Alcoholic fermentation drives the selection of Oenococcus oeni strains in wine but not in cider

Oenococcus oeni is the predominant lactic acid bacteria species in wine and cider, where it performs the malolactic fermentation (MLF). The O. oeni strains analyzed to date form four major genetic lineages named phylogroups A, B, C and D. Most of the strains isolated from wine, cider, or kombucha belong to phylogroups A, B + C, and D, respectively, although B and C strains were also detected in wine. This study was performed to better understand the distribution of the phylogroups in wine and cider. Their population dynamics were determined by qPCR all through wine and cider productions, and the behavior of the strains was analyzed in synthetic wines and ciders. Phylogroups A, B and C were all represented in grape must and throughout the alcoholic fermentation, but on the transition to MLF, only phylogroup A remained at high levels in all wine productions. In the case of cider, phylogroups A, B and C were detected in stable levels during the process. When they were tested in synthetic wine and cider, all phylogroups performed MLF, but with different survival rates depending on the ethanol content. In this sense, ethanol and fermentation kinetics are the main agent that drives the selection of phylogroup A strains in wine, while B and C strains dominates in cider containing less ethanol

Population dynamics and yeast diversity in early winemaking stages without sulfites revealed by three complementary approaches

Nowadays, the use of sulfur dioxide (SO2 ) during the winemaking process is a controversial societal issue. In order to reduce its use, various alternatives are emerging, in particular bioprotection by adding yeasts, with different impacts on yeast microbiota in early winemaking stages. In this study, quantitative-PCR and metabarcoding high-throughput sequencing (HTS) were combined with MALDI-TOF-MS to monitor yeast population dynamic and diversity in the early stages of red winemaking process without sulfites and with bioprotection by Torulaspora delbrueckii and Metschnikowia pulcherrima addition. By using standard procedures for yeast protein extraction and a laboratory-specific database of wine yeasts, identification at species level of 95% of the isolates was successfully achieved by MALDI-TOF-MS, thus confirming that it is a promising method for wine yeast identification. The different approaches confirmed the implantation and the niche occupation of bioprotection leading to the decrease of fungal communities (HTS) and Hanseniaspora uvarum cultivable population (MALDI-TOF MS). Yeast and fungi diversity was impacted by stage of maceration and, to a lesser extent, by bioprotection and SO2, resulting in a modification of the nature and abundance of the operational taxonomic units (OTUs) diversity

L’identification des levures et bactéries œnologiques par spectrométrie de masse de type MALDI-TOF

La spectrométrie de masse de type MALDI-TOF a été adaptée afin d’être utilisée comme outil innovant d’identification au niveau de l’espèce des levures et bactéries isolées d’échantillons variés (moûts, vins, boissons). L’analyse d’un grand nombre de clones permet d’apprécier la diversité des espèces de levures, bactéries acétiques et lactiques présentes dès les phases pré-fermentaires, au cours des fermentations, pendant l’élevage ou après conditionnement. Dans le cas d’altération de produits, cet outil innovant participera à une meilleure maitrise des risques microbiologiques

Two different Oenococcus oeni lineages are associated to either red or white wines in Burgundy: genomics and metabolomics insights

Oenococcus oeni is the bacterium most often associated with spontaneous malolactic fermentation (MLF) of wine. During MLF, malic acid is transformed into lactic acid and several metabolites are modified, modulating wine’s total acidity and improving its sensory properties. Previous works have suggested that certain genetic groups of O. oeni strains are associated to different kinds of products. In the present study we have spotted two groups of strains isolated mainly from Burgundy wines, one associated to red wines and the other to white wines. Sequencing 14 genomes of red and white wine strains revealed that they share a common ancestor that probably colonised two different substrates –red and white wine-associated environments–, diverging over time and disseminating to various regions. Their capacity to perform MLF and modify the volatile profile of wine was determined by fermenting a chardonnay wine and analysing its volatile fraction with a non-targeted metabolomics approach by GC-MS. The strains had a different impact on the volatile composition depending on their group of origin. These results show for the first time a correspondence between the product of origin of the strains and the volatile profile of the wines they produce. Furthermore, the genetic features that might be implied in these different phenotypes are examined

