Aureobasidium pullulans GM-R-22, an indigenous strain previously isolated from wine grape surface of the viticulture region DO (Denomination of Origin) San Rafael (Mendoza, Argentina), and a producer of cold-active pectinases under low-temperature winemaking conditions ( Merín et al., 2011; Merín & Morata de Ambrosini, 2015; Merín & Morata de Ambrosini, 2018; Merín & Morata de Ambrosini, 2020) was used in this study. This strain is conserved in the Biodiversidad San Rafael (Mendoza) Culture Collection of the Laboratory of Biotechnology (FCAI-UNCuyo) affiliated with the FELACC (Latin American Federation for Culture Collections) as institutional partner SI-66. Saccharomyces cerevisiae IOC 18-2007®, purchased from Institut Œnologique de Champagne (France), was utilised as a fermentation starter culture. The strain GM-R-22 was propagated in YPD (containing in gL ^-1: yeast extract 10, peptone 20, glucose 20, pH 4.0) broth at 25 °C for 48–72 h at least three times prior to experimental use.

Pre-adaptation of microorganisms for fermentation was as follows: Sterilised Malbec grape juice (diluted 1:2 with distilled water and adjusted to pH 3.80) was inoculated at 2 % (v/v) with a pre-culture grown in YPD broth-pH 4.0 of A. pullulans GM-R-22 in exponential phase and incubated at 20 °C for 72 h. Commercial S. cerevisiae strain was inoculated at 200 mgL ^-1 of active dry yeast and prepared according to the supplier’s specifications. All reagents were analytical grade and were purchased from Sigma-Aldrich, Merck and Oxoid.

Malbec red grapes ( Vitis vinifera L.) were obtained from a commercial vineyard located in Rama Caída district (34.66° South latitude and 68.38° West longitude) in DO San Rafael wine region (Mendoza, Argentina). Grapes were immediately crushed and destemmed in the experimental cellar of the Facultad de Ciencias Aplicadas a la Industria (FCAI-UNCuyo). The resulting must in the presence of skins (reducing sugar 257.7 gL ^-1, yeast assimilable nitrogen [YAN] 230 mgL ^-1, titratable acidity 3.82 gL ^-1 of tartaric acid, pH 3.92) was adjusted to 5.5 gL ^-1 tartaric acid and sulphited (50 mgL ^-1 SO ₂).

Vinifications were carried out in biological duplicates in 30 L food-grade plastic tanks containing 20 L of Malbec must per replica. Three different vinification treatments were applied: (i) co-inoculation of A. pullulans and S. cerevisiae in low-temperature fermentation for red winemaking (LTF; 17.5 ± 2.5 °C) constituting the pectinolytic treatment [LTF (Ap + Sc)], (ii) inoculation of S. cerevisiae in low-temperature fermentation for red winemaking (LTF; 17.5 ± 2.5 °C) constituting the control treatment [LTF (Sc)], and (iii) inoculation of S. cerevisiae in traditional fermentation (TF, 25.0 ± 1.5 °C) referred to as the general control [TF (Sc)].

Grape must (skins, seeds, flesh and juice) was inoculated with the pre-adapted cultures of A. pullulans and S. cerevisiae to obtain a cell concentration of around 2 × 10 ⁵ or 2 × 10 ⁶ CFU/mL, respectively. Vinifications were conducted at a controlled temperature using cold (20 °C) or room (25 °C) chambers. Progress of the alcoholic fermentation was monitored by daily measurements of temperature and mass density (gmL ^-1) at 20 °C using the gravimetric method ( Iland et al., 2000). The end of alcoholic fermentation was considered when at least a mass density of 0.995 gmL ^-1 was reached and the reducing sugar was depleted to less than 2.0 ± 0.1 gL ^-1. During the period of skin contact, cap management consisted of two daily punch downs (morning and afternoon, 1 min each). After the alcoholic fermentation had finished, the wines were racked twice, physically stabilised (4 °C) for 20 days, bottled (750-mL glass bottles) without filtration and stored at 12 °C in darkness until analysis.

Viable yeast counts were carried out by the serial dilutions method on YPD agar for total yeasts and on Lysine agar (Oxoid) for A. pullulans GM-R-22, which is unable to support S. cerevisiae growth, and where the pectinolytic strain is clearly differentiated from other microorganisms by its colony morphology. The count can be ascribed to the A. pullulans GM-R-22 strain because this species naturally occurs in sound grapes at about 10 ¹–10 ⁴ CFUmL ^-1 ( Barata et al., 2012), which counts much lower than the cell concentration inoculated in this study. The plates were incubated at 28 °C for 3–5 days.

