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…

The combined effects of climate, soils, and deficit irrigation on yield and quality of Touriga Nacional under high atmospheric demand in the Douro Region

Global warming is one of the biggest environmental, social and economic threats in several viticultural regions. In the Douro Valley, changes are expected in the coming years, namely an increase in temperature and a decrease in precipitation. These changes are likely to have consequences for the production and quality of wine.
The aim of this study was to explore the effects of different soil characteristics combined with several deficit irrigation strategies, managed throughout ETc references and predawn leaf water potentials thresholds, on physiology, yield, and qualitative attributes on the Touriga Nacional variety under years of mild to severe water and heat stress.
The studies were conducted over seven years (2015 to 2021) in two plots of a commercial vineyard located at Quinta do Ataíde (Symington Family Estates) planted in 2011 and 2014 at 170 meters elevation, growing under three water regimes: non-irrigated (NI) and two deficit irrigation strategies (30% and 60% ETc) assessed weekly by Ψpd. The site has an annual rainfall below 500 mm, with high atmospheric demand. Climate data was collected from a weather station, located on site. Berry ripening was followed weekly for fruit analysis. At harvest, yield, vigour and pruning weight per vine were determined from 90 vines by treatment. Each season at veraison the NDVI Index was accessed by a drone. The soils physic-chemistry in the experimental blocs were analysed and grouped by SWHC. Delta C-13 analyses were also performed per treatment in two years.Irrigation had a positive effect on yield per vine, mostly due to an increase in berry and cluster weight, and fertility index through the years. A significant increase in sugar content, colour and phenols was observed with deficit irrigation in some years, but vine vigour related to soil characteristics had by far the greatest impact on quality.

VineyardFACE: Investigation of a moderate (+20%) increase of ambient CO2 level on berry ripening dynamics and fruit composition

Climate change and rising atmospheric carbon dioxide concentration is a concern for agriculture, including viticulture. Studies on elevated carbon dioxide have already been on grapevines, mainly taking place in greenhouses using potted plants or using field grown vines under higher CO2 enrichment, i.e. >650 ppm. The VineyardFACE, located at Hochschule Geisenheim University, is an open field Free Air CO2 Enrichment (FACE) experimental set-up designed to study the effects of elevated carbon dioxide using field grown vines (Vitis vinifera L. cvs. Riesling and Cabernet Sauvignon). As the carbon dioxide fumigation started in 2014, the long term effects of elevated carbon dioxide treatment can be investigated on berry ripening parameters and fruit metabolic composition.
The present study aims to investigate the effect on fruit composition under a moderate increase (+20%; eCO2) of carbon dioxide concentration, as predicted for 2050 on both Riesling and Cabernet Sauvignon. Berry composition was determined for primary (sugars, organic acids, amino acids) and secondary metabolites (anthocyanins). Special focus was given on monitoring of berry diameter and ripening rates throughout three growing seasons. Compared to previous results of the early adaptative phase of the vines [1], our results show little effect of eCO2 treatment on primary metabolites composition in berries. However, total anthocyanins concentration in berry skin was lower for eCO2 treatment in 2020, although the ratio between anthocyanins derivatives did not differ.
[1] Wohlfahrt Y., Tittmann S., Schmidt D., Rauhut D., Honermeier B., Stoll M. (2020) The effect of elevated CO2 on berry development and bunch structure of Vitis vinifera L. cvs. Riesling and Cabernet Sauvignon. Applied Science Basel 10: 2486

Postveraison shoot trimming in Tannat and Merlot: preliminary results on yield components, plant balance and berry composition

