Abstract
A total of 61 samples of Galician (NW Spain) Orujo spirits, 28 from white grape pomace and 33 from red grape pomace, were analyzed by gas Chronoatography with flameionization detector (GC-FID) in order to determine which volatile compounds show significant differences (p ≤ 0.05). Orujo spirits obtained from white grape residues showed a higher concentration of methanol, hexanols, hexenols, ethyl lactate, benzaldehyde, 2-butanol, and rate 1-propanol/2-methyl-1-propanol, which indicated a high trend to the degradation by anaerobic microorganisms during the storage and alcoholic fermentation of the raw material. However, in the spirits from red grape, the concentration of ethyl esters was higher as was the concentration of the compounds that are derived from the activity of aerobic bacteria or oxygen contact with the fermented grape pomace, especially acetaldehyde and ethyl acetate.
INTRODUCTION
Grape pomace distillates are alcoholic beverages produced in most European wine producing countries. Galicia (NW of Spain) is a region with a long tradition in the production of these kinds of beverages. The European Community, in Annex II of Council Regulation (EEC 1576/89) for geographical designations, named Orujo to the grape pomace distillates produced in this Spanish region. By definition, Orujo is an alcoholic drink or spirit produced by distillation of the solid waste of the grape that is generated in the first stages of winemaking, to which can be added up to 25% (v/v) or 35% (m/m) of lees (bottoms). Spirits with similar characteristics to Orujo are produced in Italia (Grappa), Portugal (Bagaçeira), Greece (Tsipouros), Yugoslavia (Kommovica), and in Turkic (Raki).Citation[1] These European alcoholic beverages present similar chemical composition and sensorial properties due to the kind of raw material (grape pomace and lees) and the technology employed in the distillation process (still) are similar too. The six distilled beverages previously mentioned have been extensively analysed to know the concentration and origin of major and minor components responsible for the total aroma Spirits.[Citation2–7]
The variety of grape employed in the production of distillates contributes to the final aroma with an important number of volatile compounds (terpenes and C13-norisoprenoids)[Citation1,Citation8,Citation9] that are mainly responsible for floral and fruity notes. The solid waste or grape pomace goes through a process of alcoholic fermentation that, while in the case of red wines is produced with the grape juice, in white wines is carried out separately, stored in bags, stainless steel, plastic, and concrete containers.[Citation10–12] In this sense, an important difference between both types of raw material is the presence of absence of the wine lees during the distillation process, an aspect that notably affects the composition and quality of the final spirit.
For these and other reasons, the variations in raw material of white or red grape pomace will generate a different volatile composition in the resulting distillates. Volatile compounds of distilled beverages have been subjected to several studies and confirmed that higher alcohols, esters, acids, and carbonyl compounds make important contributions to the aroma profile and quality of them.Citation[13] During the distillation step, the first 0.5–1 L, at the beginning, is removed like “heads,” with ethanol content higher than 85% (v/v). The fraction used in the production of Orujo spirits is called “hearts,” with an ethanol content between 85–30% (v/v). The last fraction or “tails” content is lower than 30% (v/v) of ethanol and the volatile compounds with high boiling point. The volatile compounds are present in the three fractions of the distillate according to their volatility and solubility in ethanol/water.[Citation13–15]
During the fermentation and distillation process, the presence and concentration of this group of compounds must be preserved and the volatiles generated in these important stages (higher alcohols, ethyl esters, acetates, acids, etc.) shouldn't have overlapped the varietal aroma. In this sense, it is very important to define the adequate fermentation and storage conditions of the grape pomace and of the corresponding distillation. Nowadays, an important number of Orujo spirits present in the market are made from a single variety of grape, white (Albariño, Godello) and red (Mencia). However, some distilleries produce Orujo from plurivarietal red or white grape pomace. In both cases, the efforts of the producers must be directed towards improving quality and to promote their typicity and differentiation. In this sense, it is very important to know the behaviour of the raw material during the the fermentation process, independent of the grape variety.
In this study, several samples of Orujo spirit from red and white grape pomace were analysed by gas chromatography in order to: (i) compare the volatile composition of Galician Orujo spirits obtained from white and red grape pomace to estimate if there are significant differences between them in terms of qualitative and quantitative; (ii) evaluate the influence that the process of fermentation exercised in the above-mentioned volatile composition; (iii) estimate which of two types of grape pomace (red and white) showed a major trend to the bacterial degradation.
