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Ciência e Técnica Vitivinícola

versão impressa ISSN 0254-0223

Ciência Téc. Vitiv. vol.27 no.2 Dois Portos dez. 2012


Phenolic composition of Bobal red wines elaborated with prefermentative cold maceration

Composição fenólica dos vinhos tintos da casta Bobal feitos com maceração pré-fermentativa em frio


J.L. Aleixandre*, N. Sánchez, J.L. Aleixandre-Tudó, V. Lizama, M.J. García and I. Alvarez

Departamento de Tecnología de Alimentos, Universidad Politécnica de Valencia, Camino de Vera, s/n, 46022 Valencia, Spain.

*Corresponding author:



The technique called pre-fermentative cold maceration is used to enhance the anthocyanins diffusion from the skins to the must, increasing the pigments extraction. In this study, the influence of the pre-fermentative cold maceration treatments (3 and 5 days) and the time of maceration (7 or 14 days) on color characteristics and phenolic composition of Bobal red wines made from grapes harvested at different phenolic maturity was studied. The results showed the higher the phenolic maturity the higher polymerized tannins, ethanol index and the lower astringency. The prefermentative cold maceration enhanced berry compounds extractability, but led to wines with less polymerized anthocyanins and tannins. Moreover wines with higher tannin concentration, total polyphenols and polymerization degrees were produced with longer maceration times. On the contrary a decrease in colour and anthocyanin concentration was observed. Regarding sensory analysis, it has been highlighted the importance of grape maturity and pre-fermentative cold maceration length on the wine quality.

Key words: grape maturity, cold maceration, phenolic composition, Bobal red wines



A técnica de vinificação chamada de maceração pré-fermentativa em frio é utilizada para aumentar a difusão de antocianinas das peliculas da uva para o mosto, aumentando por sua vez a extração de pigmentos. Neste trabalho pretendeu-se estudar a influência da maceração pré-fermentativa em frio (3 e 5 dias) e o tempo de maceração (7 ou 14 dias) sobre as características da cor e da composição fenólica dos vinhos tintos de casta Bobal feitos a partir de uvas colhidas em maturação fenólica diferente. Os resultados mostraram que quanto maior a maturação fenólica, maior a quantidade de taninos polimerizados, maior índice de etanol e menor adstringência. Esta técnica permitiu aumentar consideravelmente o extração dos compostos fenólicos da uva; contudo pode levar a que haja menor grau de antocianinas e taninos polimerizados nos vinhos. Além disso os vinhos com maior concentração de taninos, polifenóistotais e graus de polimerização foram produzidos com maior tempo de maceração. Por outro lado foi observada uma diminuição da cor e da concentração de antocianina. Em relação à análise sensorial, destaca-se a importância da maturação da uva e da duração da maceração pré-fermentativa em frio na qualidade do vinho.

Palavras-chave: maturação da uva, maceração em frio, composição fenólica, vinhos tintos da casta Bobal



The grape maturation is a complex process which involves biosynthesis, transport, storage and transformations of different components. Compounds such as sugars and organic acids or phenols and flavor precursors are involved in grape quality. Therefore it is important to understand the grape development during ripening in order to define the date of harvest (Diaz-Plaza et al., 2000; Rodriguez-Vila, 2000; Souquetet al., 2000; Brenon et al., 2005; Ribereau-Gayon et al., 2006).

The prefermentative maceration is defined as the period of time from the filling into tanks with the crushed grapes to the beginning of the alcoholic fermentation. This technique, also known as cold soaking or cryomaceration, is being increasingly used by enologists worldwide in order to improve some important quality characteristics of wines, such us color and aroma (Blouin et al., 2000; Gómez-Plaza et al., 2001; Parenti et al., 2004; Koyama et al., 2007; Gómez-Miguez et al., 2007; Gil-Muñoz et al., 2009). During the prefermentative period the extraction of polyphenols from the skins to the must take place in the absence of ethanol (Feuillat, 1996). The cold needed to achieve the low temperature can be obtained in diverse manners, being probably the most widely the addition of dry ice (solid carbon dioxide) (Heredia et al., 2010).

