Measuring the oxygen consumption rate of some inactivated dry yeasts: comparison with other common wine antioxidants

The aim of the study was to evaluate the oxygen consumption kinetics of some inactivated dry yeasts in comparison with those of sulfur dioxide, ascorbic acid and glutathione. The oxygen consumption rates of three inactivated dry yeasts, sulfur dioxide, ascorbic acid and glutathione at the usual doses in a model wine solution were determined by carrying out noninvasive fluorescence measurements. The results indicate that two of the studied inactivated dry yeasts consume oxygen more effectively than sulfur dioxide. These data suggest that some inactivated dry yeasts may be useful for protecting wine against oxidation. This study shows for the first time that inactivated dry yeasts can actually consume oxygen, therefore opening up an interesting area for future research.

Yeast autolysis has been reported to have different sensory effects on wine. Polysaccharides and mannoproteins have been found to improve wine mouthfeel (Gawel et al., 2018) and, in combination with some peptides and proteins, to contribute to wine sweetness, smoothing the perception of acidity and astringency (Marchal et al., 2011). Furthermore, some amino acids, peptides and nucleotides can contribute to creating an umami taste (Vilela et al., 2016) and have been described as flavour enhancers, while lipids and amino acids have been described as aroma precursors (Styger et al., 2011). Additionally, mannoproteins seem to exert a protective colloid effect, increasing the stability of proteins (Waters et al., 1994) and tartaric acid salts (Gerbaud et al., 1997).
Lees has also been reported to consume oxygen, slowing down the oxidation of the wine (Salmon et al., 2000;Pons-Mercadé et al., 2020). The mechanism by which this occurs is not known. Some authors associate it with the oxidation of membrane lipids (Fornairon-Bonnefond and Salmon, 2003), but it may also be related to the release of glutathione by yeast (Kritzinger et al., 2013). Whatever the mechanism, oxygen consumption by lees appears to be the main factor involved in white wines aged on lees being able to age for longer than other white wines.
In short, wines aged on lees are generally richer in mouthfeel and have greater depth and flavour complexity, and white wines on lees tend to age better. The practice, however, is laborious and entails certain risks, such as the appearance of reductive characters (Dubourdieu, 1995) and Brettanomyces taints (Renouf et al., 2007). For this reason, inactivated dry yeasts (IDY) are now used, because in theory they have the same effect as lees, but without any of the drawbacks (Pozo-Bayón et al., 2009a;Del Barrio-Galán et al., 2011).
It has also been recently reported that some IDYs consume oxygen in a similar way to other common antioxidants used in wine, such as sulfur dioxide (Sieczkowski et al., 2016a, Sieczkowski et al., 2016bPons et al., 2019). However, little is known about using IDY to protect wine against oxidation. Therefore, the aim of this study was to measure the kinetics of oxygen consumption using different IDYs and to compare them with those of other common wine antioxidants (sulfur dioxide, ascorbic acid and glutathione) in order to determine their real antioxidant capacity for protecting wine from the detrimental effects of oxygen.

Experimental design
The experimental design for determining the oxygen consumption rate of IDYs and other antioxidants was an adaptation of those previously described by Navarro et al. (2016) and Pascual et al. (2017) for the oxygen consumption rate of oak chips and oenological tannins respectively. A model wine solution composed of ethanol (12 % v/v) and L-(+)-tartaric acid (4 g/L) adjusted at pH = 3.5 with sodium hydroxide was used. This solution was enriched with 3 mg of iron/L, in the form of iron (III) chloride hexahydrate, and 0.3 mg of copper/L in the form of copper (II) sulfate pentahydrate. We worked with this model wine solution rather than with real wine, because the naturally occurring phenolic compounds would have competed with the IDY or other antioxidants, making it impossible to determine their oxygen consumption kinetics. This model wine solution was saturated in oxygen (around 8.0 mg/L) by bubbling with air for 10 min just before beginning the experiment.
The usual doses of IDY (400 mg/L), potassium metabisulfite (30 mg/L), ascorbic acid (100 mg/L) or glutathione (20 mg/L) were placed in clear glass bottles (750 mL) into which a pill had previously been inserted (PreSens Precision Sensing GmbH, order code: SP-PSt3-NAU-D5-CAF; batch number: 1203-01_PSt3-0828-01, Regensburg, Germany) for the noninvasive measurement of dissolved oxygen by luminescence (Nomasense TM O2 Trace Oxygen Analyzer by Nomacorc S.A., Thimister Clermont, Belgium). The bottles were completely filled with the model wine solution and then immediately closed with a crown cap and bedule so as to minimize the headspace volume. The bottles were then gently shaken to resuspend the IDYs or to dissolve the different antioxidants: sulfur dioxide (added as potassium metabisulfite), ascorbic acid or glutathione. The bottles were maintained at 20 ± 2 °C during all this time. Oxygen was measured (Diéval et al., 2011) periodically to determine the oxygen consumption rate: oxygen measurements were made every day for the assays containing IDYs, sulfur dioxide and glutathione over two weeks, and every hour for the assay containing ascorbic acid over one day. Figure 1 is a schematic representation of the experimental design. Two types of control were prepared. Control-A bottles were filled with the oxygen-saturated model wine solution without adding any antioxidant, and Control-B bottles were filled with the same model wine solution, but after bubbling for 10 min with nitrogen to eliminate the oxygen. Control A was used to verify that the model wine solution did not consume oxygen, and Control B was used to verify the tightness of the closure system. All assays were performed in triplicate.