MATERIALS AND METHODS
Samples
White and red grape pomace spirits were obtained from a prestigious distillery in Galicia (Spain) that produces high quality Orujos inside the Geographic Denomination of the Spirits and Traditional Liquors from Galicia. An industrial distillation unit using entrainment with steam and equipped with a rectification column was employed. For each cycle, heads and tails were discarded. The samples of Orujo spirits employed in this study were 28 samples of “hearts” obtained from distillation of white grape pomace and 33 from distillation of red grape pomace. From total hearts volume obtained in each distillation, a portion of 750 mL was collected. All samples were stored under the same conditions of temperature (±5°C) until they were analysed by gas chromatography.
Reagents
Ethanol of analytical grade, was purchased from Merck (Darmstadt, Germany). 2-Butanol, 1-butanol, 1-propanol, 2-methyl-1-propanol, 4-methyl-2-pentanol (internal standard), trans-2-hexenol, cis-2-hexenol, acetaldehyde, 1,1-diethoxyethane, diethyl succinate, and ethyl mirystate were supplied by Aldrich Chemical (Schweiz, Switzerland). Methanol, allyl alcohol, 2-phenyl-ethanol, 2-methyl-1-butanol, 3-methyl-1-butanol, hexanol, benzyl alcohol, ethyl butyrate, ethyl laurate, hexyl acetate, isoamyl acetate, ethyl acetate, methyl acetate, and 2-phenylethyl acetate were purchased from Merck. Ethyl hexanoate, ethyl octanoate, ethyl decanoate, trans-3-hexenol, cis-3-hexenol, furfural, and benzaldehyde were supplied by Fluka (Schweiz, Switzerland). Ethyl lactate was purchased from Sigma (Schweiz, Switzerland). The stock solutions were prepared in ethanol/water (40% (v/v)). The internal standard 4-methyl-2-pentanol was prepared in ethanol absolute.
Gas Chromatographic Analysis
For determination of the volatile compounds (methanol, aldehydes, higher alcohols, and esters), 1 mL of an internal standard solution (5 g of 4-methyl-2-pentanol per 1 L of ethanol) was added to a 10-mL sample of spirit. An aliquot of 1 μL was injected directly into the chromatograph (Hewlett Packard 5890 Series II gas chromatograph Hewlett Packard, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an HP 6890 automatic injector. The compounds were separated in a CHROMPACK CP-WAX 57CB Wcot fused silica column (polyethylene glycol stationary phase; 50 m × 0.25 mm i.d. with a 0.25-μm film thickness (Vlarian, Agilent Technologies, Spain). Injections were made in split (1:1). The injector temperature was 250°C and the oven was programmed to 6 min at 40°C, 1.5°C min−1 up to 80°C and afterwards at a rate of 3°C min−1 to 200°C. The carrier gas was helium at a flow rate of 1.07 mL min−1; detector (FID) Ta: 260°C; H2: 40 mL min−1; air: 400 mL min−1; and auxiliary gas (N2): 30 mL min−1. Qualitative and quantitative analyses of the compounds in the spirit samples analysed were made by comparison of their retention times with those of the pure standards. In the calibration, the response factor of each compound, RFi, was calculated by RFi = (Ais/Asi).(Csi/Cis), where Ais and Asi are the peak areas of the chromatographic internal standard and of the chromatographic standard of the compound of interest, respectively, and where Cis and Csi are the concentrations of the chromatographic internal standard and of the chromatographic standard of the compound of interest, respectively. In the actual quantification, the concentration of each compound of interest, Ci, was determined via Ci = (Ai/Ais).Cis.RFi, where Ai is the areas of peak interest. Determinations were performed in triplicate.
Statistical Analysis
A computer programme, Statgraphics Plus for Windows, Version 3.1 (1997), (Statgraphics, Madrid, Spain) was used for the statistical study of the results. A multifactor variance analysis (ANOVA) was applied to establish if significant differences existed between the values obtained for the concentration of volatile compounds in the two groups of spirits analyzed (red and white grape pomace) (p < 0.05, LSD test). To interpret the results and establish the relationship between the volatile compounds and Orujo spirits, principal component analysis (PCA) was performed. PCA is used as a tool for screening, extracting, and compressing data.
