Macrowine 2021
IVES 9 IVES Conference Series 9 Proteomic and activity characterization of exocellular laccases from three Botrytis cinerea strains

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

Abstract

Botrytis cinerea is a fungus that causes common infection in grapes and other fruits. In winemaking, its presence can be both considered desirable in the case of noble rot infection or undesirable when grey rot is developed. This fungus produces an extracellular enzyme known as laccase which is able to cause oxidation of phenolic compounds present in must and wine, causing most of the times a decrease in its quality and problems during the winemaking process [1]. Material and methods: Three B. cinerea strains (B0510, VA612 and RM344) were selected and grown in a liquid medium adapted from one previously described [2]. The enzyme was isolated by tangential ultrafiltration of the culture medium using a QuixStand system equipped with a 30 KDa filtration membrane. The purity of the isolated enzymes was checked using SDS-PAGE. The characteristics (molecular weight, % of glycosylation, specific activity, activity in function of pH of the enzyme isolated from each strain were studied with ABTS as a substrate. Results: The enzymes isolated from the three strains showed the same molecular weight, 97 KDa, in good agreement with the molecular weight previously determined for B. cinerea laccase using SDS-PAGE [2]. The percentage of glycosylation was high, being estimated in 70% on weight, also similar to that described by other authors [3]. Despite similar physical characteristics of the enzymes obtained from different strains, their activity were quite different. The enzymes isolated from B0510 and VA612 strains showed similar specific activity for ABTS oxidation, being 0.3 and 0.21 mM for their Km and their Vmax were 1.28 and 1.45 mM/min per milligram of enzyme respectively. The activity for RM344 enzyme was found much lower, with values of 0.78 mM for Km and 0.13 mM/min per milligram of enzyme for Vmax. The enzyme isolated from the B0510 strain presented its highest activity at pH 2.9 while VA612 and RM344 enzymes showed the maximum activity at pH 3.3. All these values were quite lower compared to previously measured by other authors [4-6]. Those differences in the enzyme activity may be related with differences in the active center of the enzyme and could have important consequences for the winemaking process depending on the strains of the B. cinerea strain involved in the infection of grapes.

[1]P. Ribéreau-Gayon, Y. Glories, A. Maujean, D. Dubourdieu, Handbook of Enology, Volume 2: The chemistry of Wine Stabilization and Treatments, John Wiley & Sons, Ltd, 2000. [2]D. Slomczynski, J.P. Nakas, S.W. Tanenbaum, Applied and Environmental Microbiology, 61 (1995) 907. [3]C. THURSTON, Microbiology-Sgm, 140 (1994) 19. [4]I. MARBACH, E. HAREL, A. MAYER, Phytochemistry, 23 (1984) 2713. [5]I. MARBACH, E. HAREL, A. MAYER, Phytochemistry, 22 (1983) 1535. [6]M. Dubernet, P. Ribereau-Gayon, H.R. Lerner, E. Harel, A.M. Mayer, Phytochemistry, 16 (1977) 191.

Publication date: May 17, 2024

Issue: Macrowine 2016

Type: Poster

Authors

Cédric Saucier*, Anne-Sophie Walker, Christiane Auclair, François Garcia, Francois-Xavier Sauvage, Jullien Drone, Natalia Quijada-Morin, Patrick Chemardin

*Université de Montpellier

Contact the author

Tags

IVES Conference Series | Macrowine | Macrowine 2016

Citation

Related articles…

Comprehensive exploration of wine aroma-related compounds as promoted by alternative vinification procedures in case of Zelen (Vitis vinifera L.) grapes processing

Not only vintner’s decisions in the vineyard, but also winemaker’s choices of technology approaches in the cellar play a significant role in the final wine style and quality. Whereas traditional technologies within chosen terroir are quite well explored and thus somehow predictable, there is no proper knowledge available on possible outcomes in case of implementing novel, alternative winemaking strategies. To reveal their effects on wine aroma compounds and sensory characteristics, two alternative strategies
(cryoextraction or addition of whole grape berries during last stages of fermentation) were compared to classical Vipava valley winemaking approach as normally used for an autochthonous variety Zelen. After separate vinification and bottling, all the experimental wines were subjected to semiquantitative metabolic profiling of volatile compounds (VOCs) by means of GC/MS and were then also sensorialy evaluated by pre-trained panel.

Sensory and nephelometric analysis of tannin fractions obtained by ultrafiltration of red wines

The assessment of red wine mouthfeel relies primarily on the sensory description of its tannic properties. This evaluation could be improved by gaining a better understanding of the physicochemical properties of these tannins. Hence, the objectives of the present study were threefold: (1) to gain an insight into the sensory properties of subpopulations of proanthocyanidic tannins of different molecular sizes obtained through several ultrafiltration steps, (2) to quantify the kinetics of haze formation of these proanthocyanidic tannins in a dynamic polyvinylpyrrolidone (PVP) precipitation test, (3) to determine whether a correlation exists between the sensory and the precipitation data.

Grape byproducts as source of resveratrol oligomers for the development of antifungal extracts

Grape canes are a non-recycled byproduct of wine industry (1-5 tons per hectare per year) containing valuable phytochemicals of medicine and agronomical interest. Resveratrol and wine polyphenols are known to exert a plethora of health-promoting effects including antioxidant capacity, cardioprotection, anticancer activity, anti-inflammatory effects, and estrogenic/antiestrogenic properties (Guerrero et al. 2009). Additionally, resveratrol is a major phytoalexin produced by plants in response to various stresses and promotes disease resistance (Chang et al. 2011). Our project aims to develop polyphenol-rich grape cane extracts to fight phytopathogenic or clinically relevant fungi. We initiate the project with the development of analytical methods to analyze resveratrol mono- and oligomers (dimers, trimers and tetramers) from grape canes and we evaluate their potential activity against clinically relevant opportunistic fungal pathogens (Houillé et al. 2014).

WineMetrics: A new approach to unveil the “wine-like aroma” chemical feature

“The Human being has an excellent ability to detect and discriminate odors but typically has great difficulty in identifying specific odorants”(1). Furthermore, “from a cognitive point of view the mechanism used to judge wines is closer to pattern recognition than descriptive analysis.” Therefore, when one wants to reveal the volatile “wine-like feature” pattern recognition techniques are required. Sensomics is one of the most recent “omics”, i.e. a holistic perspective of a complex system, which deals with the description of substances originated from microorganism metabolism that are “active” to human senses (2). Depicting the relevant volatile fraction in wines has been an ongoing task in recent decades to which several research groups have allocated important resources. The most common strategy has been the “target approach” in order to identify the “key odorants” for a given wine varietal.

Characterization of various groups of pyranoanthocyanins in Merlot red 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.