Modelisation of the oxygen consumption kinetics
In order to quantify the real oxygen consumption ability of the IDY in comparison with that of the other common antioxidants, we applied the kinetic model proposed by Pascual et al. (2017) for estimating the oxygen consumption kinetics of oenological tannins. This model consists in displaying the inverse of consumed oxygen versus the inverse of time. According to this mathematical model, the following equation can be established: 1/[O2] = A/t + B. This equation, which describes the relationship between oxygen consumed and time, can be used to determine the oxygen consumption rate at time zero (OCRto) and also the total oxygen consumption capacity (TOCC). Figure 2A shows how the consumed oxygen can be cleared up. Figure 2B shows how the OCRto can be determined after calculating the first derivative and considering time equal to zero. Finally, Figure 2C shows how the TOCC is obtained by calculating the limit when time tends towards infinity. According to this model, the OCRt o corresponds to the inverse of the slope of the initial equation and the TOCC to the inverse of the y-intercept of the initial equation.

Determination of the glutathione content of inactivated dry yeasts
Glutathione extraction and analysis were performed according to an adaptation of the method reported by Nisamedtinov et al. (2010).

Reagents
TE8 solution: 6.05 g/L of tris(hydroxymethyl) aminomethane and 1.12 g/L of EDTA disodium salt dehydrate adjusted at pH = 8.00 with HCl 1N. This solution was stored at 4 °C. DTNB stock-solution: 39.6 mg of DTNB + 8 ml of TE8 solution + 2 ml of NaOH 0.1 N. This solution is stable for at least one month when protected from light. DTNB working-solution: 8 ml of DTNB stock-solution + 92 ml of TE8 solution; fresh solution was prepared and used daily.

Glutathione extraction
Two grams (dry weight) of each inactivated dry yeast was placed into 15 ml centrifuge tubes to which 5 ml of 0.1 N formic acid solution was immediately added. The samples were maintained at room temperature for one hour with frequent vortex shaking. After one hour had elapsed, the samples were centrifuged (Sorval RC5C) at 11.500 rpm for 10 min at 10 o C. One hundred mL of the supernatant were added to 4.9 mL of DNTB working solution. The sample was immediately vortexed and kept for 10 min at room temperature. The samples were used immediately for UPLC analysis.

Glutathione UPLC Analysis
An ACQUITY UPLC system (Waters, USA), equipped with a C18 column (AccQ Tag Ultra Column, 100 × 2.1 mm, 1.7 mm, Waters, USA) and a photo diode array (PDA) detector ACQUITY PDA 2996 was used for the chromatographic determination of reduced glutathione. A twogradient mobile phase (A: water + 0.1 % formic acid and B: acetonitrile + 0.1 % formic acid) and a flow rate of 0.3 mL min-1 was used. The standards (cysteine, ³-L-glutamyl-L-cysteine and reduced glutathione) were used for external calibration after derivatisation with DTNB. Results are expressed as mg of reduced glutathione by gram of dry weight.

Statistics
All data are expressed as the arithmetic average ± standard deviation of three replicates. One-factor analysis of variance (ANOVA) was carried out using the SPSS 15.0 software (SPSS Inc., Chicago, IL). Figure 3 shows the oxygen concentration evolution over time of the initially oxygen-saturated model wine solutions with supplements (the different IDYs and the other common antioxidants) or without supplements.

