AIM: The aim of this work was to study the inhibitory effect of the three most frequently used wine antioxidants – sulfur dioxide, ascorbic acid and glutathione – on the kinetics of browning caused by Botrytis cinerea laccase using a grape juice synthetic model in which (-)-epicatechin was the substrate. METHODS: A grape juice model solution containing 100 g/L of D-glucose, 100 g/L of D-fructose and 4 g/l of tartaric acid adjusted to pH 3.5 was used for all the browning assays. (-)-Epicatechin was used as substrate at concentrations between 0 and 0.8 mM. This media was supplemented with sulfur dioxide at concentrations of 0, 10, 20 and 30 mg/L, ascorbic acid concentrations of 0, 50 and 100 mg/L, or reduced glutathione concentrations of 0, 20, 50 and 100 mg/L in order to determine how these antioxidants inhibit browning caused by laccase. Laccase from Botrytis cinerea was purified according to Vignault et al., (2019)1. Browning reaction was started by adding 2 units of laccase activity/ml and absorbance at 420 nm was measured at time 0, 15, 30 and 45 minutes. The slope of the regression straight line was determined in order to express the intensity of browning. The Michaëlis-Menten and Hill plots were depicted for each substrate in order to determine the kinetic parameters of browning: Vmax, K0.5 and Hill number. All the experiments were performed in triplicate. RESULTS: All the three studied wine antioxidants, sulfur dioxide, ascorbic acid and glutathione reduced the laccase browning Vmax. Sulfur dioxide and glutathione also increased the K0.5, which indicates that sulfur dioxide not only decreases the Vmax of laccase browning but also the affinity for its substrate. In contrast, ascorbic acid did not change the values of K0.5, probably because it acts to reduce the availability of one of the laccase substrates (oxygen) and does not act directly on the enzyme. CONCLUSIONS: These results confirm that sulfur dioxide, ascorbic acid and glutathione really are effective in protecting grape juice against laccase browning. The effectiveness of ascorbic acid and glutathione also confirm that both antioxidants could be useful tools to reduce the doses of sulfur dioxide in winemaking, especially when grey rot is present. However, in the case of glutathione the dose needed to effectively protect grape juice against laccase browning is higher than the current maximum dose established by the OIV2. Given the safety of this compound, it would be advisable to increase its maximum dosage. FUNDING: This work was funded by CICYT (Efecto de las lacasas sobre la sensorialidad, calidad y salubridad de los vinos – project RTI2018-095658-B-C33). ACKNOWLEDGMENTS: Authors thank professors Marc Fermaud and Jean Roudet from INRAE, UMR SAVE, Bordeaux Science Agro, ISVV, France for having provided us with the B. cinerea strain.
Authors: Joan Miquel Canals – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain.,Pol Giménez – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain. Sergi Anguela – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain. Arnau Just-Borras – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain. Pere Pons-Mercadé – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain. Jordi Gombau – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain. Adeline Vignault – Unité de Recherche Oenologie, EA 4577, USC 1366 INRAE, ISVV, Université de Bordeaux, F33882 Villenave d’Ornon, France. – Laffort, 11 rue Aristide Bergès, 33270 Floirac, France Joan Miquel Canals – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona, Spain. Pierre-Louis Teissedre – Unité de Recherche Oenologie, EA 4577, USC 1366 INRAE, ISVV, Université de Bordeaux, F33882 Villenave d’Ornon, France. Fernando Zamora – Departament de Bioquímica i Biotecnologia, Facultat d’Enologia de Tarragona, Universitat Rovira i Virgili, C/Marcel.li Domingo, 1. 43007 Tarragona
Email: jmcanals@urv.cat
Keywords: laccase, botrytis cinerea, browning, inhibition, sulfur dioxide, ascornic acid, glutathione
