First Approach to the Analytical Characterization of Barrel-Aged Grape Marc Distillates Using Phenolic Compounds and Colour Parameters

Diff erent high-alcohol drinks can be obtained a er distilling some previously fermented raw material. The most popular alcoholic beverages obtained from grape marc are orujo (Spain), bagaçeira (Portugal), grappa (Italy) and tsipouro (Greece). In the majority of these cases, freshly distilled beverages are defi ned with sensory a ributes like sharp, alcoholic, rude and bi er, therefore ageing in oak barrels is essential to give them the sensory characteristics that consumers like (1,2). During the maturation process, several physicochemical reactions occur in distillates, such as extraction of wood components, loss of low-boiling-point compounds from the immatured distillate by evaporation, and interactions among components from wood and beverage. As a result of diff erent reactions such as polymerization, esterifi cation, acetalisation, hydrolysis and oxidation (3,4), the initial product modifi es its chemical composition and the sensory characteristics, visual aspect (colour and limpidity), taste and fl avour (5). Low molecular mass phenolic ISSN 1330-9862 original scientifi c paper


Introduction
Diff erent high-alcohol drinks can be obtained a er distilling some previously fermented raw material.The most popular alcoholic beverages obtained from grape marc are orujo (Spain), bagaçeira (Portugal), grappa (Italy) and tsipouro (Greece).In the majority of these cases, freshly distilled beverages are defi ned with sensory a ributes like sharp, alcoholic, rude and bi er, therefore ageing in oak barrels is essential to give them the sensory characteristics that consumers like (1,2).
During the maturation process, several physicochemical reactions occur in distillates, such as extraction of wood components, loss of low-boiling-point compounds from the immatured distillate by evaporation, and interactions among components from wood and beverage.As a result of diff erent reactions such as polymerization, esterifi cation, acetalisation, hydrolysis and oxidation (3,4), the initial product modifi es its chemical composition and the sensory characteristics, visual aspect (colour and limpidity), taste and fl avour (5).Low molecular mass phenolic compounds can be pointed out among the released components from wood during ageing, since they are not present in fresh distillates (6).The identifi cation and quantifi cation of phenols are crucial due to their infl uence on the chemical composition, sensory characteristics of the resulting aged distillate and also on their antioxidant activity (7,8).
The extension of these physicochemical changes depends on several factors, such as the botanical species and geographical origin of the wood (region, type of forest, climate, soil type, etc.), the technology of the barrel-making process (mainly the toasting intensity), the size of the barrel, their previous uses, ageing time, ageing conditions (cellar temperature and humidity) and the pH, alcohol content and total acidity of the initial wine or distillate (9)(10)(11)(12)(13)(14)(15).
Oak (genus Quercus) is the most suitable botanical species of wood for alcoholic drink maturation (16).Quercus alba (American oak), which grows in diff erent areas in the United States, Quercus petraea (sessile oak) and Quercus robur (pedunculate oak) from Europe, are the most common oak species used in barrel-making (17).French oak is the most popular wood for the maturation of beverages around the world (18), whereas Spanish oaks, mainly grown in the North of Spain, are used at a lower scale for ageing of wines and a few studies about their behaviour have been published (19)(20)(21).Some distilleries in Galicia (NW of Spain) also use oak from Q. robur grown in this area to age the grape marc distillate (called orujo).However, only a small part of the production of orujo is subjected to an ageing process, due to signifi cant expenses: wooden barrels are expensive, and this cost increases in proportion to the length of the ageing period, mainly due to alcohol evaporation.
The physicochemical parameters of these beverages are fi xed by the Regulatory Council in the control program (22).These parameters are the same as in young orujo and only change the minimum and maximum values fi xed for them, although the ageing process changes the chemical composition of young distillate and as consequence its sensory profi le (23).The infl uence of the species of oak wood and time of ageing on the mineral composition of distillate was also observed (24), and the results showed a great infl uence of the species of oak wood on the fi nal composition of the distillate.
The current study has been undertaken to obtain the descriptive phenolic and sensory a ributes of aged orujo distillates, in order to characterize these alcoholic beverages for the fi rst time.The results can also be useful to valuate the suitability of employing oak barrels grown in Spain as an alternative to the barrels bought from other countries and thus contributing to the improvement of the quality of these distillates and the a empt to reduce the high initial cost.