Traditionally made in Burgundy due to the low harvest temperatures, this technique has a considerable effect on the wine quality, enhancing the aromatic fraction of Pinot noir wines. Retali (2004) noted that prefermetative cold maceration in Nielluccio grapes led to wines with greater aromatic expression and quality, where the anthocyanins are mainly extracted at this step. However when the alcohol content increases a slightly decrease by degradation, absorption or structure modification was observed.

It is well known that this technique was designed to improve the extraction of pigments, tannins and aromas from grape skins to the wine. The underlying idea is that aqueous extraction should improve wine color although previous studies have provided contradictory results. The results obtained in several studies have shown that a cold soak alone has either no effect or a negative effect on the phenolic composition (Watson et al., 1995; Feuillat, 1996; Heatherbell et al., 1997; Okubo et al., 2003; Marais, 2003 a,b). Moreover several authors note the positive influence if this technique on the final composition and sensory quality (Parenti et al., 2004; Álvarez et al., 2006; Gómez-Miguez et al., 2007; Koyama et al., 2007; Gil-Miñoz et al., 2009; Gordillo et al., 2010; Heredia et al., 2010).

One of the main features in the red wine making process is the presence of skins and seeds. Red winemaking is thus a very complex process where an overlapping between alcoholic fermentation and maceration take place. This phenomenon, which is responsible for wine color, is not simply an extraction procedure, as numerous reactions in the grape phenolics also occur that deeply influence the color intensity and stability of the wine (Sims and Bates, 1994). The length of skin maceration is the first factor that affects this phenomenon, although maceration affects the extraction not only of phenolic compounds but also of other compounds (proteins, polysaccharides) that may participate in condensation reactions (Glories, 2001; Gómez-Plaza et al., 2001; Gónzalez-Neves et al., 2003).

In recent years, some winemaking techniques have also being tested in order to elaborate red wines (Sacchi et al., 2005). However the knowledge of the influence of these techniques on the final Bobal red wines composition and sensory quality has not been well studied yet, so only a few data can be found in the literature. For instance Gómez Gallego et al. (2012a) stated that wines made with Moravia agria or Bobal, two red single cultivars in danger of extinction in Castilla-La Mancha, are a viable alternative to traditional grape varieties, increasing the offer to the consumer, when the phenolic composition, chromatic characteristics, antioxidant activities and sensory profile were tested. Gómez García-Carpintero et al. (2011) also demonstrate that Bobal wines present a complex chemical profile with a wealth of aromas in its aromatic composition. Gómez-Gallego et al. (2012b) studied the phenolic composition and sensory properties of five minor red grape varieties from Castilla-La Mancha region (Bobal, Moravia agria, Moravia dulce, Rojal y Tortosí). The study suggested that, in order to see how the compounds studied in these grapes are transferred to the elaborated wines and affects their sensory properties; future work verifying the effect of the wine making process will be needed.

The Bobal grape variety was selected, on the one hand, because it is the most widely used in the Designation of Origin Utiel-Requena (Valencia, Spain) with more than 70% of the total grapevine area of this region and, on the other hand, because despite being used traditionally in the production of rosé and red “doble pasta” wines and being sold mostly in bulk during the last few years Bobal grapes have been used to produce high quality aged wines shown the high potential of the grapes (Méndez, 2005; Sánchez, 2008; Gómez-García Carpintero et al., 2011). The main objective of this study was to assess the prefermentative cold maceration influence on the color and phenolic composition of Bobal red wines. In this sense the definition of chemical and phenolic characteristics of the wines produced by two maceration times and at different grape maturity levels has been studied. The knowledge of the most important phenolic parameters may profit from the selection of the best winemaking procedure and the optimum date of harvest in order to increase wine quality.



Grapes of Vitis Vinifera cv. Bobal were harvested in 2008 from 40 years old vines at the Coloraos experimental vineyard located in Requena (Valencia, Spain) at two different maturity levels, spaced one week. Grapes were carefully harvested into 15 kg boxes and transported to an experimental wine production center.