Evolution of oxygen concentration over time
The oxygen concentration of Control-A (no antioxidant added) remained stable with only slight, erratic variations that are probably linked to the inherent variability of the analytical procedure. The oxygen intake in Control-B (no oxygen) was very low and can be considered negligible (data not shown). By contrast, the oxygen concentration of the model wine solutions supplemented with the different antioxidants decreased over time, with the exception of IDY-2, where the concentration remained stable.
As expected, the model wine solution containing ascorbic acid consumed all the oxygen in a few hours, confirming that it is more effective than the other antioxidants (Barril et al., 2012).
The graph clearly shows that, with the sole exception of IDY-2, all the antioxidants consumed oxygen: the most effective was IDY-3, followed in decreasing order by IDY-1, sulfur dioxide and glutathione. All data are expressed as the average of 3 replicates ± standard deviation. IDY = Inactivated Dry Yeasts.

Modeling oxygen consumption kinetics
Since Figure 3 does not provide a clear quantitative comparison of the oxygen consumption kinetics of the IDYs and the other antioxidants, we applied the kinetic model proposed by Pascual et al. (2017) to better estimate the real capacity of both IDYs to consume oxygen in comparison to that of the other common antioxidants. This model consists in displaying the inverse of consumed oxygen versus the inverse of time, thus obtaining the following equation: 1/[O2] = A/t + B. Figure  4 shows the results for ascorbic acid, sulfur dioxide, glutathione and both IDYs. In all cases, satisfactory linear regression coefficients were obtained (between 0.9752 and 0.9921), confirming that this mathematical model works reasonably well.
According to the model, the OCRto corresponds to the inverse of the slope and the TOCC corresponds to the inverse of the y-intercept of the above equation (See Figure 2).
After clearing up the oxygen, the equation [O2] = t/(A + Bt) was obtained, which can be used to compare the experimental data ( Figure 5A) with the theoretical data ( Figure 5B) obtained after applying this model.
As the figures show, the mathematical model reproduces the experimental kinetics of oxygen consumption by all the antioxidants quite well. Once again, these results demonstrate the validity of the model.

