Winemaking interventions to modulate glutathione status

Abstract

Glutathione (GSH) is a natural peptide and grape-derived antioxidant that has been proposed as an alternative to sulfur dioxide in wine. This project focused on the addition of pure GSH prior to or during fermentation. Its consumption during wine production was quantified and the impact of process variables such as grape juice nitrogen concentration and the degree of protection from oxygen on GSH concentration was assessed in both laboratory- and pilot-scale trials. It was demonstrated that low concentrations of GSH have little impact when added prior to fermentation and that higher addition rates caused undesirable sensory outcomes.

Summary

Sulfur dioxide (SO2) is the antioxidant most commonly used to protect wine and preserve quality; however, more benign alternatives are continuously being sought. Reduced glutathione (GSH) is a naturally occurring antioxidant present in many organisms and as result it has been investigated for its potential to augment the use of SO2 for the preservation of wine freshness and aroma intensity.

GSH is naturally present in both grapes and yeast and its concentration in grape juice is dependent on the conditions under which grape processing is conducted. Aside from grapes, other potential sources of GSH in wine include yeast-derived products, some of which are marketed as GSH-enriched inactive dried yeast preparations.

The work presented in this report will focus on the application of pure GSH as outlined in an International Organisation of Vine and Wine (OIV) resolution passed in 2015 that recommended GSH addition as an option during grape processing (OIV-OENO 445-2015). The addition of GSH to wine after alcoholic fermentation, as outlined in a second OIV resolution (OIV-OENO 446-2015), was not considered as part of this work and neither were sources of GSH from the grape berry or yeast-derived additives.

For this work to proceed, technical developments to methods for the quantification of GSH in grape were required. While conventional methods for quantitating reduced (GSH) and oxidised (GSSG) glutathione in human, animal and plant material existed, quantifying GSH to the low concentrations for which these methods were developed was unnecessary when determining the concentration of GSH in juice and fermenting wines to which GSH is added in the mg/L range. Therefore, a method was developed that enabled the determination of GSH and GSSG concentrations over a range from 2 to 300 mg/L in juice and fermenting wines using a simple, rapid and robust approach that required minimal sample preparation. This method was used in all subsequent fermentation-based monitoring of GSH.

A fundamental question arising from OIV-OENO 445-2015 was whether a level of juice nitrogen could be defined that prevented the metabolic consumption of GSH by yeast. To address this question, GSH was added to juices and defined media with increasing yeast assimilable nitrogen (YAN) concentrations, and the consumption of GSH was monitored throughout fermentation. Yeast strains that varied in their nitrogen demand were also evaluated. It was demonstrated that although increasing the YAN status of the juice did indeed decrease the consumption of GSH during fermentation, the loss of GSH could not be eliminated entirely. Even at a low initial GSH concentration and high initial YAN concentrations, some consumption of GSH was evident. This work raised some questions about whether the criteria for GSH usage as outlined in OIV-OENO 445-2015 could reasonably be met.

This project also sought to understand the impact of GSH addition during alternative processing regimes. Inert pressing of grapes has previously been shown to preserve both grape-derived GSH and much of the phenolic material that is commonly oxidised during more routine pressing of white grapes. Adding GSH to such juice could result in initial GSH concentrations that substantially exceed OIV guidelines. In such a scenario it was of interest to determine the likely consequences of this combination of processing and GSH addition steps. Inert pressing combined with GSH addition led to substantial formation of both 4-methyl-4-sulfanylpentan-2-one (4-MSP) and grape reaction product (GRP) in the finished wines. The concentrations of these were decreased in oxidatively produced wines when GSH was added. In the absence of GSH addition, little of either compound was detected. This demonstrated the capacity for GSH additions to interact with processing variables other than YAN to alter the wine composition.

Two pilot-scale experiments evaluated the impact of pre-ferment GSH addition in authentic winemaking circumstances. In one trial, the wines were bottled using a high and a low SO2 regime to determine whether GSH could be used to supplement SO2. Both high (250 mg/L) and OIV recommended (20 mg/L) additions of GSH, prior to or during fermentation, were trialled. High GSH addition trials were bottled with residual GSH concentrations of 50 mg/L indicating a potential consumption of 200 mg/L of GSH from all sources during wine production. This residual GSH concentration was insufficient to limit the loss of SO2 during wine ageing, however, and did not limit the appearance of oxidative characteristics which were evident in the sensory evaluation of those wines. Overall, there were no compositional or sensorial differences between wines that had GSH added prior to or after inoculation. Wines with added GSH were consistently described as ‘cat pee’, ‘flinty’ or ‘baby sick’. As the wines aged the concentration of hydrogen sulfide (H2S) increased dramatically. If low concentrations of GSH were added immediately post-pressing there were only subtle differences in the sensory characteristics of the wines, irrespective of the yeast strain used. Wines to which low concentrations of GSH had been added after pressing tended to be associated with ‘apple’, ‘pear’ and ‘stone fruit’ sensory descriptors, but there was considerable overlap among all wines within the low GSH study, in which yeast strain choice appeared to have an equal impact.

The key benefit to industry of this work is the provision of evidence-based advice about GSH as an additive and the risks and benefits associated with its use. This work was undertaken as a collaborative project between the AWRI and the University of Adelaide.