Pectinase activity was evaluated during fermentation on the must-wine centrifuged at 10,000 g (15 min, 4 °C), called enzymatic extract, by measuring the amount of reducing groups released from a pectin dispersion (0.25 % citrus pectin in 50 mM citric-citrate buffer, pH 3.8) using 3,5-dinitrosalicylic acid (DNS) reagent ( Miller, 1959). The reaction mixture (enzymatic extract/substrate ratio: 1/10, v/v) was incubated for 15 min at the corresponding enzyme-production temperature (20 or 25 °C) in each vinification technique, according to the method proposed by Merín & Morata de Ambrosini (2015). One pectinase unit (U) is defined as the enzymatic activity that releases 1 µmol of reducing groups per minute under assay conditions. Determinations were performed in triplicate.

Ethanol (alcohol content, % v/v), residual sugars (gL ^-1), total and volatile acidity (gL ^-1 tartaric acid and gL ^-1 acetic acid, respectively), pH, density (20 °C/20 °C), dry extract (gL ^-1) and glycerol (gL ^-1) of wines were determined at bottling using an ALPHA FT-IR Wine Analyser (Bruker Optik Gmbh, Ettlingen, Germany).

The effect of the pectinolytic treatment on the clarification was evaluated through the filterability of wines at bottling. Filterability was determined by measuring the time required for 25 mL of the final product to pass through a 0.45 µm filter according to the method proposed by Fernández-González et al. (2005).

Chromatic and polyphenolic parameters of wines were determined during the fermentation process, at bottling and throughout bottle storage (12 months of ageing). Samples were centrifuged (10,000 g, 5 min at 4 °C) before analyses and absorbance (A) was measured with 1-mm path-length glass cells for classical colour indices and CIELAB ( Comisión Internacional de L’Eclairage) parameters, as well as with 1-cm path-length cells for total phenols.

Classical colour indices colour intensity (CI) and tonality (T) were calculated according to Glories (1984). The CIELAB parameters, L* (lightness), a* (red colour intensity) and b* (yellow colour intensity) were obtained using MSCV® software (Grupo de Colour, Universidad de La Rioja, Spain). The CIELAB colour difference (Δ E*) was calculated by using the following equation: Δ E* = [(Δ L*) ²+(Δ a*) ²+(Δ b*) ²] ^1/2, where the difference (Δ) is calculated for each independent variable between the enzymatically treated wine and control wines.

Total anthocyanins (TA) (mgL ^-1) were determined based on the method of Puissant-León, which consists of measuring A at 520 nm after incubation of a wine sample for 30 min in 0.1 N HCl (1:40 ratio, v/v), according to Merín and Morata de Ambrosini (2015).

Monomeric (MA), copigmented (CA) and polymeric (PA) anthocyanins were determined using the colorimetric effects of SO ₂ and acetaldehyde on anthocyanins according to the method proposed by Levengood and Boulton (2004). Absorbance (A) at 520 nm was measured with 1-mm path-length glass cells after adjusting the pH to 3.6 and A _acet, A _SO2 and A ₂₀ were assessed. Colour caused by monomeric anthocyanins (MA = A ₂₀ – A _SO2), copigmented anthocyanins (CA = A _acet – A ₂₀) and polymeric anthocyanins (PA = A _SO2) was determined for each sample and expressed as absorbance units at 520 nm referred to 1-cm of optical path-length.

The total polyphenolic index (TPI) was determined according to Glories (1984), after measuring the absorbance at 280 nm of a 100 times diluted wine sample using deionised water. Additionally, total polyphenols content (TPC) was determined according to the classic Folin–Ciocalteu (FC) method ( Chen et al., 2015) and expressed in mg gallic acid equivalents (GAE) per L of sample, at 12 months of bottle storage.