The grapes were destemmed and crushed, and the must was homogenized and distributed to 50 L stainless steel tanks. Sulfur dioxide was added (5 g/hL) prior to carry out the different vinification methods. Alcoholic fermentation was induced by inoculation with Saccharomyces cerevisiae (2056 Uvaferm Rhone) at the rate of 20 g/hL. To ensure the development of malolactic fermentation selected Oenococcus oeni lactic acid bacteria (Starin 31 ITV France Lallemand) were inoculated at the end of alcoholic fermentation.

Cold prefermentative maceration process consisted of two stages: a first stage where the refrigeration of the grapes takes place in a cold storage-room at 10 ºC for 3 or 5 days, followed by 7 or 14 days of traditional maceration-fermentation (between 20-25 ºC) - Figure 1. After the cold maceration period was completed the temperature of the tanks was rapidly brought to 20 ºC to allow the starting of alcoholic fermentation. Punching was carried out once a day during the fermentation process. For the wine conservation 30 mg/L sulfur dioxide was added. At the end of fermentation the wines were raked and stored in a cold room at 5 ºC for two months. All wines were elaborated in triplicate.



Analytical essays

Enological parameters such as degree Beaumé (ºBé), pH or total acidity (mg/L tartaric acid) or alcohol (% v/v) were determined according to official EU methods (Regulamento 440/2003).

Color intensity (CI) and total polyphenol index (TPI) was determined by spectophotometric methods (Blouin 1992). Quantification of anthocyanins was done by high performance liquid chromatography (HPLC) using the modified method as described by Boido et al. (2006). Concentrations were determined using the external standard method and their factor responses were deduced from their respective calibration curves. Quantification of condensed tannins were done by precipitation with methyl cellulose (Sarneckis et al., 2006). Polyvinylpolipyrrolidone (PVPP) and astringency was estimated by the method referenced by Llaudy et al. (2004). Hdrochloridric (HCl), ethanol (EtOH), polymerization, dyalisis, and ionization (Glories, 1984), and DMACH (Vivas et al., 1994) indexes were determined. Each analysis was performed in triplicate.

Sensory analysis

The sensory panel consisted in 9 judges with considerable experience in sensory analysis. Sensory profile was determined using six descriptors (color, aroma intensity and aroma quality, taste intensity and taste quality and final assessment). The panelists used a 10-point scale to rate the intensity of each attribute. Assessment took place in a standard sensory analysis chamber, equipped with separate booths and wine tasting glasses.

Statistical analysis

To study the influence of the several factors analysis of variance (ANOVA) was performed. Principal Component Analysis (PCA) was carried out and was applied to discriminate among the means of phenolic and traditional parameters in the wines according to the different winemaking techniques.

Analysis of variance (ANOVA) and Principal Component Analysis (PCA) were applied, in order to evaluate whether significant differences among the samples exist as well as to select the variable that most influence the differences between them. LSD test was used to separate the means (P-value < 0.01) when the ANOVA test was significant. For the statistical treatment of the data the Statgraphics Plus 5.1 software was used.



General composition of must

Means values of must standard parameters for maturity 1 (October 15th) and maturity 2 (22th October) respectively are: degree Beaumé (13.0±0.05 and 14.1±0.07), pH (3.40 ± 0.02 and 3.48 ± 0.03), total acidity (6.18 ± 0.20 and 6.10 ± 0.15 g/L tartaric acid). The data obtained was concordant with previous studies carried out in Bobal grapes (Gómez García-Carpintero et al., 2011). These analyses revealed that the grapes were in good conditions to be harvested and elaborated. As was to be expected total acidity and pH of musts was higher and lower, respectively, in the grapes harvested first (Blouin and Gimberteau, 2004). With regards to the results it should be pointed out that, despite the high degree Beaumé observed in both dates of harvest, the cultivar is able to keep higher values of acidity displaying the great results of this variety in the Utile-Requena region edaphoclimatic conditions.

