Wine microbiome : A dynamic world of microbial interactions

International audience; Most fermented products are generated by a mixture of microbes. These microbial consortia perform various biological activities responsible for the nutritional, hygienic, and aromatic qualities of the product. Wine is no exception. Substantial yeast and bacterial biodiversity is observed on grapes, and in both must and wine. The diverse microorganisms present interact throughout the winemaking process. The interactions modulate the hygienic and sensorial properties of the wine. Many studies have been conducted to elucidate the nature of these interactions, with the aim of establishing better control of the two fermentations occurring during wine processing. However, …

Truncated internalin A and asymptomatic Listeria monocytogenes carriage: in vivo investigation by allelic exchange

ABSTRACT Allelic exchange of the region coding for the C terminus of InlA between one epidemic (with an 80-kDa InlA) and one asymptomatic (with a 47-kDa InlA) carriage Listeria monocytogenes strain confirmed the need for this region for internalin entry in vitro. Interestingly, restoration of internalin A functionality did not result in full virulence in chicken embryo assays.

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…

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…

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable populat…

Indigenous Listeria spp. in biowastes and during composting

International audience

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…

Isolation and characterisation of new Gram-negative and Gram-positive atrazine degrading bacteria from different French soils

The capacity of 12 soils to degrade atrazine was studied in laboratory incubations using radiolabelled atrazine. Eight soils showed enhanced degradation of this compound. Twenty-five bacterial strains able to degrade atrazine were isolated by an enrichment method from 10 of these soils. These soils were chosen for their wide range of physico-chemical characteristics. Their history of treatment with atrazine was also variable. The genetic diversity of atrazine degraders was determined by amplified ribosomal restriction analysis (ARDRA) of the 16S rDNA gene with three restriction endonucleases. The 25 bacterial strains were grouped into five ARDRA types. By sequencing and aligning the 16S rDN…

Influence of organic amendments on diuron leaching through an acidic and a calcareous vineyard soil using undisturbed lysimeters.

9 pages; International audience; The influence of different organic amendments on diuron leaching was studied through undisturbed vineyard soil columns. Two composts (A and D), the second at two stages of maturity, and two soils (VR and Bj) were sampled. After 1 year, the amount of residues (diuron þ metabolites) in the leachates of the VR soil (0.19e0.71%) was lower than in the Bj soil (4.27e8.23%), which could be explained by stronger diuron adsorption on VR. An increase in the amount of diuron leached through the amended soil columns, compared to the blank, was observed for the Bj soil only. This result may be explained by the formation of mobile complexes between diuron and water-extrac…

Yeast–yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts

International audience; There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the vola…

Diversité des Brettanomyces et de leur résistance au SO2. Les nouvelles avancées vers une meilleure gestion du SO2 en vinification.

International audience; Des recherches ont été menées par le Groupe National « Lutte contre Brettanomyces » et plus particulièrement sur la relation SO2 et Brettanomyces bruxellensis afin d’approfondir les connaissances sur le comportement de la levure et d’apporter des données essentielles à une bonne gestion du risque.Une grande diversité de la levure Brettanomyces a été mise en évidence (identification de différents groupes génétiques) ainsi que des comportements différents vis-à-vis du SO2 : sensibles, tolérants ou résistant. Grâce à la mise au point d’un outil prédictif (TYP \ Brett), les professionnels pourront connaître le groupe génétique pour mieux intervenir.Ces travaux ont égalem…

Altérations de la couleur des vignes : maladies et ravageurs contribuent à la palette

National audience

Plasmid localisation of atrazine-degrading genes in newly described Chelatobacter and Arthrobacter strains

Abstract In a previous study, we isolated a collection of atrazine-degrading bacteria from various soils. The aim of this study was to localise the atrazine-degrading genes in these 25 atrazine-degrading strains. In the case of the Gram-negative strains of Chelatobacter heintzii, six to seven plasmids were observed. The atzABC and trzD genes were located on two or three plasmids with variable molecular masses. For the Gram-positive strains of Arthrobacter crystallopoietes, the atzBC genes were located on a single plasmid of 117 kb. The organisation of atrazine-degrading genes seems to be highly variable between the strains studied. We have shown by a specific PCR the occurrence of IS1071-li…

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 …

High-throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation

Abstract We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods …

Membres du comité d'organisation scientifique

Inoculation of an atrazine-degrading strain, Chelobacter heintzii Cit1, in four different soils : effects of different inoculum densities

International audience

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…

Diversité taxonomique, génotypique et physiologique des souches bactériennes dégradant l'atrazine dans les sols. Impacts d'apports de boues de station d'épuration et essai de bioremédiation

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…

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…

Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring

Abstract In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (106 CFU ml−1) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis …

Analyse de la biodégradation accélérée dans un sol cultivé avec du maïs

National audience

Long-term survival of pathogenic and sanitation indicator bacteria in experimental biowaste composts

ABSTRACT For economic, agricultural, and environmental reasons, composting is frequently used for organic waste recycling. One approach to limiting the potential risk from bacterial food-borne illnesses is to ensure that soil amendments and organic fertilizers are disinfected. However, more knowledge concerning the microbiological safety of composted substrates other than sludge and manure is necessary. Experimental in-vessel biowaste composts were used to study the survival of seeded Listeria monocytogenes , Salmonella enterica subsp. enterica serotype Enteritidis, and Escherichia coli . Four organic waste mixtures, containing various proportions of paper and cardboard, fruits and vegetabl…