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Dès les années 1950, B. bruxellensis apparait dans les inventaires de levures de moûts et de vins, et rapidement, on lui attribue des altérations du vin. Beaucoup plus tard, les phénols volatils produits par ces levures sont identifiés comme la cause de déviations sensorielles. Leur présence est de plus en plus prégnante, surtout pendant l’élevage en barriques. Le sulfitage est le moyen idéal d’éradication, mais s’avère quelquefois insuffisant. Particulièrement bien adaptée au vin, B. bruxellensis est aujourd’hui au centre des préoccupations des vinificateurs et des microbiologistes.

Aims: The aim of this study was to develop a colorimetric test to determine Oenococcus carbohydrate consumption profile. Methods and results: A semi-defined growth medium which enabled efficient bacterial growth and medium acidification in the presence of glucose was developed. The inoculum size and the presence of citrate in the medium were optimized. Acidification of the medium was revealed by adding bromocresol green and was shown to be perfectly correlated with D-glucose, D-fructose, L-arabinose, D-xylose, L-rhamnose, D-cellobiose or D-trehalose consumption by 23 distinct O. oeni strains and one O. kitaharae strain (confirmed with HPLC analysis). Significance and impact of the study: Efficient bacterial growth is essential before malic acid degradation occurs in wine. The method developed here will facilitate the comparison of numerous O. oeni strains and their ability to grow by using the various carbohydrates present in wine prior to malolactic fermentation.

Aims: This work reviews the latest knowledge concerning the role of Brettanomyces bruxellensis in red wine alteration. Results and conclusion: The origin of this yeast species and its place in the wine microbial consortium are discussed as well as microbial equilibriums with the other species, notably Saccharomyces cerevisiae and lactic acid bacteria. As a consequence, fermentations are described as key steps in Brettanomyces development management. Furthermore, the influence of ageing through the use of traditional winemaking practices is explained. Significance and impact of study: Finally, this paper emphases the need for a better understanding of chemical and microbial analysis together in order to better control this undesirable yeast and prevent the production of volatile phenols.

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 rpoΒ 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.

Yeast, bacteria, species and strains play a key role in the winemaking process by producing metabolites, which determine wine sensorial qualities. Therefore microbial population enumeration, species identification and strain discrimination from berry surface at harvest to storage in bottle are fundamental. The microbial diversity and significance of its variation according to the estate localization have not really been thoroughly considered in literature. This is the focus of this work. That should be of great interest because the spontaneous microbial population dynamics associated with a wine producing estate provide information on what might be considered as the method to obtain specific terroir typed wine. The both use of conventional microbiological methods like microbial population enumeration on nutritive selective media and efficient molecular tools of species identification like PCR-RFLP for yeasts and PCR-DGGE for bacteria and strains discrimination have demonstrated significant microbial differences between different estates localized in the Bordeaux area. Theses results appeared very interesting since certain microbial species are clearly specific of certain estates, in particular the bacterium Pediococcus parvulus, and also some strains of Saccharomyces cerevisiae, of Oenococcus oeni and Brettanomyces bruxellensis. Within an estate this specificity persists from one year to another. These differences observed suggest that the indigenous winemaking processes can contribute to the specificity of the wines produced on the various estates.. That concludes to the hypothesis of a microbial part in wines speficities.

The alcoholic fermentation is a crucial winemaking step. Its failure is problematic. In spite of several studies to understand and elucidate these problems wine global microbial ecology has never been considered. Using conventional microbiological methods and sensitive molecular tools we monitored the alcoholic fermentations of different red grape varieties in several cellars located in Bordeaux area. These observations were made during three successive vintages in different oenological conditions. The effect of the addition of commercial active dried yeast and of initial cold maceration was studied. All these factors were compiled and their effects on microbial changes were investigated. Some of them acted directly on the microbial population of berries surface at the harvest time and should have impact on alcoholic fermentation. They could modify the microbial changes which in some cases could lead to sluggish fermentation. In these cases, we focused on the Brettanomyces bruxellensis spoilage problem. The risk of further contamination was discussed according to the alcoholic fermentation development.

