## 2 Materials and Methods
### Analytical Determinations  
#### 2.3.1 UV-Vis Spectrophotometric Determinations  
Spectrophotometric determinations (total polyphenols--TPFs, total catechins--TCs, total tannins--TTs, total anthocyanins--TAs, and antioxidant activity--AA) of the musts and wines were conducted using an Analytik Jena Specord 205 UV/VIS spectrophotometer (Analytik Jena GmbH, Jena, Germany) equipped with 1 cm path length glass and quartz cells. Total polyphenols (TPFs) were determined via the colorimetric Folin-Ciocalteu method, using gallic acid as a reference standard [33; 34], measuring the maximum absorbance at 760 nm. In a 100 mL volumetric flask, the following were inserted in order, wine sample (1 mL of white wine and 1 mL red wine diluted 1/5, respectively), 50 mL of distilled water, 5 mL of Folin-Ciocalteu reagent, and 20 mL of sodium carbonate 20% (\(m/v\)) solution, and these were brought to the mark with distilled water. We then stirred in order to achieve homogenization. After 30 min, the absorbance was measured at 760 nm against a blank prepared with distilled water instead of wine. TPF quantification was based on the standard curve obtained with a serial dilution of a gallic acid standard solution in the range of 50-1000 mg/L of gallic acid (r\({}^{2}\) value of the standard curve: 0.9900). The values were expressed as mg gallic acid equivalents (GAE)/L of must or wine. Catechins (TCs) are monomer flavourolic units (proanthocyanidins), and total catechins are determined via a proanthocyanin-specific test using 1% alcoholic vanillin solution; when a reaction occurs, that leads to the appearance of the red color, which then becomes stable in concentrated solutions of HCl [35]. Briefly, 10 mL of wine or must were combined with 10 mL of 11.5 N HCl and 5 mL of 1% vanillin solution, and after 20 min, the absorbances were measured at 500 nm against ultrapure water. A calibration curve using standard catechin solutions covering the range of 0.02-0.1 mg/L was used to express the quantitative values in mg/L (r\({}^{2}\) value of the standard curve: 0.9846). Tannins (TTs) were determined based on their property to change into cyanidin in a strongly acidic environment combined with a high temperature [35]. In two tubes, 2 mL of wine/must was combined with 4 mL of a 27.75% HCl solution, and then one of the tubes was boiled for 45 min while the other was kept at 20 \({}^{\circ}\)C. After cooling the tube, the color was stabilized with 0.5 mL of concentrated H\({}_{2}\)SO\({}_{4}\), and then the optical density of the boiled and unboiled solutions was measured at 520 nm. The results were calculated using the following formula:  
\[\text{TT (mg/L)}=((15.7\times\Delta\text{OD520)}\times\text{V} \tag{1}\]  
where the reading of \(\Delta\text{DO520 nm}\) (the difference between the boiled and the unboiled sample) was at wavelength 520, and V denotes the volume of must or wine. Anthocyanins (TAs) determination is based on their property to react with SO\({}_{2}\), forming colorless products. We prepared the mixture using the following instructions: add 1 mL of filtered wine or must, 1 mL of HCl 0.1%, and 20 mL of HCl 2%. From this mixture, two solutions were prepared which were put into two tubes with the following composition: solution 1 (5 mL of mixture and 2 mL of distilled water) and solution 2 (5 mL of mixture and 2 mL of solution of sodium metabisulphite 16%). After 20 min, the optical densities of solutions 1 and 2 were measured against distilled water at a wavelength of 520 nm in 1 cm path length glass cells [36]. The quantitative results were calculated using the following formula:  
\[\text{TA (mg/L)}=\Delta\text{DO520}\times 875 \tag{2}\]  
where \(\Delta\text{DO520}\) is the difference in the two solutions at wavelength DO520. DPPH Assay (AA). The antioxidant activity was determined using the method reported by [PERSON] et al. [37] with some modifications. An aliquot of 0.1 mL of must/wine was mixed with 3.9 mL of DPPH\(\bullet\) methanolic solution (2.5 \(\times\) 10\({}^{-2}\) mg/L methanolic DPPH solution).