Front Microbiol

Brettanomyces bruxellensis is the main spoilage microbial agent in red wines. The use of fungal chitosan has been authorized since 2009 as a curative treatment to eliminate this yeast in conventional wines and in 2018 in organic wines. As this species is known to exhibit great genetic and phenotypic diversity, we examined whether all the strains responded the same way to chitosan treatment. A collection of 53 strains of was used. In the conditions of the reference test, all were at least temporarily affected by the addition of chitosan to wine, with significant decrease of cultivable population. Some (41%) were very sensitive and no cultivable yeast was detected in wine or lees after 3 days of treatment, while others (13%) were tolerant and, after a slight drop in cultivability, resumed growth between 3 and 10 days and remained able to produce spoilage compounds. There were also many strains with intermediate behavior. The strain behavior was only partially linked to the strain genetic group. This behavior was little modulated by the physiological state of the strain or the dose of chitosan used (within the limits of the authorized doses). On the other hand, for a given strain, the sensitivity to chitosan treatment was modulated by the chitosan used and by the properties of the wine in which the treatment was carried out.Recherches sur l’origine et les effets secondaires des propriétés stabilisantes du chitosane fongique dans le vi

Traitement des vins au chitosane fongique. Origine de la variabilité des résultats pour l’élimination de Brettanomyces bruxellensis

International audienceB. bruxellensis est le principal agent d’altération des vins rouges dans lesquels elle produit des phénols volatils pouvant déprécier la qualité du vin. Les levures de cette espèce sont particulièrement adaptées aux contraintes du vin (nutriments disponibles, pH, présence d’alcool, dioxyde de soufre), capables de se développer même à basse température et peuvent persister dans différents lots de vins d’un même château plusieurs années de suite (Cibrario et al., 2019 a, b ; 2020 a, b). Le sulfitage reste le moyen de lutte le plus utilisé pour prévenir le développement récurrent de cette espèce de levure et ainsi limiter la production de phénols volatils. Toutefois, dans un contexte d’engouement pour les vins issus de l’agriculture biologique et de limitation générale des doses de SO2, d’autres traitements ont émergé. Ainsi, depuis 2009, le chitosane fongique est autorisé comme agent antiseptique dans les vins dits conventionnels (OIV/Oeno, 338A/2009 et Réglementation européenne EC/53/2011) et depuis 2018 pour ceux issus de l’agriculture biologique.Cet agent antimicrobien, non allergène contrairement au SO2, a fait l’objet de nombreuses études attestant de son efficacité pour réduire les populations de B. bruxellensis dans les vins (Teissedre et al., 2007, Blayteron et al., 2012, Taillandier et al., 2015). Cependant, d’autres travaux, étayés également par les retours de professionnels, modèrent l’efficacité du chitosane pour éliminer B. bruxellensis dans les vins rouges (Petrova et al., 2016). Le projet collaboratif CHITOWINE, démarré en 2017 et financé par l’Agence nationale de la recherche (ANR), vise à mieux comprendre le mécanisme d’action du chitosane et à proposer des préconisations pour un emploi raisonné. Ces travaux s’appuient sur des résultats issus d’une démarche scientifique rigoureuse, en tenant compte des dernières connaissances sur l’espèce B. bruxellensis, notamment la très large diversité génétique de l’espèce, récemment mise en évidence (Avramova et al., 2018a) ou encore la diversité des vins à traiter

Mol Biotechnol

Oenococcus oeni is the main bacterial species that drives malolactic fermentation in wine. Most O. oeni strains produce capsular exopolysaccharides (EPS) that may contribute to protect them in the wine hostile environment. In O. oeni genome sequences, several genes are predicted to encode priming glycosyltransferases (pGTs). These enzymes are essential for EPS formation as they catalyze the first biosynthetic step through the formation of a phosphoanhydride bond between a hexose-1-phosphate and a lipid carrier undecaprenyl phosphate. In many microorganisms, mutations abolishing the pGT activity also abolish the EPS formation. We first made an in silico analysis of all the genes encoding putative pGT over 50 distinct O. oeni genome sequences. Two polyisoprenyl-phosphate-hexose-1-phosphate transferases, WoaA and WobA, and a glycosyltransferase (It3) were particularly examined for their topology and amino acid sequence. Several isoforms of these enzymes were then expressed in E. coli, and their substrate specificity was examined in vitro. The substrate specificity varied depending on the protein isoform examined, and several mutations were shown to abolish WobA activity but not EPS synthesis. Further analysis of woaA and wobA gene expression levels suggests that WoaA could replace the deficient WobA and maintain EPS formation