Bioprotection using a selected strain of Metschnikowia pulcherrima as an alternative to SO2 for preventing the browning of white grape must Original language of the article: English.
Enzymatic browning of grape must still represent one of the major problems in oenology today (Li et al., 2008), and is especially serious when the grapes are affected by grey rot (Ky et al., 2012). Sulphur dioxide is the most common tool used in wineries to protect grape must against enzymatic browning. This additive is a potent inhibitor of tyrosinase and laccase (Giménez et al., 2023) and also has antimicrobial properties (Ough and Crowell, 1987). However, the wine industry is currently trying to reduce and even eliminate sulphur dioxide due to its negative effects on the environment and human health. Hence, to prevent browning, some alternatives to sulphur dioxide additives have been proposed over recent years, such as the use of inert gases, oenological tannins, ascorbic acid, reduced glutathione, inactivated dry yeasts rich in glutathione and bioprotection using some Non-Saccharomyces yeasts (Giménez et al., 2023; Windholtz et al., 2023; Bustamante et al., 2024a). Different microorganisms have been proposed for Bioprotection, but according to recent literature, Metschnikowia pulcherrima seems to be the most promising microorganism for wine bioprotection against enzymatic browning due to its high capacity for consuming oxygen (Giménez et al., 2023; Bustamante et al., 2024b). The aim of this paper is therefore to study the bioprotective effect of a selected strain of Metschnikowia pulcherrima (MP1) against enzymatic browning caused by polyphenol oxidases.
Experimental design
Muscat of Alexandria grapes were harvested, pressed and diluted 5 times with a model grape must synthetic buffer at pH = 3.50; this made it possible to monitor it with higher precision, and also to standardise the pH of the different measurements. The diluted grape must was immediately supplemented or not with 20 mg/L of SO2, 2 UA/mL of laccase activity and 250 mg/L of the selected M. pulcherrima MP1 (Level2 Initia™, Lallemand Inc, Montreal, Canada). Immediately, the samples were saturated with O2 and its concentration was noninvasively monitored overtime by luminescence (Nomasense TM O2 Trace Oxygen Analyzer by Nomacorc S.A., Thimister Clermont, Belgium)
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 the SPSS 15.0 software (SPSS Inc., Chicago, IL). Differences were considered significant when the p value was less than 0.05.
Results and Discussion
Figure 1 shows the oxygen consumption rates of the diluted grape must supplemented or not with sulphur dioxide, Metschnikowia pulcherrima strain MP1 or with the combination of the two agents, in the absence or presence of 2 U/mL of laccase.
As expected, in the absence of SO₂, the grape must actively consumed O₂, and total oxygen consumption (TOC) was significantly higher when the medium was supplemented with laccase. By contrast, TOC decreased drastically with the addition of sulphur dioxide, regardless of the presence of laccase. Inoculation with the selected M. pulcherrima MP1 strain significantly increased TOC in the absence of SO₂, confirming the strong oxygen consumption capabilities of this non-Saccharomyces yeast strain. However, in the presence of SO₂, the impact of M. pulcherrima MP1 strain on TOC was less pronounced, likely due to the inhibitory effect of SO₂ on its growth.
Figure 2 shows the absorbance at 420 nm (A420nm) and the CIEL*a*b* blue–yellow component (b*) of the samples supplemented or not with sulphur dioxide, Metschnikowia pulcherrima strain MP1 or the combination of the two, in the absence and presence of laccase as indicators of browning intensity.
The supplementation with sulphur dioxide exerted a clear protective effect against browning since the values of A420 and b* were significantly lower than in the control sample. This confirms the described protective effect of this additive for avoiding enzymatic browning
In the presence of laccase all the samples showed significantly higher values of A420 and b* than their corresponding controls without laccase. This confirms the high oxidative capacity of this polyphenol oxidase. It also indicates that it is more difficult to completely protect against browning in grapes affected by grey rot, even when using sulphur dioxide. The protective effect of M. pulcherrima on wine colour was also observed in final hydroxycinnamic acids concentration that was higher than in the control (data not shown).
