Biotechnological tools for reducing the use of sulfur dioxide in white grape must and preventing enzymatic browning: glutathione; inactivated dry yeasts rich in glutathione; and bioprotection with Metschnikowia pulcherrima

Sulfur dioxide is the most used additive today for preventing browning in grape musts and wines. However, since wine consumers are increasingly interested in healthier wines, the wine industry is keen to reduce its use. Some promising alternatives to sulfur dioxide have been proposed in recent years, including glutathione, both pure and in the form of inactivated yeasts, and Metschnikowia pulcherrima used as a bioprotective agent. Some information exists about the protective effect against oxidation of glutathione but there is very few about the use of bioprotection for that purpose. Supplementation with glutathione, regardless of the commercial form, reduced oxygen consumption and browning intensity when laccase was not present in the grape juice. Metschnikowia pulcherrima also reduced browning intensity in the absence of laccase but increased the total oxygen consumption. However, in the presence of laccase, glutathione and Metschnikowia pulcherrima were not effective enough to adequately prevent the grape juice from browning. Glutathione, both pure and in the form of inactivated yeasts, and Metschnikowia pulcherrima are interesting tools for protecting grape must against browning, and thus reducing the use of sulfur dioxide.


Introduction
One of the problems that most seriously affects oenology today is enzymatic browning [1], especially when the grapes are infected by grey rot [2]. Enzymatic browning is an oxidation process that occurs in many foods that increases the brown color [3], which often leads consumers to reject them. This problem is especially harmful in the wine industry since grape must is highly vulnerable to browning [4,5].
Enzymatic browning is caused by polyphenol oxidases, a broad family of oxidoreductases (EC. 1 class, according to the International Union of Biochemistry and Molecular Biology-IUBMB [6]). However, in the case of grape juice, browning is caused by just two enzymes: tyrosinase (EC 1.14.18.1, IUBMB [6]), which is naturally present in grapes [4,5], and laccase (EC 1.10.3.2, IUBMB [6]), which is present only when the grapes are infected by Botrytis cinerea [5,7]. Both enzymes use molecular oxygen to mainly oxidize the diphenols present in grape must and wine such as caftaric and cutaric acids, catechin and other flavanols, anthocyanin, and flavanone, though laccase is able to oxidize a wider range of substrates than tyrosinase [5,7,8].
The main consequence of enzymatic browning, irrespective of whether tyrosinase and/or laccase is the enzyme responsible, is that diphenols are oxidized to quinones, which can later polymerize through various reactions to form brown pigments called melanins [9,10]. These compounds are responsible for increasing the intensity of the brown color in white wines (browning) and for precipitating the coloring matter in red wines (oxidasic haze) [11].

3
Sulfur dioxide is the main and, until recently, only tool used by wineries to protect grape must from browning [12,13]. This additive (E-220) is widely used in winemaking, thanks to its well-known antioxidant, antioxidasic, and antimicrobial properties [14], which make it practically essential not only in winemaking but also in the manufacture of other foods [15]. However, the current trend in winemaking is to reduce and even eliminate this unfriendly additive owing to its negative effects on the environment [16], the increasing tendency toward minimal intervention [17] and health [18] since it could cause headaches in sensitive people [19].
For all these reasons, the wine sector is extremely keen to find strategies for preventing oxidation and microbiological spoilage and for reducing or even eliminating sulfur dioxide.
Some of the most promising alternative agents for protecting grape juice against browning are glutathione and several non-Saccharomyces yeasts such as Metschnikowia pulcherrima used as a bioprotective agent.
Glutathione (GSH) limits browning because it reacts with the orthoquinones produced by the enzymatic oxidation of orthodiphenols to form GRP (Grape Reaction product; 2-S-glutathionylcaftaric acid). GSH traps the orthoquinones in a colourless form, and thus restricts the formation of brown polymers [23,31]. The use of glutathione in winemaking was authorized by the International Organisation of Vine and Wine (OIV) in 2015 at a maximal dose of 20 mg/L [32,33]. However, the high price of pure glutathione led to the use of certain inactive dry yeasts especially rich in glutathione (IDY-GSH) being proposed as more economical alternatives [34,35]. The use of these yeasts in winemaking was authorized by the OIV in 2018 [36].
Bioprotection is also nowadays considered a highly promising alternative tool to sulfites in winemaking [27,37]. In fact, the OIV has authorized the use of non-Saccharomyces yeasts in winemaking for various purposes, one of which is bioprotection [38,39]. Several studies on the use of certain strains of non-Saccharomyces yeasts for preventing the development of spoilage microorganisms have been reported in recent years [40][41][42][43]. However, few of these studies have proposed their use in protecting the grape must against browning [44,45]. Specifically, certain strains of Metschnikowia pulcherrima have shown interesting results both for preventing the development of spoilage microorganisms [37,46] and for protecting against browning [27,47].
In this paper, we compare the protective effect against browning in white grape musts of some of the most promising antioxidant tools-reduced glutathione, both pure and in the form of commercial inactivated dry yeasts rich in glutathione, and a commercial strain of Metschnikowia pulcherrima-with the protective effect of classical tools such as sulfur dioxide and ascorbic acid.