HPLC analysis of anthocyanin compounds of wines was carried out at 12 months of bottle storage on a Shimadzu (Kyoto, Japan) LC10 HPLC chromatograph, equipped with a SPD-M10Avp UV/Vis Photodiode Array Detector, fitted with Shimadzu software, and a LiChrospher 100 RP-18 reversed-phase column (4.6 mm × 250 mm, 5 μm particle size) (Merck, Darmstadt, Germany) equipped with a precolumn (RP-18; 2 mm × 20 mm, 30 to 40 μm particle size) according to the protocol and procedure reported by Cabeza et al. (2009) and the quantification was performed according to Merín and Morata de Ambrosini (2020).

Antioxidant activity (AA) was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH ^●) radical scavenging method, according to the modified technique by Brand-Williams et al. (1995) at 12 months of bottle storage. The reaction mixture was prepared with 100 µL of sample (1:10 dilution) and 2.9 mL of DPPH ^● solution (0.03 mgmL ^-1), the reaction time was set at 25 min and spectrophotometric readings were performed at 515 nm. All determinations were carried out at room temperature, and the results are expressed as antioxidant capacity in mg of GAE per L of the sample.

Table 1 shows the physicochemical composition of Malbec wines obtained with the different cultures and vinification techniques at bottling. Sugars in the three fermentations were completely consumed, achieving must dryness, although a slight difference was observed between the two LTF wines, and standard ethanol concentrations (around 14.5 %, v/v) were obtained in the three wines. The wines exhibited the oenological characteristics of most regular wines, and the A. pullulans strain had no effect on density, ethanol, total acidity, pH and glycerol with respect to its control, showing the same behaviour as that in different red winemaking techniques at laboratory scale ( Merín & Morata de Ambrosini, 2018; Merín & Morata de Ambrosini, 2020). Similar Malbec wine compositions have been observed previously with other non- Saccharomyces yeasts in mixed fermentations ( Mendoza et al., 2011; Fanzone et al., 2020; Longhi et al., 2022a). A particularly interesting result was the reduction of volatile acidity, mainly due to acetic acid, in the wine made with A. pullulans compared to both controls, although not statistically significant for LTF (Sc) wine. In our previous works, a similar reduction was observed in laboratory-scale winemaking ( Merín & Morata de Ambrosini, 2018; Merín & Morata de Ambrosini, 2020). We can hypothesise that A. pullulans can metabolise acetic acid in the presence of glucose, as previously shown for some wine strains of Zygosaccharomyces bailii, S. cerevisiae ( Vilela-Moura et al., 2011) and Saccharomyces uvarum, which metabolise acetic acid faster than their production during fermentation, resulting in low acetic acid concentration ( Kelly et al., 2020).

Table 1 also presents the chromatic and phenolic indices of Malbec wines at bottling. The CI, TA and TPI were significantly higher in wine inoculated with the pectinolytic strain, LTF (Ap + Sc), than in its control, LTF (Sc), whereas no statistical differences were found for these parameters between LTF (Ap + Sc) and TF (Sc) wines (p > 0.05, LSD test). For further comprehension of the colour composition of wines, the CIELAB parameters were also examined (Table 1). L* and b* exhibited a significant decrease, whereas a* was significantly increased in LTF (Ap + Sc) wine with respect to LTF (Sc) wine. The L* value is the vertical axis and defines the lightness, the property according to which each colour can be considered equivalent to a member of the greyscale, between black and white, taking values within the range of 0−100, respectively ( Gordillo et al., 2014). This interpretation can be seen in Figure 3, which shows the position of the three wines in the colour plane ( a*b*) of CIELAB space (Figure 3A) and L* (lightness) values in the vertical axis (Figure 3B). Therefore, the LTF (Ap + Sc) wine was more vivid and displayed higher intensity due to an increase in a* and a decrease in L*, and had less contribution to yellow tonalities, indicated by lower values of b*, with respect to LTF (Sc) wine. These results can be explained by the higher levels of phenolic compounds, especially anthocyanins that are responsible mainly for red pigments, detected in LTF (Ap + Sc) wine at the end of fermentation. It has been reported previously that Malbec wines elaborated at low temperatures with A. pullulans enzyme-treatments improved their phenolic and chromatic parameters TPI, CI, L*, a* and b* ( Longhi et al., 2022a; Longhi et al., 2022b; Merín & Morata de Ambrosini, 2018; Merín & Morata de Ambrosini, 2020). Likewise, these results are in accordance with those reported for other pectinolytic non- Saccharomyces yeast co-inoculated with S. cerevisiae to improve the quality of wines, such as Metschnikowia pulcherrima ( Belda et al., 2016), Metschnikowia fructicola ( Benucci et al., 2018), Kluyveromyces marxianus ( Rollero et al., 2018) and Torulaspora delbrueckii ( Longhi et al., 2022a).

