La microbiologie des vins issus des raisins botrytisés au cours de l'élevage. Caractérisation des souches de "Saccharomyces cerevisiae" responsables de refermentations.

La fermentation alcoolique des vins liquoreux français issus de raisin botrytisés est arrêtée brutalement par ajout massif de dioxyde de soufre après qu'un certain équilibre est atteint entre la teneur en alcool formé et la concentration en sucres résiduels. Certaines souches de levures fermentaires survivent et parfois se multiplient provoquant une nouvelle fermentation alcoolique indésirable ; c'est la refermentation. Le suivi microbiologique de nombreux lots de vin a permis de montrer que des levures sont dans un état physiologique similaire à celui décrit chez les bactéries sous l'appellation de viable non cultivable. Cet état explique l'apparente stérilité du vin après le mutage. Au sein de l'espèce Saccharomyces cerevisiae, une sélection naturelle se produit, ne laissant souvent la place qu'à une seule souche de refermentation, tolérante au dioxyde de soufre. Une étude écologique a montré que seules certaines espèces fermentaires et oxydatives survivent. Les plus tolérantes au dioxyde de soufre forment de l'éthanal au cours de l'élevage, malgré un métabolisme ralenti, et augmente la combinaison du dioxyde de soufre libre. Cet éthanal vient progressivement combiner le dioxyde de soufre libre. La sortie de l'état viable non cultivable est probablement la clef des mécanismes engendrant les refermentations. L'utilisation du diméthyldicarbonate au moment du mutage a été étudiée en couplage avec le dioxyde de soufre. Des souches de Saccharomyces cerevisiae de refermentation ont été isolées. Elles exhibent des singularités de séquence de leur ADNr, les apparentant aux souches de voile de certains vins spéciaux. Ces souches surexpriment constitutivement le gène SSU1 et synthétisent rapidement une forte concentration d'éthanal en réponse à la présence de dioxyde de soufre. La présence de fortes concentrations de dioxyde de soufre sélectionne les souches les plus résistantes. La refermentation est donc le résultat d'une adaptation génétique et d'une sélection, fruit d'une multitude de paramètres microbiologiques, physico-chimiques et humains. Botrytis-affected wines microbiology during maturation. Characterization of Saccharomyces cerevisiae strains responsible for refermentation. ABSTRACT : The alcoholic fermentation of Botrytis-affected wines is stopped by addition of sulphur dioxide. Some fermenting yeast species can survive during maturation and grow in spite of high ethanol, sugars and sulphur dioxide concentrations. An undesirable new fermentation, named "refermentation", can sometimes occur. In this study, it was proved that some yeast species were able to survive in a viable but non-culturable-like state. This state explains the apparent sterility of wines during maturation. Within Saccharomyces cerevisiae species, an intraspecific selection was spontaneously operated. After some weeks, only one strain could often survive. An ecological study was realized. Some highly fermentative and oxidative species could survive. In spite of slower metabolism, they synthesized acetaldehyde during maturation. The exit from the VBNC state and the high sulphur dioxide binding power were the keys of refermentations. The use of dimethyldicarbonate to stop alcoholic fermentation was studied. The most efficient action was observed for the mixed sulphur dioxide and DMDC addition. Some Saccharomyces cerevisiae strains responsible for refermentations were isolated. These strains exhibited rDNA sequence singularities, showing that they were close to flor strains, responsible for velum formation in some special wines. Moreover, those strains constitutively over-expressed SSU1 gene and could rapidly synthesize high concentrations of acetaldehyde in response to sulphur dioxide addition. High sulphur dioxide concentrations had probably selected the most resistant strains. Refermentation is the result of genetic adaptation and selection, under the influence of microbiological, physical, chemical and human parameters

Transcriptomics unravels the adaptive molecular mechanisms of Brettanomyces bruxellensis under SO2 stress in wine condition

CITATION: Valdetara, F. et al. 2020. Transcriptomics unravels the adaptive molecular mechanisms of Brettanomyces bruxellensis under SO2 stress in wine condition. Food Microbiology, 90. doi:10.1016/j.fm.2020.103483.The original publication is available at https://www.sciencedirect.com/journal/food-microbiologySulfur dioxide is generally used as an antimicrobial in wine to counteract the activity of spoilage yeasts, including Brettanomyces bruxellensis. However, this chemical does not exert the same effectiveness on different B. bruxellensis yeasts since some strains can proliferate in the final product leading to a negative sensory profile due to 4-ethylguaiacol and 4-ethylphenol. Thus, the capability of deciphering the general molecular mechanisms characterizing this yeast species’ response in presence of SO2 stress could be considered strategic for a better management of SO2 in winemaking. A RNA-Seq approach was used to investigate the gene expression of two strains of B. bruxellensis, AWRI 1499 and CBS 2499 having different genetic backgrounds, when exposed to a SO2 pulse. Results revealed that sulphites affected yeast culturability and metabolism, but not volatile phenol production suggesting that a phenotypical heterogeneity could be involved for the SO2 cell adaptation. The transcriptomics variation in response to SO2 stress confirmed the strain-related response in B. bruxellensis and the GO analysis of common differentially expressed genes showed that the detoxification process carried out by SSU1 gene can be considered as the principal specific adaptive response to counteract the SO2 presence. However, nonspecific mechanisms can be exploited by cells to assist the SO2 tolerance; namely, the metabolisms related to sugar alcohol (polyols) and oxidative stress, and structural compounds.https://www.sciencedirect.com/science/article/pii/S0740002020300721?via%3DihubPublishers versio

