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…

Using δ13C and hydroscapes as a tool for discriminating cultivar specific drought response

Measurement of carbon isotope discrimination in berry juice sugars at maturity (δ13C) provides an integrated assessment of water use efficiency (WUE) during the period of berry ripening, and when collected over multiple seasons can be used as an indication of drought stress response. Berry juice δ13C measurements were carried out on 48 different varieties planted in a common garden experiment in Bordeaux, France from 2014 through 2021 and were paired with midday and predawn leaf water potential measurements on the same vines in a subset of six varieties. The aim was to discriminate a large panel of varieties based on their stomatal behaviour and potentially identify hydraulic traits characterizing drought tolerance by comparing δ13C and hydroscapes (the visualisation of plant stomatal behaviour as a response to predawn water potential). Cluster analysis found that δ13C values are likely affected by the differing phenology of each variety, resulting in berry ripening of different varieties taking place under different stress conditions within the same year. We accounted for these phenological differences and found that cluster analysis based on specific δ13C metrics created a classification of varieties that corresponds well to our current empirical understanding of their relative drought tolerances. In addition, we analysed the water potential regulation of the subset of six varieties (using the hydroscape approach) and found that it was well correlated with some δ13C metrics. Surprisingly, a variety’s water potential regulation (specifically its minimum critical leaf water potential under water deficit) was strongly correlated to δ13C values under well-watered conditions, suggesting that base WUE may have a stronger impact on drought tolerance than WUE under water deficit. These results give strong insights on the innate WUE of a very large panel of varieties and suggest that studies of drought tolerance should include traits expressed under non-limiting conditions.

δ13C : A still underused indicator in precision viticulture  

The first demonstration of the interest of carbon isotope composition of sugars in grapevine, as an integrated indicator of vineyard water status, dates back to 2000 (Gaudillère et al., 1999; Van Leeuwen et al., 2001). Thanks to the isotopic discrimination of Carbon that takes place during plant photosynthesis, under hydric stress conditions, it is possible to accurately estimate the photosynthetic activity. Ever since, δ13C has been widely applied with success to zonation, terroir studies and vine physiology research, but is still not widely used by viticulturists. This is quite astonishing by considering the impact of global warming on viticulture and the need to improve water management, that would justify a widespread use of δ13C.
The lack of private laboratories proposing the analysis, the cost of the technology, as well as the long analytical delays, have been detrimental to its development. Some laboratories tried to overcome the analytical difficulties of isotopic analysis by using fourier transformed infrared spectroscopy, as a fast and cheap alternative to the official OIV method (IRMS). These claimed FTIR models have never been published or peer reviewed and cannot be considered robust. In this work, thanks to the recent acquisition of IRMS technology, new modern and robust applications of δ13C for viticulture are proposed. This includes the use of the analysis to make parcel separations at harvesting, the possibility to increase the precision of hydric stress cartography and the potential cost reduction when compared with Scholander pressure bomb analysis.

Adapting the vineyard to climate change in warm climate regions with cultural practices

Since the 1980s global regime shift, grape growers have been steadily adapting to a changing climate. These adaptations have preserved the region-climate-cultivar rapports that have established the global trade of wine with lucrative economic benefits since the middle of 17th century. The advent of using fractions of crop and actual evapotranspiration replacement in vineyards with the use of supplemental irrigation has furthered the adaptation of wine grape cultivation. The shift in trellis systems, as well as pruning methods from positioned shoot systems to sprawling canopies, as well as adapting the bearing surface from head-trained, cane-pruned to cordon-trained, spur-pruned systems have also aided in the adaptation of grapevine to warmer temperatures. In warm climates, the use of shade cloth or over-head shade films not only have aided in arresting the damage of heat waves, but also identified opportunities to reduce the evapotranspiration from vineyards, reducing environmental footprint of vineyard. Our increase in knowledge on how best to understand the response of grapevine to climate change was aided with the identification of solar radiation exposure biomarker that is now used for phenotyping cultivars in their adaptability to harsh environments. Using fruit-based metrics such as sugar-flavonoid relationships were shown to be better indicators of losses in berry integrity associated with a warming climate, rather than solely focusing on region-climate-cultivar rapports. The resilience of wine grape was further enhanced by exploitation of rootstock × scion combinations that can resist untoward droughts and warm temperatures by making more resilient grapevine combinations. Our understanding of soil-plant-atmosphere continuum in the vineyard has increased within the last 50 years in such a manner that growers are able to use no-till systems with the aid of arbuscular mycorrhiza fungi inoculation with permanent cover cropping making the vineyard more resilient to droughts and heat waves. In premium wine grape regions viticulture has successfully adapted to a rapidly changing climate thus far, but berry based metrics are raising a concern that we may be approaching a tipping point.

How does aromatic composition of red wines, resulting from varieties adapted to climate change, modulate fruity aroma?

One of the major issues for the wine sector is the impact of climate change linked to the increasing temperatures which affects physicochemical parameters of the grape varieties planted in Bordeaux vineyard and consequently, the quality of wine. In some varietals, the attenuation of their fresh fruity character is accompanied by the accentuation of dried-fruit notes [1]. As a new adaptive strategy on climate change, some winegrowers have initiated changes in the Bordeaux blend of vine varieties [2]. This study intends to explore the fruitiness in wines produced from grape varieties adapted to the future climate of Bordeaux. 10 commercial single–varietal wines from 2018 vintage made from the main grape varieties in the Bordeaux region (Cabernet franc, Cabernet-Sauvignon and Merlot) as well as from indigenous grape varieties from the Mediterranean basin, such as Cyprus (Yiannoudin), France (Syrah), Greece (Agiorgitiko and Xinomavro), Portugal (Touriga Nacional) and Spain (Garnacha and Tempranillo), were selected among 19 samples using sensory descriptive analyses. Both sensory and instrumental analyses were coupled, to investigate their fruity aroma expression. For sensory analysis, samples were prepared from wine, using a semi preparative HPLC method which preserves wine aroma and isolates fruity characteristics in 25 specific fractions [3,4]. Fractions of interest with intense fruity aromas were sensorially selected for each wine by a trained panel and mixed with ethanol and microfiltered water to obtain fruity aromatic reconstitutions (FAR) [5]. A free sorting task was applied to categorize FAR according to their similarities or dissimilarities, and different clusters were highlighted. Instrumental analysis of the different FAR and wines demonstrated variations in their molecular composition. Results obtained from sensory and gas chromatography analysis enrich the knowledge of the fruity expression of red wines from “new” grape varieties opening up new perspectives in wine technology, including blending, thus providing new tools for producers.

Extreme canopy management for vineyard adaptation to climate change: is it a good idea?

Climate change constitutes an enormous challenge for humankind and for all human activities, viticulture not being an exception. Long-term strategic changes are probably needed the most, but growers also need to deal with short-term changes: summers that are getting progressively warmer, earlier harvest dates and higher pH in musts and wines. In the last 10-15 years, a relevant corpus of research is being developed worldwide in order to evaluate to which extent extreme canopy management operations, aimed at reducing leaf area and, thus, limiting the source to sink ratio, could be useful to delay ripening. Although extreme canopy management can result in relevant delays in harvest dates, longer term studies, as well as detailed analysis of their implications on carbohydrate reserves, bud fertility and future yield are desirable before these practices can be recommended.