Figure 3. CIELAB parameters of Malbec wines LTF (Sc) (♦), LTF (Ap + Sc) (▲) and TF (Sc) (●) wines at bottling. (A) a*b* diagram. (B) Lightness ( L*).

Moreover, the CIELAB colour difference ΔE* between LTF (Ap + Sc) and LTF (Sc) wines and LTF (Ap + Sc) and TF (Sc) wines were 6.27 and 7.80 units, respectively (Table 1). These values are visibly detectable because they are higher than the estimation of 2.7 CIELAB units which is the threshold for the human eye to distinguish between the colour of wines when trained tasters use standardised wine-tasting glasses ( Martínez et al., 2001).

The filterability of wines at bottling is shown in Table 1. Wine inoculated with A. pullulans evidenced a significant reduction of 24.6 % and 47.1 % regarding LTF and TF control wines, respectively. These results are in agreement with previous outcomes demonstrating reductions of 30–40 % in filtration times of red wines elaborated with A. pullulans GM-R-22 ( Merín & Morata de Ambrosini, 2018; Merín & Morata de Ambrosini, 2020), suggesting that the inoculation of this pectinolytic strain can improve clarification and filtration processes, giving a cleaner appearance to the wine. This filterability improvement is likely to occur due to the production of endo-polygalacturonase enzymes by the GM-R-22 strain, as suggested previously ( Merín & Morata de Ambrosini, 2018), which markedly decreases the grape pectin viscosity. The application of pectinolytic yeasts during the vinification process has been previously reported to significantly reduce the filtration time of the attained wines ( Fernández-González et al., 2005; Belda et al., 2016).

The development of the main polyphenolic and chromatic characteristics of Malbec wines in the presence or absence of A. pullulans from crushing to the end of fermentation is shown in Figure 4. The wines elaborated at low temperatures in the presence of the pectinolytic strain showed positive statistical differences with respect to control wine in CI, TA and TPI from the fourth day up to the end of fermentation (Figures 4A–C). These indices were significantly higher in TF (Sc) wine than in LTF wines, especially during the first week of fermentation, but the indices in LTF (Ap + Sc) wine reached statistically equal levels at the end of the fermentation process. The CIELAB parameters displayed a similar behaviour throughout fermentation, showing improved values in LTF (Ap + Sc) wine with respect to the control for L*, a* and b* at the end of the process (Figures 4D to 4F). Previously, we observed the same results for Malbec grapes fermented at low temperatures (18–20 °C) in co-culture with A. pullulans and S. cerevisiae ( Merín & Morata de Ambrosini, 2020), validating the present outcomes at the pilot scale.

Figure 4. Development of CI (A), TA (B), TPI (C) and the CIELAB parameters L* (D), a* (E) and b* (F) of Malbec wines elaborated with LTF technique in the absence (♦) or presence (▲) of A. pullulans GM-R-22, respectively, and TF technique in the absence of A. pullulans GM-R-22 (●, general control) during fermentation. Values represent the mean of two independent vinifications. Linear vertical bars represent standard deviation.

Figure 5 shows the colour and polyphenols evolution during 12 months of bottle storage in terms of CI, TA and TPI in the A. pullulans-treated wine and both control wines. After bottling, CI decreased up to the 6 ^th month maintaining the differences between wines reached at bottling (Figure 5A). From this point onwards, CI remained stable in LTF (Ap + Sc) and LTF (Sc) wines being statistically higher in the former, whereas this index showed a rising trend towards the 12 months of storage period in TF (Sc) wine. TA concentration showed a marked decrease during the first 6 months of bottle storage, especially in LTF (Sc) wine showing a significantly lower concentration (266.75 ± 3.86 mgL ^-1) than the values of the other wines, 297.70 ± 14.57 mgL ^-1 in LTF (Ap + Sc) wine and 295.88 ± 7.28 mgL ^-1 in TF (Sc) wine, at 12 months of storage (Figure 5B). Anthocyanins are the major grape pigments in red and black grapes and the compounds responsible for colour in red wine ( Echave et al., 2021). During storage, a reduction in TA concentration is expected due to oxidation reactions or pigment formation from anthocyanins such as pyranoanthocyanins and polymeric anthocyanins ( Martínez-Moreno et al., 2023). Regarding the pectinolytic treatment, it seems that pectinases contribute to the extraction of anthocyanins and other phenols from grape skin, which was related to the increment of CI. These results are in contrast to the previous ones obtained in low-temperature red winemaking in the presence of A. pullulans at the laboratory scale, in which there were no significant differences in TA and CI between this wine and its control at 9 months of bottle storage ( Merín & Morata de Ambrosini, 2020). In the present study, the pectinolytic treatment compensated for the lower total anthocyanin extraction and colour intensity obtained at lower fermentation temperature, approaching or reaching the levels of these parameters in the wine obtained with traditional red vinification.