La microbiologie des vins issus de raisins botrytisés au cours de l'élevage. Caractérisation des souches de Saccharomyces cerevisiae responsables de refermentation

La fermentation alcoolique des vins liquoreux issus de raisin botrytisés est arrêtee brutalement par ajout massif de dioxyde de soufre. Certaines sousches de levures survivent et parfois provoquent une refermentation. Ces levures survivent dans un état comparable à celui qualifié de viable non cultivable chez les bactéries. Seules certaines espèces fermentaires et oxydatives survivent. L'utilisation potentielle du diméthyldicarbonate en complément du sioxyde de soufre a été étudiée. Les souches de saccharomyces cerevisiae de refermentation isolées exhibent des singularités de séquence de leur ADNr, les apparentant aux souches de voile de certains vins spéciaux. Ces souches surexpriment constitutivement le gène ssu1 et synthétisent rapidement une forte concentration d'éthanal en réponse à la présence de dioxyde de soufre. La refermentation est le résultat d'une adaptation génétique et d'une sélection, fruit d'une multitude de paramètres microbiologiques physico-chimiques et humains.TOULOUSE-ENSIACET (315552325) / SudocSudocFranceF

Quantitative PCR: An appropriate tool to detect viable but not culturable <I>Brettanomyces bruxellensis </I>in wine

AgriwetenskappeInstituut Vir WynbiotegnologiePlease help us populate SUNScholar with the post print version of this article. It can be e-mailed to: [email protected]

Comparative characterization of endo-polygalacturonase (Pgu1) from Saccharomyces cerevisiae and Saccharomyces paradoxus under winemaking conditions

Wine strains of Saccharomyces cerevisiae have no to weak natural pectinase activity, despite their genetic ability to secrete an endo-polygalacturonase. The addition of external pectinase of fungal origin has therefore become a common step of winemaking in order to enhance the extraction of compounds located in the grape berry skins during maceration and to ease wine clarification after maturation. Recently, the strong pectinase activity of a wine strain of Saccharomyces paradoxus has been reported. In this study, the endo-polygalacturonase-encoding gene of S. paradoxus was sequenced and its activity was characterised, compared with that of S. cerevisiae and tested under winemaking conditions through overexpression of both genes individually in S. cerevisiae. A few differences in the amino acids sequences between the two proteins were revealed and the activity of the Pgu1 enzyme of S. paradoxus was shown to be weaker under winemaking conditions. Clear indicators of extracellular activity were observed in the wines made with both recombinant strains (i.e. enzyme activity in cell-free wine, higher methanol concentration and higher free-run wine), but the actual composition of the wines fermented with the mutants was only sparingly altered. Although unexpectedly found in lower concentrations in the latter wines, phenolic compounds were shown to be the most discriminatory components. Overexpressing the PGU1 gene of S. paradoxus or that of S. cerevisiae did not make much difference, showing that the higher activity of S. paradoxus strains under laboratory conditions could be due to a different regulation mechanism rather than to a different sequence of PGU1. © 2011 Springer-Verlag.Articl

Metabolic engineering of wine yeast and advances in yeast selection methods for improved wine quality and safety.

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<I>La biologie systemique, les levures et le secteur vitivinicole.</I> [Systems biology, yeast and the wine industry PUBLISHED IN FRENCH]

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PGU1 gene natural deletion is responsible for the absence of endo-polygalacturonase activity in some wine strains of Saccharomyces cerevisiae

The PGU1 gene encodes an endo-polygalacturonase enzyme in Saccharomyces cerevisiae. The literature reports that most S. cerevisiae strains possess this gene, despite a wide range of enzyme activity levels. Nevertheless, a few wine strains lack the PGU1 gene. We investigated the PGU1 locus sequence in these strains. The results indicated that the gene had been replaced by a partial Ty mobile element, whereas the gene promoter was still at the expected location. As all the strains lacking the PGU1 gene experienced the same phenomenon, it was tempting to hypothesize a common phylogenetic origin. However, fingerprints only allowed grouping of a few of them within one cluster. © 2007 Federation of European Microbiological Societies.Articl

Observation on the refermentation of sweet <em>Botrytis</em>-affected wines

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

Tracking the careers of grape and wine polymers using biotechnology and systems biology.

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