Composition of the wines

Table I shown the results obtained for the enological parameters of the different wines elaborated. Three conventional parameters and twelve spectral phenolic parameters were quantified due to their significance in red wines (Spranger et al., 2004).




Generally speaking the higher TPI, CI, anthocyanins and tannin concentration values were observed for the wines elaborated with grapes from maturity 1 (Wines 1-6). These results are not in agreement with other studies (Bautista et al., 2003; Llaudy et al., 2005) where better results were observed at higher maturity grapes level. An overall view of these data indicates probably overripened grapes where the harvest was carried out in the latest date (maturity 2).

In addition wines where prefermentative cold maceration (PCM) was applied for 3 days shown the higher phenolic concentration (TPI), however wines that reach the highest colour intensity and anthcyanin concentration were those cold soaked for 5 days. In previous studies this technique (PCM) has been successfully tested and has resulted in an improved CI (Gómez-Plaza et al., 2001; Parenti et al., 2004; Koyama et al., 2007; Gil-Muñoz et al., 2009) and anthocyanin concentration (Gómez-Míguez et al., 2007; Álvarez et al., 2009; Gordillo et al., 2010).

The prefermentative cold maceration for 3 or 5 days increase the anthocyanin concentration between 13% and 33% respectively, in concordance with other studies (Cuasnon, 1999a; Zamora-Marín, 2003) where an increase between 52% and 17% respectively was observed, although dry ice was used in the prefermentative step.

On the other hand the tannin concentration remains unaffected in the cold soaked wines, being the not cold soaked, the wines that show the highest tannin content. In red wines the target of cold prefermentative maceration is to enhance the water-soluble compounds in the absence of ethanol, since as the cap has not been formed the contact between the solid parts and the must is better (Gómez-Miguez et al., 2007), therefore a tannin, alcohol soluble compounds, extraction should not be expected (Sacchi et al., 2005).

Proanthocyanidins in grape berry are located in skin and seeds. The skin proanthocyanidins are located in the vacuoles of skin cells, then the extraction during the prefermentative maceration process, requires the cells walls to be broken, to allow their vacuole content to be extracted or to diffuse into the wine. Moreover the extraction of the proanthocyanidins of the seed needs longer maceration time, because the lipids present in the seed must be eliminated first, which is normally done by the increasing content of ethanol (Glories and Saucier, 2000). In conclusion skin tannins are early extracted and give volume and smoothness, but in the presence of alcohol, seed tannins are also extracted which provide rougness and astringency (Ribereau-Gayon et al., 2006).

The contradictory results obtained shown the extraction of the phenolic compounds in the prefermentative step was highly dependent on several factors such as grape variety and maturity, soil, climate and vinification techniques (Auw et al., 1996), as well as by the proanthocyanidin concentration, the composition of the berry cell walls, and the processing method (Busse-Valverde et al., 2010).

The wines elaborated with higher grape maturity and cold soaked shown the higher proportion of anthocyanins in colored forms (flavilium cation), measured by the ionisation index. In addition the higher grape maturity wines have higher tannin-polysaccharide molecules (ethanol index), decreasing considerably their concentration in prefermentative macerated wines.

In terms of stability, the higher grape maturity wines shown a higher percentage of anthocyanins linked with tannins (A-T), determined by the PVPP index, being found the highest values in the not cold soaked wines. The higher anthocyanin extraction in the cold soaked wines led to less anthocyanin-tannin molecules. The ratio between these compounds did not allow to achieve a good anthocyanin stabilization. Otherwise the higher maceration time the higher anthocyanins mean degree of polimerysation, measured by the polymerization index, showing the cold macerated wines the highest values, as has been reported by several authors (Gordillo et al., 2010; Heredia et al., 2010).

The wines made with more mature grapes shown higher dialysis index, stating higher proportion of, anthocyanin or polysaccharide, linked tannins molecules, suggesting polymerization reaction during ripening. These results were particularly relevant in the non prefermentative macerated wines. On the other hand these wines led to a higher tannin mean degree of polimerysation, measured by the HCL index. Finally the most astringent wines are those made with the less mature grapes.