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.

This study aims to understand the effect on micro-organism of racking when the wine is aged in barrels. According to the kind of micro-organism, the effects are different. Bacteria are stimulated by oxygen and their population increases. Yeasts are concentrated to the bottom of the barrel. Between two successive racking a yeast population gradient was established. Yeast cells which are larger and heavier than bacteria cells and they are deposited on the barrel bottom with other wine micro-particles. In some cases, the yeast population at the bottom was more than thousand times than at the wine surface. Moreover, the species identified at different heights in the barrel were different. Saccharomyces cerevisiae was the main yeast detected at the surface, whereas Brettanomyces bruxellensis was the main yeast lees. After racking yeast population decreases because they are eliminated with the lees during the operation. Among them, Brettanomyces bruxellensis was the majority. Since they are able to produce volatile phenols, their preservation in the barrel can lead to the alteration of the wine. Indeed, the ability of the lees to produce volatile phenols was clearly established. The importance of regular racking for microbial wine stabilization is evident. The risks of «sur lies» wine aging and sticking’s operations are underlined.

Botrytis-affected wines are microbiologically unstable. Fermentation can occasionally occur during maturing time or bottle-ageing. However, wines which undergo refermentations seem to be free of yeasts. This work deals with microbial aspects and ecology of wines during alcoholic fermentation, then during refermentation. Yeast survival after stopping the alcoholic fermentation by sulphur dioxide addition is considered. Results suggest that most yeasts could survive in sweet wine under the viable but non-culturable (VBNC) state. The increase of acetaldehyde these wines in response to sulfiting is known for long time but not the conditions of its formation. In this work we show that it is probably the key of the refermentation phenomenon, through the increase of the binding power. During maturing successive cycles occur: acetaldehyde consumption, revival, death and/or new enter into VBNC state accompanied by acetaldehyde formation which increased the binding power. Then as molecular SO2 became too low, yeasts could start a new fermentation. In this work, an ecology study was also undertaken, to understand the effect of SO2 on yeast biodiversity. Most of the fermentative yeasts could survive especially Saccharomyces cerevisiae and Zygosaccharomyces sp., together with other yeast genera. In regard to Saccharomyces cerevisiae only which is often responsible for the refermentation phenomenon, this work concluded on the decrease of strain diversity to only one or two strains, in spite of the high degree of diversity during the alcoholic fermentation. The intra-species selection probably conducted to sulfite-resistant strains.

Four direct inoculation malolactic starters were used in this study. They conducted malolactic fermentation in a merlot wine in barrels. We compared the behaviour of these strains at multiple levels. Inoculation efficiency was evaluated, and great differences were observed among the starters. Implantation controls were also carried out. They were ail clearly positive. The rates of malate degradation were compared. They were ail different, but MLF in the inoculated barrels always finished before the non-inoculated control. Physicochemical and sensory analysis were done. No major differences were seen concerning chemical data. However, some significant differences were observed at the sensory level. Positive implantation controls allowed to conclude that these differences originated from the starter strains used in this study.

Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods. European Journal of Clinical Nutrition (2010) 64, S95-S100; doi: 10.1038/ejcn.2010.218 Keywords: biogenic amines; lactic acid bacteria; risk assessment; food fermentation; acid stress;toxicological effects, Introduction Lactic acid bacteria (LAB) can produce metabolic energy and/ or increase their acid resistance by using catabolic pathways that convert amino acids into amine-containing compounds referred to as biogenic [...]