Figure 5. Evolution of CI (A), TA (B) and TPI (C) of Malbec wines elaborated with LTF technique in the absence (♦) or presence (▲) of A. pullulans GM-R-22, respectively, and TF technique in the absence of A. pullulans GM-R-22 (●, general control) during 12 months of bottle storage. Values represent the mean of two independent vinifications. Linear vertical bars represent standard deviation. The arrow indicates the bottling time.

Regarding TPI, the significant difference observed at bottling in LTF (Ap + Sc) wine with respect to its control was reduced at the 6 ^th month, but this difference was significantly higher at 12 months of storage (Figure 5C). TPI in TF (Sc) wine was the highest during the ageing period studied. Higher temperatures generally lead to increased phenolic extraction due to the increased permeability of the hypodermal cells and solubility of certain phenolics ( Sacchi et al., 2005). The higher TPI values of wines obtained at low temperatures in the presence of A. pullulans were likely to be achieved because pectinolytic and related polysaccharidase enzymes favour tissue degradation and dissolution of grape cell wall contents, including anthocyanins and other phenolic compounds such as flavan-3-ols. Therefore, the pectinolytic treatment at least partially counterweighted the lower phenolic extraction obtained at the lower fermentation temperature. These outcomes are similar to own previous results obtained at the laboratory scale in terms of relative values and differences between wines ( Merín & Morata de Ambrosini, 2020); nevertheless, the absolute TPI values on the larger scale assessed in this study were significantly higher. In contrast, Martín and Morata de Ambrosini (2014) and González-Neves et al. (2010) found in laboratory trials that pectinases produced a high extraction of certain phenolic compounds, especially anthocyanins, whereas the extraction was not sufficient to have a significant effect on TPI. It seems that small volumes of must subjected to small-scale winemaking increased the fixation of polyphenols on grape skins and seeds ( González-Neves et al., 2010).

Figure 6 exhibits the evolution of monomeric (MA), copigmented (CA) and polymeric (PA) anthocyanins in the three wines during fermentation and 12 months of storage. At bottling, MA was significantly higher in LTF (Ap + Sc) wine than in both controls (Figure 6A). These outcomes put in evidence the effect of pectinases and related polysaccharidase enzymes on the extraction of free anthocyanins from skin cells by degrading the cell wall components during winemaking, thus reaching the highest levels in LTF (Ap + Sc) wine at the end of fermentation. During the bottle ageing period, MA declined constantly in the three wines maintaining the significant differences between the A. pullulans-treated wine and its control, and resulting statistically equal to the level of the traditionally fermented wine. Monomeric anthocyanins are unstable and, during wine maturation and ageing, their concentration in red wines decreases continuously due to different mechanisms, such as their adsorption by yeast, their degradation and oxidation, their precipitation with proteins, polysaccharides or condensed tannins, and the progressive and irreversible formation of more complex and stable anthocyanin derived pigments ( Martínez-Moreno et al., 2023).

CA in LTF (Ap + Sc) wine was the highest at bottling, but it displayed a slight drop in the 6 ^th month of storage from which it started to increase up to the 12 ^th month, exhibiting the highest level, statistically the same as that of the TF (Sc) wine and greater than that of the LTF (Sc) wine (Figure 6B). These results showed that CA slightly declined during the storage period studied. The colour of young red wines is stabilised due to the interaction of anthocyanins with other colourless wine polyphenols through non-covalent copigmentation reactions ( Escribano-Bailón et al., 2019). Consequently, the pectinolytic treatment positively impacted the concentration of copigmented anthocyanins, which may be considered as a storage form of anthocyanins to form future polymeric pigments.

