sp., Shigella sp.). La majorite de ces especes ne tolerent pas les caracteristiques physico-chimiques du vin. Par ailleurs, le bois des barriques constitue un reel reservoir pour les micro-organismes du vin: Saccharomyces cerevisiae et Oenococcus oeni et des especes d'alteration, Brettanomyces bruxellensis, Pediococcus parvulus et Gluconobacter oxydans. Meme si ces micro-organismes residuels ne semblent pas capables de contaminer un vin recemment entonne dont la charge microbienne est equilibree, les soutirages doivent etre l'occasion de les eliminer. Des lavages successifs a eau chaude de preference, sous pression et diriges sur l'ensemble de la surface interieure du bois des barriques sont un bon moyen. L'interet de ce nettoyage est encore plus important si le vin entonne est depourvu de micro-organismes, comme par exemple apres une filtration ou un traitement thermique, car dans ce cas les micro-organismes du bois disposent d'un ecosysteme vacant pour se multiplier.

International audience; Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.

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; Microbial species present on the surface of grape berries at harvest play an important role in winemaking, thus counting and identifying them is of great importance. The use of conventional microbial techniques and molecular methods allowed a quantitative and qualitative inventory of the different microbial species present on the grape berries. These experiments were carried out in several areas of the Bordeaux region on the red grape varieties Merlot, Cabernet Sauvignon and Cabernet Franc. Populations and species clearly varied according to berry development stage. The most widespread yeast species at berry set, Aureobasidium pullulans was never detected at harvest. Fermentative yeasts were detected at harvest and not in the first stage of grape growth. Oenoccocus oeni was detected on immature as well as on mature berries. Gluconobacter oxydans was detected mainly at harvest. Detection of Pediococcus parvulus, was dependent on the vineyard. Veraison appeared to be a key stage for yeast colonisation and the increase in population involved a change in the proportion of each species. The number of A. pullulans fell significantly at veraison as it was superseded by fermentative yeasts. Microbial populations peaked at harvest when the berry surface available for adhesion was largest and no agrochemical treatments had been applied for some weeks. Soil, grape variety and grapegrowing practices may also influence this microbial ecosystem. Based on these and published data, we formulated hypotheses to describe this microbial ecosystem, thus enabling us to develop the concept of a microbial biofilm.

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; Aims: The presence of Brettanomyces bruxellensis is an important issue during winemaking because of its volatile phenols production capacities. The aim of this study is to provide information on the ability of residual B. bruxellensis populations to multiply and spoil finished wines during storage in bottles. Methods and Results: Several finished wines were studied. Brettanomyces bruxellensis populations were monitored during two and a half months, and volatile phenols as well as chemical parameters regularly determined. Variable growth and volatile phenols synthesis capacities were evidenced, in particularly when cells are in a noncultivable state. In addition, the volatile phenol production was clearly shown to be a two-step procedure that could strongly be correlated to the physiological state of the yeast population. Conclusions: This study underlines the importance of minimizing B. bruxellensis populations at the end of wine ageing to reduce volatile phenols production risk once the wine in bottle. Moreover, the physiological state of the yeast seems to have an important impact on ethyl-phenols production, hence demonstrating the importance of taking into account this parameter when analysing wine spoilage risks. Significance and Impact of the Study: Little data exist about the survival of B. bruxellensis once the wine in bottle. This study provides information on the alteration risks encountered during wine storage in bottle and reveals the importance of carrying on further studies to increase the knowledge on B. bruxellensis physiology.

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.

PCR-RFLP analysis of rpoB sequences were used to identify lactic acid bacteria (LAB) species commonly isolated from wine. Strains of seven cocci and 12 lactobacilli species could be identified after single digestion with AciI for the cocci and two or three digestions (AciI, HinfI and MseI) for the rods and preceded by colonies isolation on solid selective medium and microscope observation to distinguish cocci and rods cells.

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.

International audience; Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.

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.