Obtaining the samples of grape must
Muscat of Alexandria grapes were handpicked in the vineyard of the Rovira i Virgili University (Mas dels Frares, Constantí, Tarragona: 41°08′44.1′′N 1°11′51.0′′E) during the 2022 vintage harvest. The grapes were pressed in a nitrogensaturated hand-press and the must was collected in a bottle also saturated with N 2 .

Synthetic buffer
A solution of 4 g/L of L-( +)-tartaric acid, 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 adjusted to pH 3.5 with sodium hydroxide was used for all experiments.

Inactivated dry yeasts rich in glutathione and Metschnikowia pulcherrima strain
A commercial inactivated dry yeast (IDY-GSH) rich in glutathione (Glutastar ™ , Lallemand Inc, Montreal, Canada) and a strain of Metschnikowia pulcherrima (MP) selected for its high oxygen consumption capacity (Level2 Initia ™ , Lallemand Inc, Montreal, Canada) were used. Both the IDY-GSH (400 mg/L) and the MP (250 mg/L) were hydrated in ten times their weight of distilled water 10 min before the beginning of the measurements. The water temperature was room temperature for the IDY-GSH and 30 °C for the MP.

Extracellular laccase production and enzymatic activity measurement
Active laccase extracts were obtained from the Botrytis cinerea isolate 213 strain following the methodology reported by Vignault et al. [48]. This laccase extract was treated with 0.16 g of PVPP/mL for 10 min and centrifuged at 7500 rpm for 10 min, and the supernatant was subsequently dialysed with 3.5 KDa cellulose membrane for 2 days in a 0.3 M ammonium formate solution and for 2 more days in distilled water. The laccase activity of this extract was determined using an adaptation of the syringaldazine test method [49].
Thirteen mL of grape must and 52 mL of buffer were added to each flask, to which various antioxidant agents or combinations of them had previously been added. This dilution of the grape must was performed because the pure grape juice consumes oxygen so fast that it is nearly impossible to monitor correctly the oxygen concentration of all the samples. The antioxidants used were: sulfur dioxide (20 mg/L in the form of potassium disulfite), ascorbic acid (100 mg/L), glutathione (20 mg/L), a commercial inactivated dry yeast rich in glutathione (400 mg/L), and a selected commercial strain of Metschnikowia pulcherrima (200 mg/L). A control without any addition was also prepared. These assays were also performed by combining sulfur dioxide with each of the other antioxidant agents with and without the addition of 2 UA of laccase/mL. Table 1 shows all the experimental conditions and provides abbreviations for each one. All these assays were performed in triplicate.
The bottles were immediately hand-shaken for a few seconds to saturate the solution in oxygen (around 7-8 mg O 2 /L), and the oxygen concentration was measured [50] periodically until reaching an asymptotic behavior (around 5 h) to determine the oxygen consumption kinetics. All measurements were taken in an air-conditioned laboratory at 22 ± 2 °C. The total oxygen consumption capacity (TOCC) was calculated using the mathematic model previously reported by Pons-Mercadé et al. [24]. Once the oxygen concentrations were below 1 mg/L or its consumption reached an asymptotic behaviour, the samples were supplemented with 50 mg of sulfur dioxide/L to stop color evolution.