Effect of grape maturation

Mean and standard deviation values of the phenolic parameters according to the different date of harvest appear in the Table II. The statistical analysis was realized considering all the wines made from grapes harvested in both dates tested. Independent of the vinification technology used the effect of the grape maturity in wine composition was assessed. In these wines, the effect of the grape ripeness produces a significant increase in TPI, CI, anthocyanin and tannin concentration and gelatin index in wines harvested first. The results show that the maturation level has a great impact on the phenolic composition of Bobal wines. Saint Cricq de Gaulejac et al., (1998) mentioned that the optimal grape maturity level occurs when the anthocyanins decrease start i.e. at the beginning of the overripening period when the degradation of the berry cells starts.



Anthocyanins synthesis starts during veraison and remains active throughout grape ripening. For this reason anthocyanins accumulate in the skins during ripening (Fernández-López et al., 1992; Lanaridis and Bena-Tzourou, 1997). However anthocyanin concentration may decrease slightly during overripening (González-San José et al., 1990; Cacho et al., 1992). The concentration of some anthocyanin monoglucosides such as malvidin-3-glucoside and peonidin-3-glucoside usually increase during maturation, while the others anthocyanins monoglucosides tend to decrease during ripening. Simultaneously acylated anthocyanins tend to increase throughout ripening, although in some cases they decrease at the end of the process (Canals et al., 2005). Theoretically wines obtained from well-ripened grapes have more color and are richer in anthocyanins than those obtained from unripened grapes.

Generally speaking the proanthocyanidin concentration is highest at veraison. Later, proanthocyanidin concentration decrease after some time before complete ripeness, when it remains relatively constant. At the same time the main degree of polymerization increases throughout ripening (Canals et al., 2005). The significant differences obtained in the wines made from second harvested grapes could be due by a varietal factor where overripened grapes were used, although the evolution of the phenolic composition of Bobal grapes has not been researched yet. The decrease in the anthocyanin and tannin concentration led to a decrease in the TPI and therefore to the CI, parameter highly related to the phenolic composition of the elaborated wines.

No significant difference in the HCL, polymerization and DMACH indexes was found between both dates of harvest. Otherwise higher anthocyanins that contribute to the wine color (Ionisation index), and linked anthocyanin-tannin compounds (PVPP index) were found in the wines from more mature grapes. It has been well reported that the higher A-T molecules the higher color stability, keeping the anthocyanins in solution, avoiding precipitation phenomenon (Boulton, 2001), but several studies have recently questioned the stability of this reactions (Cheynier et al., 2006). Ethyl linked anthocyanins are less stable than anthocyanins in aqueous solution at wine pH because of acid-catalyzed cleavage of their ethyl bounds. Reactions of the cleavage products led to flavanyl-pyranoanthocyanins, which change color from red to tawny and increase resistant to sulfite bleaching.

The more mature grapes have shown the higher dialysis and ethanol indexes values. As was to be expected greater amounts of high polymerized tannins were observed in the wines made from grapes harvested last. Polysaccharides are present in concentrations ranging from 0.5 to 1.5 g/L. Wine polysaccharides play an important role in mouthfeel properties of wine even by providing increased viscosity or by modulating astringency. More specifically there is evidence that polysaccharide fractions are likely to contribute a fullness sensation to wine inducing a greater decrease on the attributes associated with astringency (Vidal et al., 2004).

Effect of pre-fermentative cold maceration

The use of prefermentative cold maceration allows better structured wines, richer in phenolic and aromatic compounds, thus preventing a strong connection with the area of production (Álvarez et al., 2006). Moreover this technique can potentially promote a selective and significant extraction of phenols, these results in a less aggressive taste and reduces de use of sulphur dioxide as anti-microbic and anti-oxidant (Amati et al., 1992).