Six malolactic starters were experimented. The objective was an evaluation of the capacity of each one to settle in different wines. First we obtained the genomie DNA fingerprinting with Not I a rare cutting enzyme after pulse field gel electrophoresis (PFGE). Previous results with Leuconostoc oenos strains of our collection showed that this enzyme was the most appropriated for identification at the strain level. However, we did not obtain six different patterns, three starters showed the same. Therefore Apa I and Sfi I patterns were also established. Contrary to the expected results A, B and C finally had the same fingerprinting with the three enzymes. Another investigation was conduced using the Southem hybridization of EcoRI restricted DNA with the φMC10 prophage DNA as probe. Our again all three starters showed the same pattern. They probably were prepared with the same strain. Wines in vat or barrels were inoculated just after racking. Malolactic fermentation had already begun in some of them before inoculation. However malic acid was degraded more quickly in all the inoculated wines than in the controls. The PFGE patterns were obtained from bacteria isolated from wines at the end of malolactic fermentation. In 15 cases of 21, the pattern of the starter appeared alone or superimposed with other bacteria. This proved that, even in the worst conditions of this experimentation, where the indigenous microflora was very abundant and active malolactic starters could survive and predominate. The influence of these starters on the sensorial quality of wines needs to be studied.

Copper fungicides are widely used in viticulture. Due to its large spectrum of action, copper provides an efficient control over a great number of vine pathogens. Previous studies showed that, high levels of cupric residues can impact grape-berry microbiota, in terms of the size and population structure, reducing the diversity and the abundance. Due to the importance of grape-berry bacterial in crop health, and the potential impact of copper fungicides over the microbiota, we determined Minimum Inhibitory Concentration (MIC) of different copper formulations for bacterial species isolated from grape berries. We study the Minimum Inhibitory Concentration (MIC) of different copper formulations (copper sulphate (CuSO4) pure, Bordeaux mixture (CuSO4 + Ca(OH)2), copper oxide (Cu2O), copper hydroxide (Cu(OH)2)) over 92 bacterial strains isolated from grape berries in different stages of the ripening process. The results of MIC measurements revealed that the different copper formulations have a variable inhibitory effect and among the different isolates, some species are the most resistant to all copper formulations than others. This study confirm that usage of cupric phytosanitary products should be reasonable independently of the farming system; they also provide evidence of the importance of the choice of which copper formulations are to be used regarding their impact on the grape berry bacterial microbiota.

Plasmids in lactic acid bacteria occasionally confer adaptive advantages improving the growth and behaviour of their host cells. They are often associated to starter cultures used in the food industry and could be a signature of their superiority. Oenococcus oeni is the main lactic acid bacteria species encountered in wine. It performs the malolactic fermentation that occurs in most wines after alcoholic fermentation and contributes to their quality and stability. Industrial O. oeni starters may be used to better control malolactic fermentation. Starters are selected empirically by virtue of their fermentation kinetics and capacity to survive in wine. This study was initiated with the aim to determine whether O. oeni contains plasmids of technological interest. Screening of 11 starters and 33 laboratory strains revealed two closely related plasmids, named pOENI-1 (18.3-kb) and pOENI-1v2 (21.9-kb). Sequence analyses indicate that they use the theta mode of replication, carry genes of maintenance and replication and two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tauE) and a NADH:flavin oxidoreductase of the old yellow enzyme family (oye). Interestingly, pOENI-1 and pOENI-1v2 were detected only in four strains, but this included three industrial starters. PCR screenings also revealed that tauE is present in six of the 11 starters, being probably inserted in the chromosome of some strains. Microvinification assays performed using strains with and without plasmids did not disclose significant differences of survival in wine or fermentation kinetics. However, analyses of 95 wines at different phases of winemaking showed that strains carrying the plasmids or the genes tauE and oye were predominant during spontaneous malolactic fermentation. Taken together, the results revealed a family of related plasmids associated with industrial starters and indigenous strains performing spontaneous malolactic fermentation that possibly contribute to the technological performance of strains in wine.

