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…

The modification of cultural practices in grapevine cv. Syrah, does it modify the characteristics of the musts?

The work shows the results of a year of experimentation (2020) in a Syrah variety vineyard in La Roda (Castilla-La Mancha, Spain). The trial approach was on a randomized block design with two factors: Irrigation (I) and Pruning (P).
Irrigation schedules were adjusted to apply amounts close to 1,500 m3/ha. With this provision, 2 different irrigation treatments were proposed: I1) Start of irrigation from pea-sized grape to post-harvest (providing at least 20 % of the total amount of irrigation water to be provided post-harvest); I2) Start of irrigation from pea-sized grape to harvest (usual irrigation practice in the study area). Pruning was proposed with two treatments, one at the end of January (P1), which is pruning on a conventional date; and P2) pruning carried out at the beginning of budding. In total, 4 repetitions were designed with 4 elementary plots, each one of them representing one of the proposed treatments (I1P1; I1P2; I2P1; I2P2). In total, 16 plots were worked on and each elementary plot consisted of 30 strains, distributed in 3 lines.
The productive response was evaluated with the yield results of the harvest harvested at 23 ºBrix. The qualitative response was measured in the musts through the indices of technological (acidity, pH and potassium) and phenolic maturity and aromatic compounds in free and glycosylated fractions. The treatments tested had, in general, an effect on the different variables analyzed.

Measurement of redox potential as a new analytical winegrowing tool

Excell laboratory has initiated the development of an analytical method based on electrochemistry to evaluate the ability of wines to undergo or resist to oxidative phenomena. Electrochemistry is a powerful tool to probe reactions involving electron transfers and offers possibility of real-time measurements. In that context, the laboratory has implemented electrochemical analysis to assess oxidation state of different wine matrices but also in order to evaluate oxidative or reduced character of leaf and soil. Initially, our laboratory focused on dosage of compounds involved in responses of plant stresses and we were also interested in microbiological activity of soils. These analyses were compared with the measurement of redox potential (Eh) and pH which are two fundamental variables involved in the modulation of plant metabolism. Indeed, the variation of redox states of the plant reflects its biological activity but also its capacity to absorb nutriments. The Eh-pH conditions mainly determine metabolic processes involved in soil and leaf and our goal is to determine if this combined analytical approach will be sufficiently precise to detect biological evolutions (plant health, parasitic attack…).

Different soil types and relief influence the quality of Merlot grapes in a relatively small area in the Vipava Valley (Slovenia) in relation to the vine water status

Besides location and microclimatic conditions, soil plays an important role in the quality of grapes and wine. Soil properties influence…

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.

Adaptability of grapevines to climate change: characterization of phenology and sugar accumulation of 50 varieties, under hot climate conditions

Climate is the major factor influencing the dynamics of the vegetative cycle and can determine the timing of phenological periods. Knowledge of the phenology of varieties, their chronological duration, and thermal requirements, allows not only for the better management of interventions in the vineyard, but also to predict the varieties’ behaviour in a scenario of climate change, giving the wine producer the possibility of selecting the grape varieties that are best adapted to the climatic conditions of a certain terroir. In 2014, Symington Family Estates, Vinhos, established two grape variety libraries in two different places with distinctive climate conditions (Douro Superior, and Cima Corgo), with the commitment of contributing to a deeper agronomic and oenological understanding of some grape varieties, in hot climate conditions. In these research vineyards are represented local varieties that are important in the regional and national viticulture, but also others that have over time been forgotten — as well as five international reference cultivars. From 2017 to 2021, phenological observations have been made three times a week, following a defined protocol, to determine the average dates of budbreak, flowering and veraison. With the climate data of each location, the thermal requirements of each variety and the chronological duration of each phase have been calculated. During maturation, berry samples have been gathered weekly to study the dynamics of sugar accumulation, between other parameters. The data was analysed applying phenological and sugar accumulation models available in literature. The results obtained show significant differences between the varieties over several parameters, from the chronological duration and thermal requirements to complete the various stages of development, to the differences between the two locations, confirming the influence of the climate on phenology and the stages of maturation, in these specific conditions.