RESULTS AND DISCUSSION
shows the mean concentration for each volatile compound determined in the samples of spirits from red and white grape pomace. A multivariate statistical analysis (ANOVA) was also applied to determine whether or not significant differences exist between the mean concentration of each compound as a function of the type of grape pomace (95% confidence limit).
Table 1 Mean concentration (mg L−1) and standard deviation of volatile compounds in distillates from white and red grape pomace. ANOVA results are also showed
In both kinds of distillates, the alcoholic content of the hearts was high due to the reflux generated during distillation in this type of equipment with a rectification column. The mean concentration of ethanol did not present significant differences between the types of distillates (p < 0.05). So, it can be concluded that the ethanol recovery of both types of raw material is similar, at least when the grape pomace was stored in good conditions. Methanol is a volatile compound with high toxicity, so its content in the alcoholic beverages must be lower than 1000 g HL−1 a.a. The final concentration of methanol in the distillates depends on varied parameters, such as the storage conditions of grape pomace, the distillate fraction, and the grape variety.[Citation5,Citation12] Methanol was formed from pectin by the action of pectin-methyl esterases. The percentage of solid parts was major in the white grape pomace and, therefore, the presence of certain volatile compounds, in which origin was associated with activities of certain enzymes, are present in major proportion in skins and stalks.Citation[16] This aspect justified that methanol is more abundant in distillates from white grape pomace.
Higher alcohols are a group of volatile compounds with a higher molecular mass and boiling point than ethanol and they are also important precursors to ester formation. Higher alcohols were the most important group of volatile substances in distillates.Citation[17] All of them, except 2-butanol, are formed from amino acids during the course of alcoholic fermentation and their concentration depended on several factors, such as raw material, yeast strains, and fermentation conditions.Citation[18] The results obtained in this study showed that, in general, the higher alcohols, from 1-propanol to 3-methyl-butanol, present major concentrations in the distillates obtained from red grapes except for 2-butanol. The concentration of 2-butanol was not significantly different in the samples of white grapes. Its concentration increased by the microbiological processes that take place during the ensilage of the grape pomace.[Citation5,Citation19] The results confirmed a higher trend to degradation in the case of the white grape residue. However, only significant differences were observed for the concentrations of 2-methyl and 3-methyl-butanol (p < 0.05). The relationship between 1-propanol/2-methyl-1-propanol, proposed by Cantagrel et al.Citation[20] as an indication of bacterial degradation, is higher for the white grape pomace distillate (0.605) than the red one (0.37).
The concentrations of hexanol and hexenols were significantly more abundant in the white grape distillates. These compounds, that impart herbaceous aromas,Citation[21] increase their concentration during the storage of the raw material.Citation[22] High concentrations of hexanols and hexenols in the spirit could be originated during the destemming and pressing of the grape, especially if both vinification processes were at high temperatures, as well as to a long contact of the solid parts of the cluster; this last aspect justifies that these volatile compounds were in higher concentration in samples distilled from white grape pomace. No significant differences were found for the concentration of allyl alcohol between both types of distillates, though its content was slightly higher in the spirit from red grapes. The concentration of 2-phenyl ethanol was significantly higher in red distillates, and contributed to the aroma with floral nuances. Benzyl alcohol content was similar in both types of distillates. This last compound is released by yeast during alcoholic fermentation.Citation[23]
The concentrations of methyl and ethyl acetate were higher in distillates from red grape pomace probably due to the presence of oxygen in contact with the mass of the red residue in the container. Ethyl acetate may be formed by esterification between acetic acid and ethanol during the alcoholic fermentation or during the distillation process. Qualitatively, ethyl esters are the most important group of volatile compounds in wine and distilled drinks. Although the isoamyl acetate was in high concentration in distillates from red grape pomace, it did not reach significantly different concentrations with regard to those of white residue. Hexyl acetate was in higher concentration in distillates from red grapes, whereas ethyl butyrate prevailed in spirits of white residue. According to Nykänen,Citation[24] ethyl hexanoate, ethyl octanoate, and ethyl decanoate are considered important contributors to the aroma of alcoholic distillates. Ethyl hexanoate showed a significantly higher concentration in the spirits from red grape pomace. It was verified that, in general, the ethyl esters were present in higher concentration in spirits from red grapes, probably due to the presence of wine lees in contact with the grape pomace during the alcoholic fermentation. Nevertheless, the concentration of ethyl lactate was significantly higher in distillates from white grape pomace; this result confirmed, again, its relation with the bacterial degradation.