Samples
Ten commercial grape marc distillates from Galicia (orujo) aged in 225-litre wooden barrels from the species Quercus robur (pedunculate oak; origin: Limousin, France and Galicia, Spain), Quercus alba (American oak) and Quercus petraea (origin: Allier, France) were analysed.These samples correspond to all the types of barrels currently present on the market.They were collected and bo led in glass bo les a er an ageing period between 1 and 6 years.General information about aged orujo (species of oak wood, time of ageing, etc.) was supplied by the producers.In Table 1 all the information concerning the analysed samples is summarized.

Total phenolic content
According to Spigno et al. (25), total phenols were determined applying two methods: (i) Singleton and Rossi procedure (26) using Folin-Ciocalteu reagent, the best method to determine total phenols, including tanins, and (ii) direct measure of the absorbance of each sample at 280 nm (A 280 nm ) employing a one-centimetre quartz cuve e (27).All samples were previously diluted 20 or 100 times.Total phenolic index (TPI) was determined by the following equation: The quantifi cation of both parameters was carried out using a calibration curve with known concentrations of gallic acid and expressed as gallic acid equivalents (GAE).

Colour intensity and hue
Colour intensity and hue, determined by absorbance measurements of diluted or undiluted samples at the wavelengths of 420 (yellow), 520 (red) and 620 nm (violet), were also evaluated employing one-centimetre polystyrene cuve es (28,29).The equations for the calculation of colour intensity (CI) and hue (h) were as follows: CI=A 420 nm +A 520 nm +A 620 nm /2/ and h=A 420 nm /A 520 nm /3/ Spectrophotometric analyses were performed using a UV-VIS Cintra 6 spectrophotometer (GBC Scientifi c Equipment, Madrid, Spain).

HPLC-MWD analysis
All samples of aged grape marc spirits were fi ltered through 0.45-µm pore membranes of cellulose acetate (Sartorius, Goe ingen, Germany) before the analysis using high-performance liquid chromatography (HPLC).An Agilent Technologies 1200 series system consisted of a quaternary pump (G1311A), an injector, a degasser (G1322A), a multiple wavelength detector (MWD, UV/VIS; Agilent, Palo Alto, CA, USA) and a Zorbax SB-Aq reversed-phase column, 5 µm, 150 mm×4.6 mm i.d.(Agilent) with a guard column.Samples of 20 µL of aged spirit or calibration standards were injected into the column and eluted with the following gradient: solvent A (methanol) and solvent B (2.5 % formic acid in Milli-Q water, by volume) at a fl ow rate of 1 mL/min.Zero time conditions were 100 % B, a er 35 min the pumps were adjusted to 52 % B and 48 % A, at 56 min to 100 % A until the end of the analysis at 65 min.Detection was carried out at (276±4) nm.The identifi cation of each compound was done by comparing the retention times with those of pure standards.All determinations were made in duplicate.

Sensory analysis
The panel for sensory analysis consisted of eight assessors, fi ve males and three females aged from 35 to 55, all of them members of the offi cial panel of Geographic Denomination of Spirits and Liqueurs from Galicia, expert tasters in sensory analysis of this kind of alcoholic beverages.The selected judges worked directly for wineries and distilleries either as winemakers or in marketing.The sensory analysis was performed in a laboratory, containing 20 independent tasting booths, and designed according to the International Organization for Standardization, standard ISO 8589 (30).
Before evaluation, during three training sessions (12 h), a collection of six representative samples was tested by the panellists in order to generate relevant appearance and taste a ributes.All samples were commercially available.The aim of these sessions was to develop a common vocabulary for the description of the sensory a ributes of aged orujo samples.In the fi rst phase of this training, the judges identifi ed thirty-three descriptors (13 for appearance and 20 for taste).A er a round-table discussion and by consensus, the panel selected and refi ned the a ributes that best describe their perceptions.Synonymous, hedonic and irrelevant descriptors were also eliminated by using statistical methods described in ISO 11035 standard (31).Finally, the generated a ributes were reduced to eight (three for appearance and fi ve for taste).
In the formal session, the intensity of each a ribute was rated on a fi ve-point scale, where 0 indicated that the descriptor was not perceived, 1=low, 2=low-medium, 3=medium, 4=medium-high and 5=high.Each sample was previously coded and presented to judges in random order.The sensory evaluation was carried out at room temperature, using the offi cial glasses of the corresponding Regulation Commission.Tasting was carried out in the morning during fi ve sessions on diff erent days to avoid fatigue of the tasters due to the high degree of alcohol in the aged grape marc distillates (37.5-50 %, by volume).

