Non-canonical regulation of glutathione and trehalose biosynthesis characterizes non- Saccharomyces wine yeasts with poor performance in active dry yeast production

Abstract

Several yeast species, belonging to Saccharomyces and non-Saccharomyces genera, play fundamental roles during spontaneous must grape fermentation, and recent studies have shown that mixed fermentations, co-inoculated with S. cerevisiae and non-Saccharomyces strains, can improve wine organoleptic properties. During active dry yeast (ADY) production, antioxidant systems play an essential role in yeast survival and vitality as both biomass propagation and dehydration cause cellular oxidative stress and negatively affect technological performance. Mechanisms for adaptation and resistance to desiccation have been described for S. cerevisiae, but no data are available on the physiology and oxidative stress response of non-Saccharomyces wine yeasts and their potential impact on ADY production. In this study we analyzed the oxidative stress response in several non-Saccharomyces yeast species by measuring the activity of reactive oxygen species (ROS) scavenging enzymes, e.g., catalase and glutathione reductase, accumulation of protective metabolites, e.g., trehalose and reduced glutathione (GSH), and lipid and protein oxidation levels. Our data suggest that non-canonical regulation of glutathione and trehalose biosynthesis could cause poor fermentative performance after ADY production, as it corroborates the corrective effect of antioxidant treatments, during biomass propagation, with both pure chemicals and food-grade argan oil.

Keywords: active dry wine yeasts; antioxidant defense; food-grade argan oil; non-Saccharomyces yeasts; oxidative damage.

Figure 1. FIGURE 1: Effects of argan oil supplementation on physiological performance and oxidative damage.

(A) Biomass yield, measured as OD at 600 nm. (B) Fermentative capacity measured as the volume of CO₂ produced per 10⁷ dry cells. (C) Lipid peroxidation in dry cells was expressed as the amount of MDA per mg of cells. (D) Protein carbonyl in dry cells was expressed as Ci/ Pi, where Ci is the protein carbonyl content quantified by an image analysis and Pi is the total protein from coomassie-stained membranes. Error bars correspond to the SD value of three independent experiments. (*) significantly differed from the control (non-supplemented molasses) with p < 0.05.

Figure 2. FIGURE 2: Analysis of the predictive biomarkers in ADY after argan oil supplementation during biomass propagation.

(A) Trehalose content after drying. (B) Increment in glutathione reductase (GR) activity after drying. (C) Increment in catalase activity after drying. Error bars correspond to the SD of three independent experiments. (*) significantly differed from the control with a p < 0.05.

Figure 3. FIGURE 3: Analysis of the glutathione levels in ADY after argan oil supplementation during biomass propagation.

(A) Oxidized glutathione after drying. (B) Total glutathione after drying. (C) The GSH/GSSG ratio after drying. Error bars correspond to the SD of three independent experiments. (*) significantly differed from the control with a p < 0.05.

Figure 4. FIGURE 4: Principal components (PCA) statistical analysis of the argan oil effects on the physiological and biochemical biomarkers with represented total variance of 79%.

Component 1 reflects 39.55% total variance (with a positive correlation with biomass yield, trehalose levels and glutathione reductase activity) and Component 2 reflects 30.45% total variance (with a positive correlation with GSSG levels). Lines belong to the variance of the dependent variables or the biochemical biomarkers measured (biomass yield, fermentative capacity, lipid peroxidation, protein carbonylation, protective metabolites and enzymatic activities) arranged in two dimensions according to Components 1 and 2. Study strains and conditions (control and argan oil supplementation) are labeled with different symbols: T73 (dark blue); C. stellata (pink); T. delbrueckii (green); P. fermentans (yellow); H. osmophila (red); and H. guilliermondii (blue), and are associated with the dependent variable, which differs from the other strains and conditions.

Figure 5. FIGURE 5: Fermentation parameters for vinifications with T73 and H. osmophila pure cultures and with mixed cultures.

Sugar consumption (A and C) and viability (B and D) profiles during natural must fermentation inoculated with the ADY obtained from molasses in the absence (control) and presence of argan oil (argan). Panel E shows sugar consumption in vinifications conducted by mixed starters where S. cerevisiae T73 ADY was obtained from molasses in the absence (control) or presence of argan oil (argan) and the H. osmophila ADY was always obtained from molasses supplemented with argan oil. Fermentations were considered complete when the sugars concentration went below 2 g/L.