Macrowine 2021
IVES 9 IVES Conference Series 9 Evaluating analytical methods for quantification of glutathione in grape juice and wine

Evaluating analytical methods for quantification of glutathione in grape juice and wine

Abstract

AIM: Glutathione (GSH) is a powerful natural antioxidant, considered as a promising molecule against oxidative damage of aroma during winemaking and storage. His concentration in the grape juice is variable. Enological practice in the cellar can promote the preservation of the GSH in the grape juice. Recently the prescription of OIV allows to add glutathione rich substances to the must. This new practice creates an increasing interest in the quantification of GSH. Several analytical methods were published to measure GSH[1,2,3,4] and his dimer (GSSG) separately[5] or together as total glutathione[6] content. In this work we compared two analytical methods for the analyses of grape juice and wine samples.

METHODS: The first method is an enzymatic assay (EA), based on the reaction of thiol with DTNB in the presence of glutathione reductase enzyme to measure the total glutathione content. This method was automatized to allow high through-put measurements in the concentration range of 5-100mg/l. The second method, using UPLC-MS/MS, is more sensitive (LOD = 0.5mg/l) and permits simultaneous quantification of GSH, GSSG and additionally the sulfonated form of glutathione (GSSO3H).

RESULTS: The best results were obtained with 2.5g/l ascorbic acid. Using the two analytical methods, we found a strong correlation (R2=0.98) between the total glutathione (EA) and the sum of GSH and GSSG (UPLC-MS/MS) in grape juice samples (n>100), where the GSSO3H concentration was low (0-8 mg/l) comparing to the GSH and GSSG (5-100mg/l). In wine samples the total glutathione concentration was low (2-7mg/l) and the GSSO3H was more important (5-9mg/l) due to the combination of SO2 with the glutathione. View the high reactivity of GSH, a special attention should be accorded to the preparation and the storage of grape juice samples. We compared the effect of different concentration of SO2 and ascorbic acid as additives and found that 2.5g/l ascorbic acid gave the best results.

CONCLUSION

Based on our results the enzymatic assay is an economic alternative to measure the total glutathione concentration of grape juice. However for wine the UPLC-MS/MS method is recommended, to reach the necessary sensitivity and to analyze all glutathione species.

DOI:

Publication date: September 7, 2021

Issue: Macrowine 2021

Type: Article

Authors

Ágnes Dienes-Nagy

Agroscope, Nyon, Switzerland,Frédéric VUICHARD, Agroscope, Nyon, Switzerland Marie BLACKFORD, Agroscope, Nyon, Switzerland Fabrice LORENZINI, Agroscope, Nyon, Switzerland

Contact the author

Keywords

glutathione, enzymatic assay, uplc-ms/ms

Citation

Related articles…

Investigating the impact of grape exposure and UV radiations on rotundone in Vitis vinifera L. Tardif grapes under field trial conditions

Rotundone is the main aroma compound responsible for peppery notes in wines whose biosynthesis is negatively affected by heat and drought. Through the alteration of precipitation regime and the increase in temperature during maturation, climate change is expected to affect wine peppery typicality. In this context there is a demand for developing sustainable viticultural strategies to enhance rotundone accumulation or limit its degradation. It was recently proposed that ultraviolet (UV) radiations could stimulate rotundone production. The aim of this study was to investigate under field trial conditions the impact of grape exposure and UV treatments on rotundone in Vitis vinifera L. Tardif, an almost extinct grape variety from south-west France that can express particularly high rotundone levels. Four different treatments were compared in 2021 to a control treatment using a randomised complete block design with three replications per treatment. Grape exposure was manipulated through early or late defoliation. Leaf and laterals shoots were removed at Eichorn Lorenz growth stages 32 or 34 on the morning-sun side of the canopy. During grape maturation, UV radiations were either reduced by 99% by installing UV radiation-shielding sheets, or applied four times using the Boxilumix™ non thermal device (Asclepios Tech, Tournefeuille) with the aim of activating plant signalling pathway. Loggers displayed in solar radiation shields were used to assess the effect of such shielding sheets on air temperature within the bunch zone. The composition of grapes subjected to these treatments will be soon analysed for their rotundone content and basic classical laboratory analyses. Grapes will be harvested to elaborate wines under standardized small-scale vinification conditions (60kg) that will be assessed by a trained sensory panel.

Impact of changes in pruning practices on vine growth and yield

A gradual decline in vineyards has been observed over the past twenty years worldwide. This might be explained by the climate change, practices change or the increase of dieback diseases. To increase the longevity of vines, we studied the impact of different pruning strategies in four adult and four young vineyards located in France and Spain. In France, vineyards were planted with Cabernet franc on 3309C while Spanish trials were planted with Tempranillo grafted on 110R. Vegetative expression, yield, quality of berries and wood vessels conductivity were measured. The distribution of vegetative expression, yield and berry composition between primary and secondary vegetation were quantified. Finally, tomography was used to evaluate the implication of the treatments on sap flows.
First results show that i) the respectful pruning leads to an increase of 30 to 50% more secondary shoots than the aggressive pruning in France and between 15 and 20% in Spain, ii) there is no major effect on the yield over the first two years following the implementation of the new pruning practices, although the proportion of clusters from suckers is higher on the respectful pruning method. On young vines, the development of the trunk according to a respectful pruning leads to a loss of harvest 2 years after planting. This is due to the removal, on the future trunk, of the green suckers which carrying bunches. This operation carried out in spring rather than during winter pruning, would promote a better leaf / fruit balance when the plant comes into production, and could lead to better hydraulic conduction in the vessels of the trunk. Maintaining these trials for several years will provide more robust data to assess the impact of these practices on the vines over the long term.