Statistical analysis
The obtained results were analysed using XLSTAT-Pro (Addinso , Paris, France).One-way analysis of variance (ANOVA) was applied to establish whether significant diff erences (p<0.05)existed between the values obtained for the mean concentration of each compound in the analysed aged grape marc distillates.The multiple range test (LSD) was applied to confi rm the obtained results.Pearson's correlations among all identifi ed phenolic compounds, between sensorial descriptors and phenolic compounds, and between sensorial a ributes and colour parameters were also calculated.In order to determine the infl uence of the oak species on the composition of grape marc distillates, a multivariate principal component analysis was carried out.

Spectrophotometric parameters of aged orujo distillates
Table 2 lists all phenolic indices and chromatic characteristics of the analysed samples.Taking into account only the species of oak, no signifi cant diff erences were observed in the Folin-Ciocalteu index among the samples; however, the values of other spectrophotometric parameters were signifi cantly diff erent.Higher contents of total phenols were observed in the group of distillates aged in Q. robur (Galicia), showing signifi cantly diff erent value with respect to the other analysed distillates, including those aged in the same oak species of other geographical origin.Total phenolic index (TPI) determined in each group of distillates showed higher values than the corresponding total phenols evaluated with Folin-Ciocalteu method.
Orujo aged in Q. robur (Galicia) showed the highest values of colour intensity, whereas the samples aged in Q. alba showed the lowest.Hue value was lower in the samples aged in Q. robur (Limousin); however, other samples did not show signifi cant diff erences.The results showed a great infl uence of the wood origin on the chromatic characteristics of the samples, since signifi cant diff erences were found among distillates aged in Quercus robur from diff erent areas.

Validation of the HPLC method
The validation of the method was done based on linearity and analytical limits (limits of detection and quantifi cation).

Linearity
The study of the linearity was performed using the HPLC analysis of seven standard solutions containing increasing concentrations of their respective standards covering the range of linearity.These solutions were prepared in triplicate in ultrapure water with 40 % (by volume) of absolute ethanol and fi ltered through 0.45-µm pore membranes of cellulose acetate.Data in Table 2 show that linearity is satisfactory in almost all cases, with the correlation coeffi cient (R 2 ) ranging from 0.9712 (4-hydroxybenzaldehyde) to 0.9999 (coniferaldehyde).

Analytical limits of detection and quantifi cation
The limit of detection (LOD) and limit of quantifi cation (LOQ) were determined from the parameters of the analytical curves.Both limits were calculated according to the following mathematical relationships: where S.D. is the estimation of the standard deviation of the regression line, and m is the slope of the calibration curve (32).The LOD and LOQ were low for all determined phenolic compounds (Table 3).