There is currently a trend towards the production of wines with low alcohol content. To achieve this, grapes with low sugar content must be used. There are techniques at the vineyard level that can delay ripening and avoid excessive sugar accumulation without, a priori, affecting the final polyphenol content. Postveraison shoot trimming (PVST) is experimentally evaluated for these purposes, but its impact under Uruguayan climatic conditions with high interannual variability is not known. The aim of this work is to assess the PVST in Tannat and Merlot cultivars and their impact on yield components, plant balance and berry primary composition. In this study, two commercial vineyards of 10 years old Tannat and Merlot (grafted on SO4) at Canelones Department were selected. During the 2020-201 growing season, grapevines were submitted to PVST when grapes reached 15º Brix. In a randomized block, trimmed (T) and control (C) plants were evaluated with three repetitions each cultivar. Evaluation of the evolution of primary berry composition during ripening, measurement of yield components and plant balance were performed. For both cultivars, PVST did not affect yield components. Merlot reached 5.4 kg per plant and Tannat 7.1 kg, with not statistical significance between treatments. However, statistical differences were observed in terms of plant balance. In Merlot Ravaz Index reached a difference of 5.3 (12.0 in T and 6.7 in C) meanwhile Tannat reached 3.5 of statistical difference (13.7 in T and 10.2 in C). The tendency to imbalance for the treated plants had an impact on the final grape composition. Merlot grapes showed statistical difference in final total acidity (0.3 g of difference between treatments) while treatments impact final sugar content on Tannat grapes (10.0 g of difference between treatments). Further studies are needed to assess the impact of different canopy management techniques in our conditions.

Use of a new, miniaturized, low-cost spectral sensor to estimate and map the vineyard water status from a mobile 

Optimizing the use of water and improving irrigation strategies has become increasingly important in most winegrowing countries due to the consequences of climate change, which are leading to more frequent droughts, heat waves, or alteration of precipitation patterns. Optimized irrigation scheduling can only be based on a reliable knowledge of the vineyard water status.

In this context, this work aims at the development of a novel methodology, using a contactless, miniaturized, low-cost NIR spectral tool to monitor (on-the-go) the vineyard water status variability. On-the-go spectral measurements were acquired in the vineyard using a NIR micro spectrometer, operating in the 900–1900 nm spectral range, from a ground vehicle moving at 3 km/h. Spectral measurements were collected on the northeast side of the canopy across four different dates (July 8th, 14th, 21st and August 12th) during 2021 season in a commercial vineyard (3 ha). Grapevines of Vitis vinifera L. Graciano planted on a VSP trellis were monitored at solar noon using stem water potential (Ψs) as reference indicators of plant water status. In total, 108 measurements of Ψs were taken (27 vines per date).

Calibration and prediction models were performed using Partial Least Squares (PLS) regression. The best prediction models for grapevine water status yielded a determination coefficient of cross-validation (r2cv) of 0.67 and a root mean square error of cross-validation (RMSEcv) of 0.131 MPa. This predictive model was employed to map the spatial variability of the vineyard water status and provided useful, practical information towards the implementation of appropriate irrigation strategies. The outcomes presented in this work show the great potential of this low-cost methodology to assess the vineyard stem water potential and its spatial variability in a commercial vineyard.

Long-term drought resilience of traditional red grapevine varieties from a semi-arid region

In recent decades, the scarcity of water resources in agriculture in certain areas has been aggravated by climate change, which has caused an increase in temperatures, changes in rainfall patterns, as well as an increase in the frequency of extreme phenomena such as droughts and heat waves. Although the vine is considered a drought-tolerant specie, it has to satisfy important water requirements to complete its cycle, which coincides with the hottest and driest months. Achieving sustainable viticulture in this scenario requires high levels of efficiency in the use of water, a scarce resource whose use is expected to be severely restricted in the near future. In this regard, the use of drought-tolerant varieties that are able to maintain grape yield and quality could be an effective strategy to face this change. During three consecutive seasons (2018-2020) the behavior in rainfed regime of 13 traditional red grapevine varieties of the Spain central region was studied. These varieties were cultivated in a collection at Centro de Investigación de la Vid y el Vino de Castilla-La Mancha (IVICAM-IRIAF) located in Tomelloso (Castilla-La Mancha, Spain). Yield components (yield, mean bunch and berry weight, pruning weight), physicochemical parameters of the musts (brix degree, total acidity, pH) and some physiological parameters related with water stress during ripening period (δ13C, δ18O) were analysed. The application of different statistical techniques to the results showed the existence of significant differences between varieties in their response to stressful conditions. A few varieties highlighted for their high ability to adapt to drought, being able to maintain high yields due to their efficiency in the use of water. In addition, it was possible quantify to what extent climate can be a determinant in the δ18O of musts under severe water stress conditions.