Mean and standard deviation values of the phenolic parameters according to the prefermentative cold maceration treatment appear in the Table III. Independent of the vinification technology used the effect of prefermentative cold maceration technique in wine composition was assessed. Two different maceration periods were assayed: 3 and 5 days. A wine elaborated following the traditional vinification method, as a control, was also carried out. The influence of this technique on the final phenolic composition led to significant changes in most of the parameters studied. According to these variables, wines were affected in general by the prefermentative treatment used. These results are similar to those found by several authors (Cuasnon, 1999b; Zamora, 2004; Casassa et al., 2005).



The TPI and the anthocyanin concentration shown the higher content in wines after 5 days of skin maceration, where significant differences between both cold maceration treatments appeared for the anhtocyanin content. Moreover the CI only shown significant differences where the prefermentative treatment is extended to 5 days. Otherwise the cold soaked wines shown lower values for the tannin concentration where the not macerated wines have significant differences displaying the higher amounts of these compounds. These results are in concordance with Retali (2004), who found no differences between the tannin concentration in cold soaked Niellucio wines, while an increase in CI, anthocyanin concentration and TPI was detected.

During the vinification of red grapes, the length of the maceration step, together with the effect of other parameters, such as temperature, stirring of the fermentative medium and wine making process have a considerable influence of the final phenolic content of the wine. Moreover it is known that the maceration temperature greatly affects the transfer of polyphenols from the skins to must (Gil-Muñoz et al., 1999). The results suggest higher maceration temperatures are needed in Bobal wines on order to obtain higher tannins concentration.

It is well known that wine color is not only due to free monomeric anthocyanins. New pigments formed after condensation or copigmentation reactions between anthocyanins and other polyphenols are also responsible for the color of young wines (Francia-Aricha et al., 1997; Darias-Martín et al., 2001). Only in the 5 days cold macerated wines, where the anthocyanin concentration is higher, the CI has significant differences, stating that a long prefermentative period is required in order to decisively influence the Bobal wines chromatic characteristics. As it was reported above several authors found better color where prefermentative maceration was applied, being probably one of the most important features of this vinification technique.

In the prefermentative step the extraction of the phenolic compounds takes place in the absence of ethanol because this low maceration temperature prevent yeast from starting the fermentation process. Anthocyanins and tannins are the compound most directly responsible for the color in red wine. Anthocyanins are easily soluble in water and are dissolved from the moment maceration begins, whereas tannins are more soluble in alcohol so, their extraction depends on fermentation. Therefore, the longer cold prefermentative maceration treatment the higher anthocyanin concentration. Five days cold maceration led to wines with better chromatic characteristics.

A significant negative effect was found in the prefermentative macerated wines. The control wines have shown higher ethanol, gelatin, PVPP and dialysis indexes, where an increase in the cold maceration length led to wines with significant lower values. By contrast the cold soak gives wines where highly colored anthocyanins concentration was present. A higher mean degree of tannin polymerisation, determined by the DMACH index, was also detected.

Unlike it was observed by several authors, who found a better phenolic interaction when prefermentative techniques were assayed (Álvarez et al., 2009, Gordillo et al., 2010; Heredia et al., 2010), in the Bobal wines elaborated this technique did not provide an increase in the interactions between the compounds that decisively influence the chromatic and sensory wine properties. This behavior could be done by the low tannin concentration observed in the prefermentative cold macerated wines. An optimum ratio between anthocyanins and tannins is needed in order to achieve a better color stability (Zamora, 2003).

During storage and ageing, wine color changes from bright red to a reddish brown hue. This is attributed to the formation of new, more stable, polymeric pigments proceeding from reaction between anthocyanins and other phenolic compounds, including, flavan-3-ol monomers and polymers (Castillo-Sánchez et al., 2008). It is well known that the formation of polymeric pigments between anthocyanins and flavanols allow the preservation of wine color (Thorngate and Singleton, 1994; Rivas Gonzalo et al., 1995; Bakker and Timberlake, 1997). Accordingly the low tannin concentration observed in the wines do not led to high anthocyanin stabilization. The longer the prefermentative cold maceration period the lower anthocyanin-tannin interaction.