Color measurements
Measurement of the yellow color (A420nm) and the CieL*a*b* coordinates of the samples were determined according to Ayala et al. (1997) [51]. Data processing was performed with MSCV software [52]. The total color difference (∆Eab*) was calculated as the Euclidian distance between two points in the CieL*a*b* space using the following formula: ∆Eab* = ((L 1 -L 2 ) 2 + (a* 1 -a* 2 ) 2 + (b* 1 -b* 2 ) 2 ) 1/2 , where L* is the lightness; a* is the color's green-red component, and b* is the color's blue-yellow component. It is generally considered as a criterion that two samples of wine could be distinguished by the human eye through the glass when ΔEab* ≥ 3 units [21,57].

Statistics
All data are expressed as the arithmetic average ± standard deviation of three replicates. One-factor analysis of variance (ANOVA F test) was conducted using SPSS 15.0 software (SPSS Inc., Chicago, IL). Significant differences were considered when p value was less than 0.05.

Results and discussion
Oxygen consumption kinetics Figure 1A and B shows the oxygen consumption kinetics of the diluted grape juice under the experimental conditions with or without supplementation with laccase. Figure 1A clearly shows that the control sample-the diluted grape juice without modification (C)-initially consumed oxygen very quickly. This oxygen consumption rate (OCR) subsequently moderated, probably due to depletion of the substrates, reaching values below 1 mg of O 2 /L after 5 h. This figure also shows that when the sample was supplemented with sulfur dioxide (SO 2 ), the OCR decreased. To statistically compare these curves, a previously reported kinetic model [24,50]   The results for TOCC are shown in Fig. 2, which confirms what was indicated in Fig. 1A, i.e., that the TOCC of the control sample (C) was significantly higher than that of the sample supplemented with sulfur dioxide (SO 2 ). These data confirm the well-known inhibitory effect of this additive on tyrosinase activity [8,[12][13][14][15]. Supplementation with glutathione (GSH) and with inactivated dry yeasts rich in glutathione (IDY-GSH) also led to a significant decrease in TOCC. This inhibitory effect observed in both experimental conditions may be due to the fact that glutathione reacts with the orthodiquinones produced by the enzymatic oxidation of hydroxycinnamic acids to form the grape reaction product (GRP) [23,31,55,56]. This process can reduce the concentration of orthodiphenols and lead to their depletion, especially in our experimental conditions where the grape must was diluted five times. The lack of substrates for polyphenol oxidases may, therefore, justify the TOCC reduction in our experimental conditions, though in a grape must without dilution, this reduction would probably not be so great. However, these data clearly confirm GSH's protective effect against oxidation, both pure and in the form of IDY rich in GSH.
On the other hand, supplementation with Metschnikowia pulcherrima (MP), and especially with ascorbic acid (AA), increased TOCC with respect to the control (C). Ascorbic acid is known to react efficiently with oxygen to produce dehydroascorbic acid and hydrogen peroxide [5,57]. It can, therefore, compete with polyphenol oxidases [8,48,57] for oxygen and prevent grape juice from browning. However, ascorbic acid must be used in combination with sulfur dioxide to eliminate the hydrogen peroxide formed and prevent subsequent oxidation [5,57]. The highest TOCC observed in the presence of MP probably occurred because this non-Saccharomyces yeast directly consumes oxygen very effectively. In fact, use of this yeast has been recommended for that purpose [44,45]. It should be noted that the used grape must was not sterilized, and therefore the presence of some yeasts, Saccharomyces or non-Saccharomyces could also have contributed to the observed oxygen consumption albeit presumably to a lesser extent than the inoculated MP.
The combined supplementation of all studied antioxidants with sulfur dioxide (GSH + SO 2 , IDY-GSH + SO 2 , AA + SO 2 and MP + SO 2 ) led to a reduction in TOCC with respect to their corresponding references (GSH, IDY-GSH, AA and MP). Note that the TOCC of GSH + SO 2 and IDY-GSH + SO 2 were very similar to that of SO 2 alone. These results may be considered logical because in those conditions, tyrosinase is completely inhibited and the possible inhibitory effect of glutathione cannot act because no orthodiquinones are produced. The TOCC of MP + SO 2 was also similar to that of SO 2 alone and much lower than that of MP alone. These results may indicate that the presence of sulfur dioxide inhibits this non-Saccharomyces yeast. The TOCC of AA + SO 2 was higher than that of SO 2 alone and lower than that of AA alone because in those conditions, oxygen consumption was only due to the direct reaction of this antioxidant with oxygen and not to polyphenol oxidase activity.
As expected, supplementation with laccase significantly increased the TOCC of the control sample. This was probably because total polyphenol oxidase activity was higher and because laccase can oxidize a wider range of substrates than tyrosinase [5,7]. This trend was also observed in the samples supplemented with laccase and containing AA, GSH, IDY-GSH, and MP in relation to their corresponding samples without laccase.
The samples supplemented with GSH and IDY-GSH in the presence of laccase showed significantly lower values of TOCC than the control sample supplemented with laccase but significantly higher values of TOCC than the corresponding samples without laccase. A possible explanation for these data is that laccase can oxidize more substrates than tyrosinase, especially since this polyphenol oxidase can oxidize GRP [23]. In any case, the reduction in TOCC produced by supplementation with GSH or IDY-GSH suggests that glutathione may perform an antioxidant role even when the grape berries are affected by grey rot, though this protection is not as effective as it is in healthy grapes.
Supplementation with sulfur dioxide inhibited TOCC in all experimental groups in a similar way to the corresponding samples without laccase. These data confirm, as previously described in the literature [4,5,8,48,57], that sulfur dioxide is also a powerful inhibitor of laccase.
Determining TOCC is necessary to understand which antioxidant agent is the most effective in trapping the oxygen in the grape must and preventing its consumption by polyphenol oxidases. However, since TOCC does not provide direct information about the browning intensity, the final color of the samples was measured by spectrophotometry. Figure 3A and B shows the absorbance at 420 nm (A420nm) and the CIEL*a*b* blue-yellow component (b*) of the various samples as indicators of browning intensity.