Acetaldehyde and acetal were determined in order to give an idea of the storage conditions in anaerobiosis and the correct distillation process.Citation[3] Acetal is formed during the fermentative process or during the distillation via the reaction between acetaldehyde and ethanol. Acetaldehyde, when present at low concentration, contributes with a fruity notes (apple), however, a high content is perceived like a sharp smell.Citation[25] The red grape pomace spirits was the group that showed the highest concentration of acetaldehyde and acetal. A correct fermentation and distillation of the raw material is very important to limit the concentration of both volatile compounds and improve the quality of the final drink.
During the raw material storage, the contact with the oxygen is negative, but, especially in the case of grape pomace from red grapes, if it is not distilled immediately after the alcoholic fermentation, the aerobic bacteria will produce degradation, with a reduction of 20–30% of the alcohol and the formation of large quantities of acetic acid, ethyl acetate, acetaldehyde, and acetal.Citation[26,Citation27] Furfural is a volatile compound that originates from the thermal degradation of the sugars,Citation[28] so its content does not depend on a type of grape pomace, but on the type of distilling equipment used. According to Versini et al.,Citation[19] the formation of benzaldehyde is associated with microbial development during ensilage of the grape pomace, and is favoured by the high pH values in the medium. Benzaldehyde was detected in significantly higher concentration in the distillate from white grape pomace. This result confirmed again the trend to microbiological degradation in grape pomace from white grapes. shows the differences between both types of distillates according to the total concentration of volatiles from each family. Methanol, C6 compounds, and ethyl esters presented higher concentrations in white distillates while higher alcohols, acetates, aldehydes, and acetal prevailed in red ones.
Figure 1 Mean and standard deviation of volatile compounds families.
PCA was performed on the concentration of the nine volatile compounds analysed in Orujo spirits, with significant differences (). The two first principal components, PC1 and PC2, accounted for 100% of total variance (72.23 and 27.7%, respectively). The first component (PC1) was characterised by major levels of 2-methyl-1-butanol (A), 3-methyl-1-butanol (B), ethyl hexanoate (E), ethyl octanoate (F), ethyl decanoate (G), and ethyl lactate (H). For the second principal component (PC2), the attributes cos-3-hexenol (C), trans-2-hexenol (D), and benzaldehyde (I) showed high and positive values. The samples of white grape pomace were situated at positive values for PC1 and PC2 and characterized by four volatile compounds (D, I, C, and H). The samples of red grape pomace were located at positive values for PC1 and the negative values for PC2. This group was correlated with five volatile compounds (A, B, F, G, and E).
Figure 2 Principal component análisis (PCA) of Orujo spirits volatile composition (color figure available online).
CONCLUSIONS
The differences between Orujo spirits from red and white grape pomace were more quantitative than qualitative. The Orujo spirits obtained from white grape residues showed a higher concentration of methanol, hexanols, and hexenols than those from red grapes, but this aspect depends on the quality of the raw material, too. Also, the Orujo spirits from white grapes showed higher values for ethyl lactate, benzaldehyde, 2-butanol, and the ratio between 1-propanol and 2-methyl-1-propanol, which indicated a high trend to the degradation of this residue by anaerobic microorganisms. In the spirits from red grapes, the concentration of ethyl esters was higher as well as the concentration of the compounds that derive from the activity of aerobic bacteria, principally, ethyl acetate and acetaldehyde. The results obtained allowed others to advise that, in general, it will be necessary to remove more volume of tails during the white grape pomace distillation and heads in those of red pomaces, in order to obtain a high-quality product.
ACKNOWLEDGMENT
S. Cortés was supported by the doctor INIA-CCAA program, financed by the European Social Fund.
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