Genotypic variability in root architectural traits and putative implications for water uptake in grafted grapevine

Root system architecture (RSA) is important for soil exploration and edaphic resources acquisition by the plant, and thus contributes largely to its productivity and adaptation to environmental stresses, particularly soil water deficit. In grafted grapevine, while the degree of drought tolerance induced by the rootstock has been well documented in the vineyard, information about the underlying physiological processes, particularly at the root level, is scarce, due to the inherent difficulties in observing large root systems in situ. The objectives of this study were to determine genetic differences in the root architectural traits and their relationships to water uptake in two Vitis rootstocks genotypes (RGM, 140Ru) differing in their adaptation to drought. Young rootstocks grafted upon the Riesling variety were transplanted into cylindrical tubes and in 2D rhizotrons under two conditions, well watered and moderate water stress. Root traits were analyzed by digital imaging and the amount of transpired water was measured gravimetrically twice a week. Root phenotyping after 30 days reveal substantial variation in RSA traits between genotypes despite similar total root mass; the drought-tolerant 140Ru showed higher root length density in the deep layer, while the drought-sensitive RGM was characterised by shallow-angled root system development with more basal roots and a larger proportion of fine roots in the upper half of the tube. Water deficit affected canopy size and shoot mass to a greater extent than root development and architectural-related traits for both 140Ru and RGM, suggesting vertical distribution of roots was controlled by genotype rather than plasticity to soil water regime. The deeper root system of 140Ru as compared to RGM correlated with greater daily water uptake and sustained stomata opening under water-limited conditions but had little effect on above-ground growth. Our results highlight that grapevine rootstocks have constitutively distinct RSA phenotypes and that, in the context of climate change, those that develop an extensive root network at depth may provide a desirable advantage to the plant in coping with reduced water resources.

How can historical cultivars mitigate the effects of climate change?

IFV, INRAe and the national network “Partenaires de la Sélection Vigne” representing 37 organizations from the different wine regions, have been working increasingly closely over the last 2 decades towards the preservation of the French varietal patrimony. There are approximately 600 patrimonial varieties according to INRAe and SupAgro Montpellier experts, including ancient cultivars (400) and intravarietal crossbreeds obtained since the 19th century. In the context of a drastic reduction in such varieties from the mid 1980’s in favor of mainstream varieties, it was essential to carry out an inventory of old vines and vineyards. INRAe Vassal collection plays a key role here as it holds the largest diversity available, along with a rich bibliography and herbariums, offering us the opportunity to document and double check the identity of a cultivar, consolidating the expertise of ampelographers. The work is carried out in several stages, from verifying the existence of a variety in a small region, through to rehabilitation. During this session, the authors present the process that leads to the official registration of a variety. After this, IFV selection center takes over to initiate the process of selection and propagation. A specific focus within regions such as the Alps, Champagne and the South-West will provide details of the full procedure. Bia, Bouysselet, Chardonnay rose, Mecle and the aptly named Tardif, are some of the cultivars that have followed this procedure. Furthermore, a recent regulation established by INAO on “varieties of interest for adaptation purposes” might boost uptake by growers. Since 2006, 36 historical cultivars have been registered. Most of these have been neglected in the past due to late maturity, lack of sugar and high titratable acidity at harvest time. Such characteristics are today considered as positive qualities, not only in mitigation of the effects of climate change, but also as an opportunity for restoring diversity…

Assessment of climate change impacts on water needs and growing cycle on grapevine in three DOs of NE Spain

This study assessed the suitability of grapevine growing in three DOs (Empordà, Pla de Bages and Penedès) of Catalonia (NE Spain) over the 21st century. For this purpose, an estimation of water needs and agroclimatic and phenological indicators was made. Climate change impacts were estimated at 1 km pixel resolution using temperature and precipitation projections from several general circulation models (GCM) and two climate change scenarios: RCP 4.5 (stabilization scenario) and RCP 8.5 (worst-case scenario). Potential crop evapotranspiration (following FAO procedure) and a daily water balance considering soil water holding capacity were used to estimate actual evapotranspiration of vines and, finally, water needs. Dynamics would be similar in the three DOs studied although the magnitude of impact differs. Water needs would be 2 and 3 times greater (ranging from 0 to more than 1500 m3/ha) than current water needs at both climate change scenarios. Moreover, blooming date would advance from 3 to 6 weeks, harvest date from 1 to 2.5 months, resulting in growing cycles from 10 to 80 days shorter. It should also be noted that frost risk would decrease from 6 to 76%, the number of days with temperatures above 30ºC during ripening would rise from 48 to 500% and tropical nights (minimum temperature >20ºC) at ripening would increase from 28 to 150%, depending on the scenario and the DOs. The impacts of climate change in the three DOs could result in significant limitations for grapevine cultivation and wine production if adaptive strategies are not applied. This result could serve as a basis for the design of specific and particular adaptation strategies to improve and maintain vineyards in the DOs studied and could be extrapolated to similar DOs and regions.