Browning intensity
As expected, supplementation with sulfur dioxide had a clear protective effect against browning since the values of A420 and b* were significantly lower than in the control sample. This effect was also observed when the samples were supplemented with laccase, which confirms that sulfur dioxide not only inhibits the polyphenol oxidase present in healthy grapes (tyrosinase) but also has a powerful inhibitory Fig. 3 Influence of laccase, SO 2 , ascorbic acid, glutathione, inactivated dry yeast rich on glutathione and M. pulcherrima on browning intensity action on the laccase present in the grapes infected with grey rot [8,[12][13][14][15].
In contrast, the sample supplemented with AA showed significantly higher values of A420nm and b* than the control without supplementation although it consumed oxygen faster than the control. This trend, which was also observed when the sample was supplemented with laccase, is probably due to the fact that ascorbic acid produces hydrogen peroxide when it reacts with oxygen [5,57]. However, when these samples were also supplemented with sulfur dioxide, the A420nm were similar to that of the sample supplemented only with SO 2 and the CIEL*a*b* coordinate b* was even significantly lower. These data confirm that ascorbic acid must be applied together with sulfur dioxide so that it reacts with hydrogen peroxide to nullify its negative effect on browning [22,57].
The A420 nm of the samples supplemented with GSH, IDY-GSH, and MP were also significantly lower than those of the control sample. This reduction in A420 nm was highly relevant, though the values were slightly but significantly higher than that of the sample treated with sulfur dioxide. The CIEL*a*b* coordinate b* of the samples supplemented with GSH, IDY-GSH, and MP showed a similar trend to the A420 nm but, in this case, no significant differences were found with the sample treated with sulfur dioxide. These data, therefore, confirm that glutathione, both pure and in the form of inactivated dry yeast, and Metschnikowia pulcherrima really do protect grape juice against browning.
This protective effect of GSH, IDY-GSH, and MP was also present in the samples supplemented with laccase but the efficiency was much lower. However, when sulfur dioxide was also added, the values of A420 and b* were similar to those of the sample protected only with SO 2 . These data indicate that these alternative tools to sulfur dioxide cannot be applied when the grapes are infected with Botrytis cinerea.
Note that browning intensity generally has a certain parallelism with the TOCC we observed. In nearly all samples, the higher the TOCC, the higher the browning intensity, with the only exception being the samples supplemented with MP in the presence or absence of laccase. The explanation for this different behavior may be that since Metschnikowia pulcherrima consumes oxygen very effectively [27,44], some of the initially dissolved oxygen is not consumed by the polyphenol oxidases.