Concentration of phenolic compounds in grape marc distillates aged in diff erent wooden barrels
Fig. 1 shows the molecular structures of the compounds extracted from the wood.The phenolic aldehydes are produced by thermodegradation of the terminal monomer units of lignin: the cinnamic aldehydes convert to benzoic aldehydes, and then they are oxidized to phenolic acids (19).Table 4 reports the concentrations of the quantifi ed phenolic compounds in the analysed aged distillates.The concentration of gallic acid in aged beverages depends on the toast level of barrels, since gallic acid is degraded at high temperatures (33).Consequently, this compound is more abundant in distillates aged in barrels with light or medium toast levels (7).Besides gallic acid, the concentration of ferulic and vanillic acids also decreased with higher temperature during the toasting process (34).The content of gallic acid was signifi cantly higher in distillates aged in Quercus robur from Galicia (>50 mg/L).This compound was also present, but at low concentration, in distillates aged in Quercus petraea from Allier.Vanillic acid can be directly extracted from oak wood or be formed by oxidation of vanillin during the ageing process, whereas syringic acid is formed during toasting by the oxidation of the corresponding aldehyde.Both compounds were present at higher concentrations in distillates aged in Quercus robur from Galicia.In contrast, distillates in the same species from Limousin showed lower values of these compounds.
Vanillin and syringaldehyde are phenolic compounds related to lignin.Vanillin was detected in all analysed samples, with signifi cantly lower concentration in the samples aged in Quercus robur from Limousin and higher in distillates aged in the same species from Galicia.Vanil-lin is the phenolic aldehyde which greatly infl uences the aroma of distillates because of its low threshold value (320 µg/L) and adds positive vanilla notes (35).In the analysed samples, syringaldehyde was the most abundant phenolic aldehyde.
Thermal degradation of lignin leads to the formation of some phenolic alcohols (19).The concentration of vanillyl alcohol was higher in the spirit aged in Quercus robur (Galicia), whereas this compound was not present in distillates aged in Quercus robur (Limousin) or Quercus alba.
Ferulic acid was used in this study as a discriminating compound of the oak wood species because it can only be quantifi ed in Quercus robur (Galicia).Similar results were obtained by Canas et al. (7), showing that the brandies aged in Portuguese oak (Quercus pyrenaica Willd.)contain higher values of this compound than other brandies aged in other oak species.The geographical proximity and similar climatic conditions of both areas (Galicia and northern Portugal) may be the main reasons to explain these similarities, despite the fact that the spirits aged in two diff erent species of Quercus.
The presence and concentration of benzoic and cinnamic aldehydes, which arise from lignin degradation, depend on the temperature applied during toasting (16,36,37).Results in this study showed that coniferalde- hyde and sinapaldehyde were present at low concentrations in samples from Quercus robur (Limousin) and Quercus alba.In all cases, their individual values were higher than the corresponding benzoic aldehydes, 4-hydroxybenzaldehyde and vanillin.
Fig. 2 shows the diff erences between the four groups of grape marc distillates according to the total concentration of phenols from each family.Most of the identifi ed phenolic compounds were at higher concentrations in the orujo samples aged in Quercus robur (Galicia) than in the samples aged in the same species of Quercus from Limousin.Between the other two species, orujo distillates aged in Quercus alba had lower concentrations of most of the determined phenolic compounds than the distillates aged in Quercus petraea.
A signifi cant eff ect of oak wood species was observed on 11 of the 15 studied compounds (Table 4).Benzoic acid, gallic acid and syringaldehyde were the main low molecular mass compounds in the analysed samples.The results are in agreement with those previously obtained by dos Anjos et al. (32) in a study about cachaça.The concentration of minor compounds such as vanillin, syringic and ferulic acids was signifi cantly higher in distillates aged in Quercus robur from Galicia.Ferulic acid was only quantifi ed in these samples.Distillates aged in Quercus robur from Limousin had the lowest concentration of all determined phenolic compounds.In the majority of cases, their concentrations were lower than their corresponding detection and quantifi cation limits.