Effect of the maceration time

The phenolic compounds present in the grape berry are transferred to the must during alcoholic fermentation process. Not only an extraction procedure takes place, as a numerous reactions in the grape phenolics also occur that deeply influence the color intensity and stability of the wine. The length of skin maceration is the first factor that affects this phenomenon, although maceration affects the extraction not only of phenolic compounds but also of other compounds (proteins and polysaccharides) that may participate in condensation reactions.

The concentration of phenolic compounds and spectral data of the wines where different maceration times were tested are shown in Table IV. The increase of the maceration time from 1 week to 2 weeks allowed to wines with significant higher TPI and tannin concentration, but otherwise a significant decrease in the color and anthocyanin concentration was observed in the longer macerated wines. Moreover no significant differences were found in the parameters which determine the reactions between the main wine phenolic compounds. As it was expected only the 2 weeks macerated wines shown a higher polymerisation index, stating a polymeric pigments increase due to the anthocyanin reactions, with other structures since anthocyanins are implicated in copigmentation complexes, chemical reactions (oxidation, covalent linking) and polymerization reactions of that transform the monomeric anthocyanins into polymeric pigments (Álvarez et al., 2009).



Longer contact time is related to a greater concentration of anthocyanins in wines, although some studies have found no direct relationship between the length of maceration and the final anthocyanin content, as a part of the anthocyanins may be fixed in the solids or maybe reduced to a colorless forms. Flavan-3-ol and tannin concentration also increase during skin fermentation, and it has been found that tannins continue to be extracted after anthocyanin extraction has reached a maximum (Gómez-Plaza et al., 2001)

Principal Component Analysis

In order to verify the results obtained a Principal Component Analysis (PCA) was tested. This procedure extracts the dominant patterns in the data matrix in terms of a complementary set of scores and loading plots. This treatment permits us a reduction of dimensionality, a data exploration finding relationships between objects, estimating the correlation structure of the variables and investigating how many components are necessary to explain the greater part of variance with a minimum loss of information. When PCA is performed on auto-scaled matrix data the principal component loading are eigenvectors of the correlation matrix.

PCA was performed on the autoscaled data (8 samples and 15 variables) using the Statgraphics software package in order to provide partial visualization of the data set in a reduced dimension, and four principal components with eigenvalues higher than one accounting for 84.24% of total variance were obtained. From the loadings of the variables (Table V) mainly alcohol (%v/v), pH, ethanol and PVPP index are the positive dominant featuresin the PC1, by contrast TPI, CI and anthocyanin and tannin concentration are the main negative ones, accounting for 43.89% of the total variability. HCL, PVPP, DMACH and Dialysis indexes dominate in the PC2, being pH, polymerization and ionization indexes the dominant in the negative side, accounting the second component for 24.05%.



In Figure 2, where the scores of each wine samples were examined in a two dimensional plot of the two first principal components (67.93% of the total variability) a complete separation of samples into two groups was found according to different date of harvest. The first group, in the positive part of the PC1 is mainly composed by samples from the earliest date of harvest. The second group in the negative part of the graph is made up of samples where grapes were harvested last.



The PCA shown a better phenolic concentration in the wines harvested first, where the presence of the main phenolic compounds were significantly higher and also a deeply increase in the wine color was found. Moreover higher astringency was detected in these wines. Is needed to be pointed out the importance of the date of harvest in the phenolic composition and chromatic characteristics of Bobal red wines, being the overripening a potential wine quality key factor. On the other hand due to the varietal factor complementary enological techniques might be needed in other to reduce the wine astringency and make wines well accepted by the consumers. It is well known by the Utiel-Requena enologists the unripe, aggressive, harsh tannins that Bobal grapes usually shown even in a perfect phenolic maturity conditions.

In addition a complete separation of samples into two groups was found according to prefermentative maceration technique. The first group, in the positive part of the PC2 is composed by the not cold macerated control wines. The second group in the negative part of the PC2 is made up of samples where the prefermentative cold maceration was applied for 3 or 5 days. The PCA do not let to a separation between the length of the prefermentative step.