Total color difference (∆Eab*)
The total color difference (∆Eab*) between the various samples and the sample supplemented with sulfur dioxide without laccase was calculated to determine whether the browning intensity of the samples can be distinguished by potential consumers.
The results are clear and highly indicative of the protective effect of the various antioxidants. Figure 4A shows the ∆Eab* of all samples without supplementation with Fig. 4 Influence of laccase, SO 2 , ascorbic acid, glutathione, inactivated dry yeast rich on glutathione and M. pulcherrima on the total color difference (∆Eab*) laccase and the corresponding sample with only the addition of sulfur dioxide. As expected, the control sample showed an ∆Eab* above 3 units, which indicates that it was much more affected by browning than the sample protected with SO 2 . The ∆Eab* of the sample supplemented with only AA was even higher than the control, whereas when sulfur dioxide was also present the ∆Eab* was below 3 units. These data confirm that using ascorbic acid alone leads to greater oxidation and that it must, therefore, be used in the presence of SO 2 [5,57].
In contrast, the samples protected with GSH, IDY-GSH, and MP with or without SO 2 showed values below this threshold. However, when the samples were also supplemented with laccase, supplementation with GSH, IDY-GSH, and MP was not effective enough to protect the samples against browning because the values of ∆Eab* were above 3 units. These results agree with previously reported data [8,23,27,31,34,35,44,46] and confirm that using glutathione, pure or in the form of inactivated dry yeasts, and Metschnikowia pulcherrima can really protect grape juice against browning at least in healthy grapes.
Since a picture is worth a thousand words, Fig. 5 shows the photographs of the different samples after the experimental process. In them, it can be clearly seen the browning developed in each one of the experimental conditions and, therefore, the protective effect of some of the treatments, which visually corroborates all the experimental data previously shown.

Conclusion
In this study, we analyzed how various antioxidants influence oxygen consumption kinetics and browning intensity in two scenarios. The first scenario was grape must from healthy grapes while the second was grape must enriched with laccase to reproduce what occurs when the grapes are infected with Botrytis cinerea. Our experimental design measured oxygen consumption kinetics and browning intensity caused by polyphenol oxidases, tyrosinase, and laccase. As expected, our results confirm that grape must consumes oxygen and browns very quickly and that the presence of laccase accelerates both of these processes. Our results also confirm that sulfur dioxide is highly effective in preventing browning even in grape musts with high levels of laccase activity. On the other hand, using only ascorbic acid leads to higher oxygen consumption and browning, which indicates that this antioxidant must be used in association with sulfur dioxide.
The other alternative antioxidants-glutathione, both pure and in the form of inactivated dry yeasts, and the non-Saccharomyces yeast, Metschnikowia pulcherrima used as a bioprotective agent-can be interesting tools for protecting Fig. 5 Influence of laccase, SO 2 , ascorbic acid, glutathione, inactivated dry yeast rich on glutathione and M. pulcherrima on the final color of the samples grape juice against browning and perhaps for reducing the use of sulfur dioxide, at least in healthy grapes. Specifically, glutathione and inactivated dry yeast rich in glutathione reduced oxygen consumption and reduced the intensity of browning when no laccase was present in the medium. However, their effectivity was reduced in the presence of laccase. The mechanism by which glutathione protects against enzymatic browning and reduces oxygen consumption is probably its capacity to combine with the orthodiquinones formed by the action of the polyphenol oxidases in stopping the browning process and depleting the medium on substrates for these enzymes.
Metschnikowia pulcherrima also reduced browning intensity but its action mechanism is different from that of glutathione. This non-Saccharomyces yeast protects because it consumes oxygen very efficiently, and therefore reduces its availability for the polyphenol oxidases.
More studies are needed to further investigate these promising alternatives to sulfur dioxide since many consumers are searching for healthier wines, and the wine industry is very keen to reduce this unfriendly additive.
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