Figure 6. Evolution of monomeric anthocyanins (A), copigmented anthocyanins (B) and polymeric anthocyanins (C) of Malbec wines elaborated with LTF technique in the absence (♦) or presence (▲) of A. pullulans GM-R-22, respectively, and TF technique in the absence of A. pullulans GM-R-22 (●, general control) during 12 months of bottle storage. Values represent the mean of two independent vinifications. Linear vertical bars represent standard deviation. The arrow indicates the bottling time.

PA showed a similar level in the three wines at bottling (Figure 6C), from which LTF (Ap + Sc) wine exhibited a continuous increase along the ageing period, reaching a statistically equal level to that of TF (Sc) wine, and a significantly higher value than that of LTF (Sc) wine at the end of storage. Ageing transforms the wine colour from dark to bright red by alterations and polymerisations of anthocyanins evolving towards more stable structures ( Echave et al., 2021). Polymerisation results in the chromophore of the anthocyanin being protected from water and nucleophilic attack, oxidation or other chemical modifications, such as pH changes in the wine and the bleaching of sulphur dioxide ( Echave et al., 2021). As the red wines aged, monomeric anthocyanin concentrations shifted into polymeric stable pigment compounds during the 12 months in the bottle. However, the pectinolytic treatment enhanced MA, CA and PA in the wine elaborated at low temperatures, equalling the parameters of traditionally fermented wine.

The global phenolic and colour parameters in Malbec wines at 12 months of bottle storage showed that, in general, the differences between wines were maintained throughout the storage time (Table 2). Significant differences were observed for TPI, in concordance with TPC, TA and ΔE* between LTF (Ap + Sc) wine and LTF (Sc) wine after ageing. Regarding antioxidant activity (AA), there were no significant differences between wines studied at 12 months of bottle storage. These results suggest that the difference in TPI and TPC could be attributed more to anthocyanins than to other polyphenolic compounds commonly associated with antioxidant capacity, such as stilbenes ( Longhi et al., 2022a).

The most important anthocyanin compounds identified in wine samples by HPLC-DAD at 12 months of bottle storage are summarised in Table 3. They were grouped into non-acylated glucosides (5), acetyl-glucosides (2), and coumaroyl-glucosides (2). In all the wines analysed, malvidin-3-O-glucoside, its derivatives and petunidin-3-O-glucoside were the predominant anthocyanins with concentrations of malvidin-3-O-glucoside around 75 %. LTF (Ap + Sc) wine showed the maximum level of the sum of anthocyanins and a significant increase in most of the anthocyanins identified (7 out of 9) with respect to the control wines. Among them, all the 3-O-glucosides, except for cyanidin-3-O-glucoside, were higher in LTF (Ap + Sc) wine than in LTF (Sc) wine, with increments varying from 11 to 33 %, and acetylated and p-coumaroylated derivatives of peonidin-3-O-glucoside were larger with increments of 19 and 44 %, respectively, and showed even higher increments with respect to TF (Sc) wine. It seems that the enzymatic treatment extracted more anthocyanins, especially peonidin-3-O-glucoside and its derivatives, similar to previous results obtained with this strain ( Merín & Morata de Ambrosini, 2020). The effect of pectinolytic treatment on anthocyanins extraction from grape skin is in accordance with earlier studies where pectinases have been applied ( Cabeza et al., 2009; Martín & Morata de Ambrosini, 2013; Osete-Alcaraz et al., 2022).

It can be seen that a higher content of anthocyanins in LTF (Ap + Sc) wine than in LTF (Sc) wine, determined by HPLC (Table 3), was correlated with significantly higher levels of TA and MA (see Figure 5 and Figure 6). However, the significant difference observed in the sum of anthocyanins determined by HPLC compared to TF (Sc) wine did not correlate with the TA or MA measured spectrophotometrically. This behaviour is in agreement with that observed in the study by Fanzone et al. (2020), which could be explained by the fact that HPLC-DAD analysis detects only free anthocyanins, whereas spectrophotometric analysis overestimates their total amount, including other red pigments ( Canals et al., 2008). Curiously, the lack of correlation between TA and MA with anthocyanins by HPLC-DAD was observed in TF (Sc) wine, but not in LTF wines. Probably, the overestimation of anthocyanins could be greater in the wine obtained with traditional red winemaking, because at higher fermentation temperatures the red pigments involved in this measurement are higher than those extracted at lower temperatures.