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…

Use of chitosan as a secondary antioxidant in juices and wines

Chitosan is a polysaccharide produced from the deacetylation of chitin extracted from crustaceous and fungi. In winemaking chitosan is mainly used in the clarification of grape juice and wine, stabilization of white wines, removal of metals and to prevent wine spoilage by undesired microorganisms. The addition of chitosan to model wine systems was able to retard browning, reduce levels of metallic ions (Fe and Cu) and to protect varietal thiols due to its antiradical activity1. The present experiment was planned in order to evaluate the use of chitosan as a secondary antioxidant at three different stages of Sauvignon blanc fermentation and winemaking. Sauvignon blanc juices from three different locations were obtained at a commercial winery in Marlborough, New Zealand. One lots of grapes was collected from a receival bin and pressed into juice with a water-bag press, and a further juice sample was collected from a commercial pressing operation. Chitosan (1 g/L, low molecular weight, 75 – 85% deacetylated) was added to the juice after pressing, after cold settling, after fermentation, or at all these stages. Controls without any chitosan additions were also prepared.

Identification of green, aggressive and hard character of wines by a chemo-sensory directed methodology

With climate change, it is progressively more often to obtain grapes with an acceptable content in sugars or acids but with immature tannins described as green, aggressive or hard (noted as GAH onwards). During winemaking, the oenologist has to make decisions related to the elaboration of such grapes based mainly on empirical experience, given the lack of objective criteria to this concern. An increase in the chemical and sensory knowledge of immature tannins would allow managing this GAH character of grapes with the maximum possible efficiency during winemaking processes. The present work aims at isolating and identifying the group of compounds responsible for the GAH character present in wines.

Impact of glutathione and elemental sulphur juice addition on the volatile thiol production in South African Sauvignon blanc wine

Three compounds, 3-mercaptohexanol (3MH), 3-mercaptohexyl-acetate (3MHA) and 4-mercapto-4-methylpentan-2-one (4MMP), also known as varietal thiols, have been identified to contribute positively to wine aroma and are responsible for the distinct gooseberry, grapefruit, guava and box tree character found in Sauvignon blanc wines. Certain volatile thiol compounds though, can cause off-aromas of onion, garlic, rubber and rotten egg, this group of molecules is known as reductive sulphur compounds (RSC). This study looks into how the addition of sulphur-compounds to Sauvignon blanc juice contributes to the varietal thiol (3MH and 3MHA) concentration and reductive sulphur compound concentration in South African Sauvignon blanc wine.

Crown procyanidin: a new procyanidin sub-family with unusual cyclic skeleton in wine

Condensed tannins (also called proanthocyanidins) are a widely distributed throughout in plants kingdom and are one of the most important classes of secondary metabolites, in addition, they are part of the human diet. In wine, they are extracted during the winemaking process from grape skins and seeds. These compounds play an important role in red wine organoleptic characteristics such as color, bitterness and astringency. Condensed tannins in red wine are oligomers and polymers of flavan-3-ols unit such as catechin, epicatechin, epigallocatechin and epicatechin-3-O-gallate. The monomeric units can be linked among them with direct interflavanoid linkage or mediated by aldehydes.

Assessing the effect of oak derived aromas on mouthfeel perception in Chardonnay wine

Mouthfeel is an important quality parameter for Chardonnay wines, particularly those aged in oak. While research on mouthfeel has traditionally focused on the impact of non-aromatic compounds, the role of aroma compounds has largely been over looked. However, in wine as well as other food interactions between retronasal aroma and mouthfeel have been noted. The goal of this research was to investigate the impact of wine aroma on the perception of mouthfeel. Because of the importance of oak aging in the development of Chardonnay mouthfeel, the impact of oak aromas on perceived mouthfeel was explored. Aroma compounds associated with oak (ethyl palmitate, eugenol, furfural, isoeugenol, syringaldehyde, vanillin and whiskey lactone) were added to two different Chardonnay wines; one with no oak influence and one fermented in neutral oak. Low and high concentrations of the compounds were added based on concentrations typically found in barrel aged Chardonnay wine.