From the loading of the variables (Table V) the PCA shown a better phenolic interaction in the not cold soaked wines, where the HCL, PVPP, DMACH and Dialysis indexes are the main dominant features. Otherwise pH and ionization and polymerization indexes are the main dominant features in the cold soaked wines. These results indicate the higher anthocyanin extraction in the cold soaked wines which led to an increase in the anthocyanins, which contributes to the wine color, with an increase in the reactions involving these compounds.

Better tannin extraction is required in cold soaked Bobal wines in order to increase the optimum ratio tannin-anhtocyanin. The higher anthocyanin concentration reached needs a higher tannin presence when better color stabilization was desirable. High tannin levels may stabilize the anthocyanins and wine color but on the other hand may contribute to excessive astringency (Sims and Bates, 1994; Gómez-Plaza et al., 2001).

Finally the PCA do not allow to separate the wines according to the length of the skin maceration. No separation of the samples was found; therefore it has no sense to extend the maceration step, avoiding future problems in wine quality. On the other hand higher tannin concentration was observed when samples were subjected to analysis of variance (ANOVA). Suggesting better tannin concentration obtained by extending the maceration length with probably future better anthocyanin stabilization. Bobal variety is characterized in the Utiel-Requena wine region by their high amount of anthocyanins that led to good chromatic characteristics, and then even losing some anchocyanins content, greater color stability could be accomplished by extending the maceration period.

Sensory evaluation of wines

The sensory panel did not observe significant differences in the wines after malolactic fermentation for sensorial aroma intensity and quality and taste intensity, where wines from two different dates of harvest were assessed (Table VI). Wines from grapes harvested first achieved a significant greater score for the sensory color as was observed above. Otherwise wines from grapes harvested last obtained better scores for the attributes taste quality and final assessment where better phenolic interactions took place. Finally, we suggest that a slightly phenolic concentration loose could be assumed in order to obtain wines better rated by the consumers.




Not significant differences in wines macerated for 1 or 2 weeks were observed by the sensory panel. As we reported above better tannin concentration can be achieved in order to increase phenolic stability, although it has no sense increase the maceration time in wines that are not going to be aged, because wines with shorter maceration time led to better chromatic characteristics.

Only the attribute taste quality shown significant higher values when the prefermentative cold maceration was applied for 5 days. Moreover the cold macerated wines shown the highest values for the final assessment, where the longer the prefermentative step the higher the scores achieved. No differences in the sensory wine color were observed, despite the higher antchocyanin concentration in the cold soaked wines. Blouin et al. (2000) reported not differences between the prefermentative macerated and the control in a sensory evaluation of Merlot and Cabernet Sauvignon wines, stating that this technique does not always achieve good results in the sensory evaluations.



The maturation level has a significant impact on the phenolic composition of Bobal wines. Higher phenolic concentration and wine color was observed in wines harvested earlier, moreover these wines shown higher astringency. Otherwise better interactions between polyphenols were also found in the wines from more mature grapes. An overripenes should be taken into account in order to avoid phenolic material decrease, and an additional enological treatment may be necessary with the objective to reduce wine astringency. Prefermentative maceration technique led to wines with lower tannin concentration although an increase in total polyphenols and wine color was also observed. A significant negative effect was found in the parameters that state the phenolic reactions in the cold soaked wines. The results suggest higher maceration temperatures are needed in Bobal wines on order to obtain higher tannins concentration which probably increase the reaction between polyphenols. The maceration length did not highly affect the polyphenol interaction, moreover an increase on the tannin concentration was also observed in the 2 weeks macerated wines. If a slight decrease in the anthocyanin concentration can be assumed an extended maceration could be done in order to improve tannin concentration and future possible phenolic stability. The wines better valued by the sensory panelist were those from grapes harvested last and pre-fermentative cold macerated.



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(Manuscrito recebido em 10.07.2012. Aceite para publicação em 11.12.2012)