Macrowine 2021
IVES 9 IVES Conference Series 9 Kinetic study of browning caused by laccase activity using different substrates

Kinetic study of browning caused by laccase activity using different substrates

Abstract

AIM: To our knowledge all the studies about laccase kinetics and its inhibition have been performed with substrates and conditions very different from those of real grape juice. Moreover, none of these researches really measure enzymatic browning, since they have not taken into account what happens after the oxidation of o-diphenols in o-diquinones and their subsequent polymerization to form melanins1. For that reason, the aim of this research was to develop a new model to measure the kinetics of browning caused by Botrytis cinerea laccase under conditions much closer to those of grape juice and using the substrates naturally present in it.

METHODS: A grape juice model solution containing 100 g/L of D-glucose, 100 g/L of D-fructose and 4 g/L of tartaric acid adjusted to pH 3.5 was used for all the browning assays. Five phenolic compounds including one triphenol: gallic acid; three orthodiphenols: caftaric acid, (+)-catechin and (-)-epicatechin; and one monophenol: 4-hydroxybenzoic acid were used at concentrations between 0 and 0.8 mM. Laccase from Botrytis cinerea was purified according to Vignault et al., (2019)2. Browning reaction was started by adding 2 units of laccase activity/mL and absorbance at 420 nm was measured at time 0, 15, 30 and 45 minutes. The slope of the regression straight line was determined in order to express the intensity of browning. The Michaëlis-Menten and Hill plots were depicted for each substrate in order to determine the kinetic parameters of browning: Vmax, K0.5 and Hill number. All the experiments were performed in triplicate

RESULTS: The results indicate that o-diphenols are better substrates for laccase browning than triphenols and that monophenols, or at least 4-hydroxybenzoic acid, do not appear to be reactive. Moreover, of the o-diphenols, (+)-catechin showed the greatest browning intensity, followed in decreasing order by (-)-epicatechin and caftaric acid.

CONCLUSIONS: This research proposes a synthetic model for measuring laccase browning in a matrix close to real grape juice that makes it possible to study how laccase browning acts in the presence of different possible substrates. Further studies are needed to verify the efficiency of the proposed model on other laccase substrates such as anthocyanins, flavonols and proantocyanidins, and also to determine the inhibitory effect toward laccase browning of the most frequently used antioxidants – sulfur dioxide, ascorbic acid and glutathione – and other possible inhibitors of laccase browning such as oenological tannins.

FUNDING:

This work was funded by CICYT (Efecto de las lacasas sobre la sensorialidad, calidad y salubridad de los vinosproject RTI2018-095658-B-C33).

ACKNOWLEDGMENTS:

Authors thank professors Marc Fermaud and Jean Roudet from INRAE, UMR SAVE, Bordeaux Science Agro, ISVV, France for having provided us with the B. cinerea strain.

DOI:

Publication date: September 28, 2021

Issue: Macrowine 2021

Type: Article

Authors

Fernando Zamora, Pol Giménez, Sergi Anguela, Arnau Just-Borras, Pere Pons-Mercadé, Jordi Gombau, Adeline Vignault,  Joan Miquel Canals, Pierre-Louis Teissedre, Fernando Zamora

Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain.
Unité de Recherche Oenologie, EA 4577, USC 1366 INRAE, ISVV, Université de Bordeaux, F33882 Villenave d’Ornon, France. – 11 rue Aristide Bergès, 33270 Floirac, France,

Contact the author

Keywords

laccase, botrytis cinerea, browning, kinetics

Citation

Related articles…

Understanding graft union formation by using metabolomic and transcriptomic approaches during the first days after grafting in grapevine

Since the arrival of Phyloxera (Daktulosphaira vitifolia) in Europe at the end of the 19th century, grafting has become essential to cultivate Vitis vinifera. Today, grafting provides not only resistance to this aphid, but it used to adapt the cultivars according to the type of soil, environment, or grape production requirements by using a panel of rootstocks. As part of vineyard decline, it is often mentioned the importance of producing quality grafted grapevine to improve vineyard longevity, but, to our knowledge, no study has been able to demonstrate that grafting has a role in this context. However, some scion/rootstock combinations are considered as incompatible due to poor graft union formation and subsequently high plant mortality soon after grafting. In a context of climate change where the creation of new cultivars and rootstocks is at the centre of research, the ability of new cultivars to be grafted is therefore essential. The early identification of graft incompatibility could allow the selection of non-viable plants before planting and would have a beneficial impact on research and development in the nursery sector. For this reason, our studies have focused on the identification of metabolic and transcriptomic markers of poor grafting success during the first days/week after grafting; we have identified some correlations between some specialized metabolites, especially stilbenes, and grafting success, as well as an accumulation of some amino acids in the incompatible combination. The study of the metabolome and the transcriptome allowed us to understand and characterise the processes involved during graft union formation.