Oxygen consumption rate at time zero and total oxygen consumption capacity.
As explained above, the kinetic model of Pascual et al. (2017) can be used to determine two parameters that define the kinetics of oxygen consumption: OCRto and TOCC. Table 1 shows the resulting values for both IDYs and the other tested antioxidants. It also shows the oxygen consumption rate at time zero (% ROCRto) and the total oxygen consumption capacity (% TOCC) of the different antioxidants compared to those of sulfur dioxide at the usual doses.
Sulfur dioxide, which is by far the most widely used antioxidant in wine production, showed the lowest OCRto of all the studied antioxidants, indicating that its direct reaction with oxygen is slow. However, sulfur dioxide is mainly used as an antioxidant because of its ability to react with hydrogen peroxide formed by oxidation of polyphenols (Danilewicz, 2015), its effectiveness to inhibit polyphenol oxidases (Vignault et al., 2020), and its capacity to react with ethanal and All data are expressed as the average of 3 replicates ± standard deviation. IDY: Inactivated Dry Yeasts. therefore eliminate its unpleasant smell (Sheridan and Elias, 2016). The TOCC obtained for sulfur dioxide using this model was lower than the corresponding stoichiometric value of 30 mg of SO 2 /L (2.27 mg/L versus 7.50 mg/L respectively). This difference can probably be attributed to the fact that the reaction is very slow, and that after 15 days not all of the sulfur dioxide had been consumed.
The % ROCRto results indicate that ascorbic acid initially consumes oxygen around 350 times faster than sulfur dioxide, and the % TOCC indicates that it can consume around 4.2 times more oxygen than sulfur dioxide at the usual doses. These data confirm the high effectiveness of ascorbic acid reported in the literature (Barril et al., 2012). However, the fact that ascorbic acid generates hydrogen peroxide after consuming oxygen must be taken into account, as its use in wine may cause subsequent oxidations (Gibson, 2006;Oliveira et al., 2011) which can affect the sensory quality of the wine. For this reason, ascorbic acid must be always used in the presence of sulfur dioxide to prevent wine oxidation (Barril et al., 2016). The TOCC obtained using this model was very  close to the corresponding stoichiometric value of 100 mg of ascorbic acid/L (9.52 mg/L versus 9.08 mg/L respectively).
The % ROCRto of glutathione is around 4.7 times greater than that of sulfur dioxide but the % TOCC is around 5 times lower. These data indicate that glutathione reacts faster with oxygen than sulfur dioxide; however, the maximal dose of this antioxidant (20 mg/L) authorised by the International Organization of Vine and Wine (OIV, 2015) can only directly consume small amounts of oxygen. To our knowledge, there are no data on the kinetics of oxygen consumption by glutathione in a medium similar to wine. There is extensive literature, however, on its protective effect against browning (El Hosry et al., 2009;Kritzinger et al., 2013) and the loss of certain aromas (Roussis et al., 2007;Rodríguez-Bencomo et al., 2014), especially volatile thiols (Ugliano et al., 2011;Nikolantonaki et al., 2018). The mechanism by which glutathione manifests these protective effects seems to be more closely related to its ability to block the orthoquinones that form grape reaction product (GRP) than to its direct reactivity to oxygen (Nikolantonaki et al., 2014;Webber et al., 2017). The orthoquinones formed by the reaction of oxygen with orthophenols can subsequently react with volatile thiols to form adducts that are no longer volatile, or with other phenols to form melanins that cause browning. Consequently, its blockage by glutathione prevents the loss of volatile thiols and browning. The TOCC obtained using this model was also very close to the corresponding stoichiometric value of 20 mg of ascorbic acid/L (0.49 mg/L versus 0.52 mg/L). Table 1 also shows the oxygen consumption kinetic parameters of IDY-1 and IDY-3. No data are shown for IDY-2, because this inactivated dry yeast did not show any effects (see Figure 3). The % OCRto indicates that oxygen is consumed 3.3 times faster by IDY-1 and 5.4 times faster by IDY-3 than sulfur dioxide. Furthermore, the TOCC values for both IDYs were also higher than those of sulfur dioxide: comparison of the % TOCC values shows that IDY-1 and IDY-3 can consume around 1.2 and 1.8 more oxygen respectively. These TOCC values were similar to those reported by Sieczkowski et al. (2016aSieczkowski et al. ( , 2016b for an equivalent dose of IDY. Table 2 shows the glutathione content of the different IDY. The highest glutathione concentration was found in IDY-3 followed in decreasing order by IDY-1 and IDY-2. Table 2 also shows the corresponding glutathione concentration corresponding to the total glutathione released to the model wine solution by the different IDYs and the corresponding stoichiometric oxygen consumption capacity (SOCC).

Glutathione content of the inactivated dry yeasts
Two clear conclusions can be drawn from these results: first, the glutathione concentration released by all the IDYs is much lower than the maximal authorised dose of pure glutathione; second, the IDYs better able to consume oxygen were precisely those that released more glutathione (IDY-1 and especially IDY-3). However, the levels of glutathione released by the different IDY does not justify all the oxygen consumption. Figure 6 displays the total oxygen consumption capacity (TOCC) of pure glutathione, and the different IDYs versus their corresponding values of the  This figure indicates a reasonably good correlation between TOCC and SOCC for the three studied IDYs. It also shows that IDY-2 does not consume oxygen and that IDY-1 and IDY-3 consume much more than the corresponding stoichiometric oxygen consumption capacity of the glutathione they are able to release. Therefore, these data indicate that the oxygen consumption by IDYs must be related to mechanisms other than glutathione release.

CONCLUSION
This work showed that nearly all the studied antioxidants directly consume oxygen in a model wine solution. Ascorbic acid consumed oxygen much faster than the other antioxidants, followed in decreasing order by IDY-3, glutathione, IDY-1 and sulfur dioxide at the usual doses. Ascorbic acid also showed the highest total oxygen consumption capacity, followed in decreasing order by IDY-3, IDY-1, sulfur dioxide and glutathione.
Furthermore, these results confirm that some inactivated dry yeasts are more effective than sulfur dioxide at directly consuming oxygen; they could therefore be a very useful tool for protecting wine against oxidation. However, the direct oxygen consumption by IDYs cannot be justified by their content in glutathione since the corresponding stoichiometric consumption is much lower than their real oxygen consumption capacity. Further research is needed to investigate the mechanisms by which some inactivated dry yeasts consume oxygen, and to determine whether they can be used to reduce the dose of sulfur dioxide while still preventing spoilage due to oxidation.