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…

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.

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.

Climate change impacts on Douro Region viticulture and adaptation measures

Climate has a significant impact in the success of any agricultural system, with a direct influence on the crops suitability to a given region, interfering on yield and quality and also with the economic sustainability of the productive activity. In the Douro Demarcated Region (RDD), as in most regions of the Mediterranean climate, the scarce precipitation (33% has less than 600 mm per year), and your high variability, associated with high rates of evapotranspiration during the summer, is usually one of the fundamental factors that limit the grapevine development, as well as the production and quality of the harvest. Thus, facing the scenario in temperature changes for the next decades (1.5-2.5°C) and confirming the predictions of precipitation decreases and/or great variability in the occurrence of heat waves and intense rainfall, the consequences for slope stability in mountain viticulture and sustainability of all operations involved, are risks to be taken into account. In this way, a deepest and sustained knowledge regarding the adaptation measures to adverse environmental conditions is of a crucial importance, enabling a more efficient adaptation of plant growth conditions and the optimization of production and quality of the grapevines. The development of this work, carried out in two commercial vineyards, one located in Soutelo do Douro, São João da Pesqueira, Cima Corgo sub-region, and another located in Numão, Vila Nova de Foz Côa, Douro Superior sub-region, it seeks to establish a relationship between climatic elements and physiological, productive and qualitative parameters, as well as to evaluate the effectiveness of adaptation measures, including different types of deficit irrigation (2002-2019) and the application of shading nets (2019-2020) in the physiological, viticultural and oenological behavior in the Touriga Nacional and Moscatel Galego Branco varieties, respectively. The results showed that the application of deficit irrigation allowed to significantly reduce the impact of the adverse weather conditions at key moments in the development of the grapevine, particularly in the period immediately before veráison and maturation, reducing the negative effects on the physiological processes and productivity, without compromise the must quality parameters. On the other hand, the application of shading nets significantly reduced de leaves temperature, allowing to increase the water potential, stomatal conductance and photosynthetic rate of grapes, which was reflected in the yield increase in the 2nd year of the study. For the maturation indicators, higher levels of total acidity, malic acid and assimilable nitrogen were obtained. The last measure presents a huge potential, being essential to carry out more years of trials to obtain stronger conclusions in terms of production parameters, but also in characteristics as important as the grape ripening components and the organoleptic characteristics of wines.

Effects of graft quality on growth and grapevine-water relations

Climate change is challenging viticulture worldwide compromising its sustainability due to warmer temperatures and the increased frequency of extreme events. Grafting Vitis vinifera L.

Spatial determination of areas in the Western Balkans region favorable for organic production

In problematic conditions for production of grapes and wine caused by the COVID-19 pandemic and the resulting occurrence of wine surpluses, producers are increasingly turning to the innovative viticulture and winemaking of products that are more appealing to the market and the consumers. On the other hand, consumption of the food safety or organic products, and therefore of organic grapes and wine, is increasingly common in the world, in particular in Europe. The Regional Rural Development Standing Working Group (SWG RRD), as a regional intergovernmental organization gathers actors in the viticulture and winemaking sector from states and territories of the Western Balkans (South-East Europe) in the Expert Working Group for Wine, with the aim of improving viticulture and winemaking in this region through joint activities. In accordance with the aforementioned, the SWG RRD is working on advancing organic production of grapes and wine, and on recognition of specificities of the terroir of wine-growing areas in Western Balkans. In addition, as part of the project “Facilitation of Exchange and Advice on Wine Regulations in Western Balkan Countries” helmed by the German Federal Ministry of Food and Agriculture, in addition to harmonization of relevant legislation with EU regulations, efforts are being invested towards recognition of organic wines. Within activities and project implemented by this organization, expert analyses and scientific research of the terroir of Western Balkans were carried out, and some of the results are presented in this paper.