Quality and authenticity of aged grape marc distillates determined by phenol ratios
Van Jaarsveld et al. (18) and Gimenez-Martínez et al. (38) showed that the gallic acid/vanillin ratio is infl uenced by the type of wood and it has been used to defi ne the quality of spirits.A higher gallic acid/vanillin ratio indicates medium to high quality brandy (39).According to van Jaarsveld et al. (40), the gallic acid/vanillin ratio increases with the level of toasting.In this study (Table 4) this relationship was signifi cantly higher in distillates  Other authors (39,41) also used the syringaldehyde/ vanillin ratio to evaluate the quality of aged beverages.Usually the syringaldehyde content exceeds more than twice the vanillin content in oak wood species (42).In this study, the syringaldehyde/vanillin ratio values were in all cases near 2. A relationship between syringaldehyde and vanillin in the range from 1.4 to 2.5 shows a balanced lignin composition (38).No diff erences were found in this ratio among the species of oak wood.The syringaldehyde/ vanillin ratio may also be used to evaluate the possible addition of commercial vanillin as fl avouring to increase the aroma of aged distillate.In addition, the relationship between benzoic aldehydes (vanillin and syringaldehyde) and cinnamic aldehydes (coniferaldehyde and sinapaldehyde) can be used to evaluate the authenticity of the aged distillates.Canas et al. (37) showed that the relationship between both aldehyde families allows diff erentiating the type of wood (chestnut or oak) used in the ageing process.In this study, the relationship between both groups of aldehydes is shown in Fig. 3. Orujo aged in Quercus alba had the highest ratio of benzoic/cinnamic aldehydes (1.95), whereas samples aged in Quercus petraea had lower ratio than the unity (0.79).Similar values were observed in the samples of orujo aged in Quercus robur from Galicia and from Limousin, with a ratio of 1.07 and 0.84, respectively.

Sensory analysis of aged grape marc distillates
Eight common descriptors (three for appearance and fi ve for taste) were defi ned by the judges to describe the samples.The mean intensity of each a ribute was used to defi ne the sensory profi le of the evaluated orujo samples (Fig. 4).The obtained results showed that the oujo samples aged in Quercus robur from Galicia reached the highest values of all descriptive parameters, which were close to the values for samples aged in Quercus robur from Limousin.Samples aged in Quercus alba had lower colour intensity and lower scores of most of the evaluated a ributes.Orujo samples aged in Quercus petraea and Quercus alba showed similar values in positive taste parameters (sweet, dense and oily), but the ageing process in Quercus petraea increased some negative notes, giving these distillates more spicy/pungent and alcoholic taste.Samples aged in Quercus petraea had the lowest visual score, having poor brightness and transparency.

Pearson correlation coeffi cients
Pearson correlation coeffi cients among all identifi ed phenolic compounds are shown in Table 5.The majority of the compounds were positively correlated, with values of R above 0.6.Vanillin and syringaldehyde are compounds related to lignin, but no correlation was established between them.However, vanillic and syringic acids, both products of lignin degradation, showed a high positive correlation (0.714).Both benzoic acids were also positively correlated with guaiacyl-type compounds (syringaldehyde and sinapaldehyde).Gallic acid showed high positive correlation with the majority of phenolic and cinnamic acids and with the corresponding aldehydes.Cinnamic acids (ferulic, isoferulic, p-coumaric and sinapic acids) were also highly correlated.
Pearson correlations between phenolic compounds and taste a ributes of aged grape marc distillates were also evaluated (Table 6).Strong positive correlations were found between, benzoic (gallic, syringic and benzoic) and cinnamic (ferulic, isoferulic and sinapic) acids with negative descriptors of mouthfeel (astringent and alcoholic notes).On the other hand, positive taste a ributes, sweet, dense and oily, showed strong positive correlations with the corresponding benzoic (protocatechualdehyde and syringaldehyde) and cinnamic (sinapaldehyde) aldehydes.The 4-hydroxybenzaldehyde and p-coumaric acid were negatively correlated with the positive a ributes, whereas vanillin, as alcohol, aldehyde and acid showed strong positive correlation with the alcoholic note.
Colour parameters and the three visual a ributes were also correlated (Table 7).The results showed that hue is the colour parameter that has the most infl uence on the positive valorisation of the aged distillates.