In lactic acid bacteria (LAB), amino acids and their derivatives may be converted into amine-containing compounds designated biogenic amines, in pathways providing metabolic energy and/ or acid resistance to the bacteria. In a previous study, a pathway converting tyrosine to tyramine was detected in Lactobacillus brevis and a fragment of a gene possibly involved in the production of another biogenic amine, putrescine, from agmatine, was detected in the same locus. The present study was carried out to determine if Lb. brevis actually harbours two biogenic amine-producing pathways in the same locus and to investigate the occurrence of the two gene clusters in other bacteria. Sequencing of the DNA locus in Lb. brevis revealed a cluster of six genes that are related to previously reported genes of agmatine deiminase pathways but with marked differences such as two genes encoding putative agmatine deiminases rather than one. Heterologous expression of encoded enzymes confirmed the presence of at least one active agmatine deiminase and one amino acid transporter that efficiently exchanged agmatine and putrescine. It was concluded that the Lb. brevis gene cluster encodes a functional and highly specific agmatine deiminase pathway. Screening of a collection of 197 LAB disclosed the same genes in 36 strains from six different species, and almost all the positive bacteria also contained the tyrosine catabolic pathway genes in the same locus. These results support the hypothesis that the agmatine deiminase and tyrosine catabolic pathways belong to a genomic region that provides acid resistance and that is exchanged horizontally as a whole between LAB.

La désacidification biologique est le résultat de la transformation enzymatique de l'acide L-malique. Levures et bactéries lactiques possèdent un équipement enzymatique, différent à la fois par la nature des produits de la transformation et leur affinité pour le substrat. La fermentation malolactique assurée par les bactéries lactiques est le procédé le plus répandu. Sa maîtrise passe par l'utilisation de levains bactériens, dont les plus récents sont ensemencés directement au vin après la fermentation alcoolique. Les résultats des recherches fondamentales sur l'enzyme malolactique permettent aujourd'hui d'envisager la construction de réacteurs enzymatiques et sans doute plus rapidement, le transfert de cette activité enzymatique chez les levures de vinification.

Des vins blancs liquoreux ont été mutés par traitement aux hautes pressions. La fermentation alcoolique est complètement et définitivement arrêtée lorsque les vins sont traités à 3000- 3500 bars pendant 10 min. Il ne reste plus aucune levure viable. Le traitement n'a aucun effet majeur sur la constitution du vin, au moins en ce qui concerne ses caractéristiques principales. Seule la couleur est légèrement modifiée. Les vins ainsi traités doivent être protégés contre l'oxydation. Les qualités organoleptiques ne sont pas modifiées. Ce traitement doit être évalué à plus grande échelle afin de connaÎtre ses effets sur le vieillissement de différents types de vins.

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.

A histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions is isolated and analyzed. The result describes the genes encoding a histamine-producing pathway and provides clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.

La différenciation des levures de fermentation des levures de contamination du vin par l'analyse de leurs profils en acides gras libres totaux a été évaluée. L'étude porte sur 39 souches de levures représentant 24 espèces. Les cultures se déroulent à la température de 28°C, en aérobiose et en milieu liquide. Par cette méthode, il est possible de différencier S. cerevisioe des autres genres de levures, mais il semble toutefois difficile de déterminer de façon exacte la souche responsable de la contamination. Enfin, ces résultats montrent qu'il est important d'étudier le métabolisme des acides gras, molécules indispensables à la survie des cellules afin de comprendre certaines réponses physiologiques des différentes espèces de levures.

La lyse de Leuconostoc oenos a été étudiée en suspension dans l'eau et dans une solution synthétique (pH 3,5; alcool 11 % v/v, acide tartrique 4 g/l). Elle a été mesurée par deux méthodes basées sur la libération de constituants cellulaires (mesure à 210 mm) et sur la diminution de l'opacité de la solution (mesure à 600 nm). Les cellules en phase de croissance exponentielle se lysent plus facilement que les cellules prélevées pendant les autres phases du cycle cellulaire. L'éthanol dans le milieu de suspension augmente la lyse de cellules cultivées dans le milieu standard. Par contre, le phénomène est plus limité dans la solution acide et alcoolisée que dans l'eau, sauf pour les cellules cultivées en présence d'acides gras insaturés où l'on observe le résultat inverse. La désorganisation des membranes et l'activité autolytique des cellules dépendent à la fois des conditions de culture des bactéries et du milieu dans lequel elles sont mises en suspension.

Dans cette note nous donnons une méthode pour la détermination des levures viables dans des vins pauvres en germes (au moment de la mise en bouteille), et nous comparons les résultats à ceux obtenus par la méthode de référence (comptage des colonies sur milieu nutritif).