The different intensities of the colour of the four samples after the experimental process can be clearly seen in the photographs in Figure 3.
In conditions of healthy grape must, the control sample without anything added to it has undergone a clear browning process. By contrast, the samples supplemented with sulphur dioxide, alone or combined with Metschnikowia pulcherrima strain MP1, are very light in colour, which confirms that this additive protects perfectly against browning. The sample supplemented only with Metschnikowia pulcherrima strain MP1 is slightly browner in colour than the samples protected with sulphur dioxide, but it is much clearer than the control without any additive. However, in the presence of laccase the protective effectiveness of Metschnikowia pulcherrima strain MP1, and even that of sulphur dioxide, were not enough to prevent browning. This confirms that supplementation with Metschnikowia pulcherrima strain MP1 exerts a protective effect against browning in conditions of healthy grape must.
Conclusions
In this work, we analysed how supplementation with sulphur dioxide and/or Metschnikowia pulcherrima strain MP1 influence oxygen consumption kinetics, hydroxycinnamic acids concentrations, and browning intensity in two situations. The first situation was grape must from healthy grapes, and the second was grape must supplemented with the enzyme laccase to reproduce what occurs when the grapes are infected with Botrytis cinerea. The results show that sulphur dioxide drastically reduces the oxygen consumption rate and prevents grape must from browning even in the presence of laccase, although less effectively than in the absence of laccase. By contrast, the supplementation with Metschnikowia pulcherrima strain MP1 drastically increases the oxygen consumption rate, which reduces the oxygen available for polyphenol oxidases; in the absence of laccase, this results in a decrease in browning. However, in the presence of laccase, supplementation with Metschnikowia pulcherrima strain MP1 was not effective enough to prevent browning. It can therefore be concluded that Metschnikowia pulcherrima strain MP1 can be considered as an adequate alternative tool to sulphur dioxide for preventing browning in white grape must in conditions of healthy grapes.
Acknowledgements: This research was funded by the Spanish Ministry of Science and Innovation, Inter-ministerial Commission for Science and Technology (CICYT - projects RTI2018-095658-B-C33 and PID2022-139868OB-C33). This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 945413.
Notes
- 1. Bustamante, M., Giménez, P., Just-Borras, A., Solé-Clua, I., Gombau, J., Heras, J.M., Sieczkowski, N., Gil, M., Canals, J.M., Zamora, F. (2024). Inoculation of a selected strain of Metschnikowia pulcherrima as a bioprotective alternative to sulphites for preventing browning of white grape must. Oeno One, 58, 1-10. https://doi.org/10.20870/oeno-one.2024.58.1.7830
- 2. Bustamante, M., Giménez, P., Just-Borras, A., Solé-Clua, I., Gombau, J., Heras, J.M., Sieczkowski, N., Gil, M., Canals, J.M., Zamora, F. (2024). Inoculation of a selected strain of Metschnikowia pulcherrima as a bioprotective alternative to sulphites for preventing browning of white grape must. Oeno One, 58, 1-10. https://doi.org/10.20870/oeno-one.2024.58.1.7830
- 3. Giménez, P., Just-Borras, A., Pons, P., Gombau, J., Heras, J. M., Sieczkowski, N., Canals, J. M., & Zamora, F. (2023). Biotechnological tools for reducing the use of sulphur dioxide in white grape must and preventing enzymatic browning: glutathione; inactivated dry yeasts rich in glutathione; and bioprotection with Metschnikowia pulcherrima. European Food Research and Technology, 249(6), 1491–1501. https://doi.org/10.1007/s00217-023-04229-6
- 4. Ough, CS., & Crowell, E. A. (1987). Use of sulphur dioxide in winemaking. Journal of Food Science ,52, 386–389. https://doi.org/10.1111/j.1365-2621.1987.tb06620.x
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