Principal component analysis
Fig. 5 shows the score plot of the fi rst two PCs, obtained with the individual phenols and chromatic characteristics as variables, which explain 88.32 % of the variability among the samples.Fig. 5a shows that the fi rst principal component, PC1 (77.91 %), was positively correlated with all studied variables, whereas the second principal component, PC2 (10.40 %), was mainly positively correlated with the benzoic and cinnamic aldehydes and negatively with gallic and isoferulic acids and total phenols.Four groups of samples plo ed on the plane defi ned by the two fi rst principal components can be observed in Fig. 5b.Samples from group 1 (QRG60 and QRG72) were be er characterized by all variables associated with the positive side of PC1, mainly by gallic, syringic and sinapic acids and by total phenols and colour intensity.In contrast, The PCA analysis clearly showed a good separation of the aged orujo samples according to the species and origin of the oak wood employed in the ageing process, independent of the time of ageing.

Conclusions
The results obtained in this study provide the fi rst data on the phenolic composition of the aged grape marc distillate (orujo) and contribute to the knowledge about this alcoholic beverage.Benzoic acid, gallic acid and syringaldehyde were the main low molecular mass compounds.Distillates aged in Quercus robur from Galicia showed the highest concentration of the majority of the determined phenolic compounds, whereas samples aged in Quercus robur from Limousin had the lowest corresponding values.These results showed the infl uence of the growth origin on the oak composition.Ferulic acid was only detected in orujo samples aged in Quercus robur from Galicia.Consequently, this compound can be used as a discriminant among the three oak wood species in this study.Most of the determined phenolic compounds and colour parameters were positively correlated with each other and with the sensory a ributes defi ned by the tasters.No signifi cant diff erences of the Folin-Ciocalteu indices were shown among the analysed aged orujo beverages; however, total phenols, colour intensity and hue were signifi cantly higher in orujo aged in Quercus robur from Galicia.Principal component analysis allowed the classification of the aged distillate samples according to the origin and species of oak wood.However, the results shown in this study must be completed with those obtained from an experimental design using a unique distillate aged in diff erent oak species during the same time.Other variables such as toast level and contact time (oak-distillate) must also be taken into account in deciding which type of oak is the most suitable for ageing grape marc spirits.In this research analytical characterization of orujo alcoholic beverages is presented for the fi rst time.

LOD=3
0.57 t R =retention time (min), a=slope, S.D. a =standard deviation of the slope, b=intercept, S.D. b =standard deviation of the intercept, R 2 =correlation coeffi cient of linear range, LOD=limit of detection, LOQ=limit of quantifi cation

t
R =retention time (min), a=slope, S.D. a =standard deviation of the slope, b=intercept, S.D. b =standard deviation of the intercept, R 2 = correlation coeffi cient of linear range, LOD=limit of detection, LOQ=limit of quantifi cation

Fig. 1 .
Fig. 1.Molecular structures of the phenolic compounds extracted to the grape marc distillates a er ageing in oak barrels.The structures circled with dashed line in bold are guaiacyl compounds, while the structures circled with double dashed line are syringyl--type compounds

Table 1 .
Main characteristics of the analysed orujo distillates aged in diff erent oak wood barrels

Table 2 .
Concentration of total phenols and chromatic characteristics of orujo distillates a er ageing in barrels Diff erent le ers in superscript within the same row indicate statistically signifi cant diff erences (p≤0.05) according to LSD test FCI=Folin-Ciocalteu index, TPI=total phenolic index, GAE=gallic acid equivalent, CI=colour intensity, h=hue *

Table 3 .
Family, compound, retention time, linearity parameters and analytical limits of the HPLC method for the quantifi cation of phenolic compounds in aged grape marc distillates

Table 4 .
Concentration of phenolic compounds in ten grape marc distillates aged in three diff erent oak species during diff erent periods of time (ANOVA results are also shown) *Diff erent le ers in superscripts within the same row indicate statistically signifi cant diff erences (p≤0.05) according to LSD test LOQ=limit of quantifi cation, LOD=limit of detection

Table 6 .
Pearson correlation matrix (r) among phenolic compounds and sensory a ributes of taste Correlations higher than ±0.6 are shown in bold; CI=colour intensity, h=hue

Table 5 .
Pearson correlation matrix (r) among phenolic compounds volatiles on the positive side of PC2, mainly syringaldehyde and sinapaldehyde.Group 4 was composed of distillates aged in Q. petraea (QPA30, QPA72A, QPA72B and QPA144) in the centre of the plot.