Proteomic and activity characterization of exocellular laccases from three Botrytis cinerea strains

1,1,6-trimethyl-1,2-dihydronaphtelene (TDN) evokes the odor of “petrol” in wine, especially in the variety Riesling. Increasing UV-radiation due to climate change intensifies formation of carotenoids in the berry skins and an increase of TDN-precursors1. Exploring new viticultural and oenological strategies to limit TDN formation in the future requires precise knowledge of TDN thresholds in different matrices. Thresholds reported in the literature vary substantially between 2 µg/L up to 20 µg/L2,3,4 due to the use of different methods. As Riesling grapes are used for very different wine styles such as dry, sweet or sparkling wines, it is essential to study the impact of varying ethanol and carbonation levels.

Tannin, anthocyanins and their reaction products play a major role in the quality of red wines. They contribute to their sensory characteristics, particularly colour and astringency. Grape tannins and anthocyanins are extracted during red wine fermentation. However, their concentration and composition change over time, due to their strong chemical reactivity1. It is also well known that yeasts influence the wine phenolic content, either through the release of metabolites involved in the formation of derived pigments1, or through polyphenol adsorption2,3.

Champagne or sparkling wines elaborated through the same traditional method, which consists in two major yeast-fermented steps, typically hold about 10 to 12 g/L of dissolved CO2 after the second fermentation in a closed bottle. Hundreds of molecules and macromolecules originating from grape and yeast cohabit with dissolved CO2; they are essential compounds contributing to many organoleptic characteristics (effervescence, foam, aroma, taste, colour…). Indeed, the second alcoholic fermentation and the maturation on lees (which may last from 12 months up to several years) both induce various quantitative and qualitative changes in the wine through the action of yeast, as listed hereafter: development of aromas during aging on lees, release of nitrogen compounds during autolysis and release of macromolecules (polysaccharides, lipids, nucleic acids) in wine.

In red wines, anthocyanins evolve during the wine-making process and ageing. They react with other compounds (such as vinylphenols, acetaldehyde, pyruvic acid…) to form a stable family of compounds called pyranoanthocyanins. Furthermore, the oxidation process can modify the anthocyanic profile of a red wine. It is also interesting to evaluate the occurrence of the different subclasses of pyranoanthocyanins and to characterize their chemical properties. The first objective of this study is to evaluate the occurrence of the different groups of pyranoanthocyanins in an oxidised Merlot wine by a centrifugal partition chromatography strategy. The second goal is to evaluate their relative impact in red wines from Bordeaux region by measuring their concentrations.

All techniques usually used to assay proteins was not reliable in vegetable extract due to interferences with the components included in extracts like polyphenols, tanins, pectines, aromatics compounds. Absorbance at 280nm, Kjeldhal assay, Biuret and Lowry methods, Acid Bicinchonique technique and Bradford assay give the results depending on the composition of extract, on the presence or not of detergent and on the raw material (Marchal, 1995). Another difficulty in these extracts for the quantification of proteins comes from the large amount of water included in vegetable and the low concentration of proteins. Thus in red wines, proteins are usually not taken into account due to their low concentration (typically below 10 mgL-1) and to the presence of anthocyanis and polyphenols.