Climate and the evolving mix of grape varieties in Australia’s wine regions

The purpose of this study is to examine the changing mix of winegrape varieties in Australia so as to address the question: In the light of key climate indicators and predictions of further climate change, how appropriate are the grape varieties currently planted in Australia’s wine regions? To achieve this, regions are classified into zones according to each region’s climate variables, particularly average growing season temperature (GST), leaving aside within-region variations in climates. Five different climatic classifications are reported. Using projections of GSTs for the mid- and late 21st century, the extent to which each region is projected to move from its current zone classification to a warmer one is reported. Also shown is the changing proportion of each of 21 key varieties grown in a GST zone considered to be optimal for premium winegrape production. Together these indicators strengthen earlier suggestions that the mix of varieties may be currently less than ideal in many Australian wine regions, and would become even less so in coming decades if that mix was not altered in the anticipation of climate change. That is, grape varieties in many (especially the warmest) regions will have to keep changing, or wineries will have to seek fruit from higher latitudes or elevations if they wish to retain their current mix of varieties and wine styles.

Terroir traceability in grapes, musts and wine: results of research on Gewürztraminer and Sauvignon Blanc grape varieties in northern Italy

In the study of terroir, a separate analysis of its many component factors can be of great help in accurately identifying a vineyard’s natural elements that impact wine quality and typicity. This research used a dedicated pluri-disciplinary approach to investigate the ecological characteristics, including geology and geographical features, of 14 vineyards that produce Gewürztraminer and Sauvignon Blanc cultivars in the alpine Alto Adige DOC wine region. Both the geopedological method using Vineyards Geological Identity (VGI) and the new Solar Radiaton Identity (SRI) topoclimatic classification method were used to provide analytical measurements and qualitative/quantitative characterisations. In addition, wide-ranging targeted and untargeted oenological and chemical analyses were carried out on grapes, musts and wines to correlate the soils’ geomineral and physical conditions with the biochemical properties of their fruits and wines. The research identified strong correlations between vineyard geo-identity and wine biofingerprint, confirming a mineral traceability of strontium rubidium ratio and some minerals distinctive to the local geology, such as K, Ca, Ag, Ba and Mn.  The study also discovered that particular geomineral and physical soil conditions of the studied vineyards are related to the different amount of amino acids, primary varietal aromas and polyphenols found in grapes, musts and wines. The research confirmed that winemaking technologies support oenological quality, although in some cases, human practices can overpower certain characteristic elements in wine, erasing the typical imprint left by the vineyards’ natural terroir, which becomes less traceable. Terroir abiotic ecological factors and vineyard identity can be classified in detail using the new VGI and SRI analysis methods to discover interrelationships between geo-pedological and topoclimatic conditions that impact wine quality. These methods are also helpful in identifying which ecological elements are exclusive to a particular vineyard or wine sub-region.

Variations of soil attributes in vineyards influence their reflectance spectra

Knowledge on the reflectance spectrum of soil is potentially useful since it carries information on soil chemical composition that can be used to the planning of agricultural practices. If compared with analytical methods such as conventional chemical analysis, reflectance measurement provides non-destructive, economic, near real-time data. This paper reports results from reflectance measurements performed by spectroradiometry on soils from two vineyards in south Brazil. The vineyards are close to each other, are on different geological formations, but were subjected to the same management. The objective was to detect spectral differences between the two areas, correlating these differences to variations in their chemical composition, to assess the technique’s potential to predict soil attributes from reflectance data.To that end, soil samples were collected from ten selected vine parcels. Chemical analysis yield data on concentration of twenty-one soil attributes, and spectroradiometry was performed on samples. Chemical differences significant to a 95% confidence level between the two studied areas were found for six soil attributes, and the average reflectance spectra were separated by this same level along most of the observed spectral domain. Correlations between soil reflectance and concentrations of soil attributes were looked for, and for ten soil traits it was possible to define wavelength domains were reflectance and concentrations are correlated to confidence levels from 95% to 99%. Partial Least Squares Regression (PLSR) analyses were performed comparing measured and predicted concentrations, and for fifteen out of 21 soil traits we found Pearson correlation coefficients r > 0.8. These preliminary results, which have to be validated, suggest that variations of concentration in the investigated soil attributes induce differences in reflectance that can be detected by spectroradiometry. Applications of these observations include the assessment of the chemical content of soils by spectroradiometry as a fast, low-cost alternative to chemical analytical methods.

Inhibition of Oenococcus oeni during alcoholic fermentation by a selected Lactiplantibacillus plantarum strain

The use of selected cultures of the species Lactiplantibacillus plantarum in Oenology has grown in prominence in recent years. While initial applications of this species centred very much around malolactic fermentation (MLF), there is strong evidence to show that certain strains can be harnessed for their bio-protective effects. Unwanted spontaneous MLF during alcoholic fermentation (AF), driven by rogue Oenococcus oeni, is a winemaking deviation that is very difficult to manage when it occurs. This work set out to determine the efficacy of one particular strain of Lactiplantibacillus plantarum(Viniflora® NoVA™ Protect), against this problem in Cabernet Sauvignon must. The work was carried out at commercial scale and in a winery environment and compared the bio-protective culture with the more traditional approach of reducing must pH by the addition of tartaric acid. The combination of both was also investigated. The concentration of both Oenococcus oeni and Lactiplantibacillus plantarum was determined using qPCR. The adventitious Oenococcus oeni showed the most growth during AF in the control wine, whereas in the wines treated with Lactiplantibacillus plantarum a bacteriostatic effect against this species was observed. This effect was comparable to the wines treated with tartaric acid. This has particular commercial relevance for controlling the flora in musts with high pH, or when the addition of tartaric acid is either not permitted or is prohibitive for other reasons.