Aroma composition of wine studied by different extraction methods

Aroma composition of wine studied by different extraction methods

Analytica Chimica Acta 458 (2002) 85–93 Aroma composition of wine studied by different extraction methods M. Ortega-Heras, M.L. González-SanJosé∗ , S...

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Analytica Chimica Acta 458 (2002) 85–93

Aroma composition of wine studied by different extraction methods M. Ortega-Heras, M.L. González-SanJosé∗ , S. Beltrán Department of Biotechnology and Food Science, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, Spain Received 20 June 2001; received in revised form 30 August 2001; accepted 7 November 2001

Abstract Several isolation and concentration methods have been developed for the analysis of volatile components in wine. However, it is generally admitted that none of them fulfill all the requirements for the isolation of aroma compounds and that it is necessary to combine different extraction methods to obtain the complete volatile fraction. Three extraction methods have been studied: liquid–liquid extraction, static headspace and a new method developed in our laboratory, which has the same principle as the dynamic headspace technique. The volatiles were swept along with a carrier gas and later condensed in a cold trap. The advantages and disadvantages of the three methods regarding sample preparation, component losses and artifact formation, have been evaluated. Also, the sensitivity and reproducibility have been compared. With the liquid–liquid extraction method used, a new compound has been described for the first time in wine. This compound is 5,6,7,7a-tetrahydro-4,4,7a-trimethylbenzofuran-2(4H)-one. Liquid–liquid extraction and static headspace show good reproducibility; although static headspace is useful for the analysis of highly volatile compounds, it is not able to detect trace compounds due to its lack of sensitivity. The new method allows to obtain an extract, free of artifacts and foreign substances that were not in the original wine. As no solvent is used, the extracts may be tasted and sensorially analyzed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Liquid–liquid extraction; Static headspace; Stripping with nitrogen; Wine volatiles

1. Introduction Wine is one of the most complex alcoholic beverages, its aroma providing much of such complexity. Describing the richness of the wine features and the great variety of wine aromas is far from a simple task for researchers. Some of the reasons for such complexity are: • More than 800 components have been identified in the volatile fraction of wine. ∗

Corresponding author. Tel.: +34-947-258815; fax: +34-947-258831. E-mail address: [email protected] (M.L. Gonz´alez-SanJos´e).

• Volatile components have a different chemical nature covering a wide range of polarity, solubility, volatility and pH. • An important number of the volatile components in wine can only be found at very low concentration (␮g ml−1 ). Therefore, the samples need to be highly concentrated for them to be accurately quantified. • Many of the aromatic components are unstable. They may be easily oxidized in contact with air or degraded by heat or extreme pH, giving rise to the appearance of artifacts. One of the main problems that researchers have to face when studying the compounds responsible for wine aroma is the choice of a suitable extraction

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procedure to qualitatively and quantitatively represent the wine original aroma. That is, to obtain an extract that contains all the volatile compounds contained in the original wine without they having been altered or degraded or artifacts been formed. Several methods have been developed trying to achieve that goal. All of them present some advantages and disadvantages regarding each other [1,2]. Usually, it is necessary to combine different methods to obtain the complete extraction of all the volatile compounds contained in wine without them being altered. Liquid–liquid extraction continues being the reference technique for the extraction of volatile components from wine [3–6]. In this method, all volatile compounds (low, medium and high volatility) have a high partition coefficient to the organic phase, although it requires solvent evaporation, which in some cases involves loss or degradation of some of the components and formation of some others that were not in the original wine [7]. Static headspace techniques have several advantages. They are fast and the sample is hardly manipulated. However, their main disadvantage is their low sensitivity [8,9]. Dynamic headspace techniques are becoming more and more popular and many authors have applied them for studying the aromatic composition of wine in the last decades [10–12]. In this report, a technique of extraction with an inert carrier gas, that applies the same principle as the dynamic headspace technique, has been developed. In this method, the volatiles are swept along with a carrier gas and then condensed in a cold trap. The three techniques previously described have been comparatively applied in this work to the study of the aromatic composition of wines. The advantages and disadvantages of the three methods regarding sample preparation, component loss and artifacts formation have been evaluated. Also, the sensitivity and reproducibility have been comparatively studied.

2. Materials and methods 2.1. Materials Three commercial wines (white, rose and red) were studied. All were young wines purchased in bottles of

750 ml. The white wine was elaborated from Chardonnay grapes and the rose and red wines were made from Tempranillo grapes. These samples were chosen in order to study wines with different aromatic characteristics. The dichloromethane used was of HPLC grade (99.9% purity). NaCl (PS quality from Panreac) was used for promoting the salting-out effect in the static headspace method. The gases used were: nitrogen (99.999% purity) as carrier gas for the corresponding extraction procedure, to obtain an inert atmosphere when necessary and as make-up gas for the flame ionization detector (FID), helium (99.999% purity) as carrier gas for the gas chromatograph–FID (GC–FID) and GC–mass spectrometer (GC–MS); and air (99.9995% purity) and hydrogen (99.999% purity) for the FID. All the gases were obtained from Carburos Metálicos (Spain). Chemical standards were from Fluka, Sigma and Aldrich with a purity grade >97%. 2.2. Methods Three techniques were used for extraction of the volatile components from the wine samples. 2.2.1. Liquid–liquid extraction The method proposed by Moio et al. [13] was followed. Two hundred milliliters of wine, 5 ml of dichloromethane and 50 ␮l of an internal standard (a solution of 1000 ␮g ml−1 2-octanol in ethanol) were introduced by means of a micro-tube peristaltic pump (Eyela MP-3) into a special flask [13], where air had previously been removed with nitrogen. The flask was placed in an ice bath and stirred at 500 rpm for 3 h. After phase separation, the organic phase and interphase, which is an emulsion, were taken into an Ependorff tube. The formation of an emulsion in the interface that still contains volatile compounds that must be recuperated is fairly common. For such recuperation, the emulsion was broken by centrifugation at 9588 g during 15 min and at a temperature <5 ◦ C, in order to avoid possible alterations of the extract. The organic extract so obtained was introduced into a 2 ml vial that was stored at −80 ◦ C until analysis. Every sample was extracted at least twice.

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2.2.2. Static headspace NaCl (1.25 g), wine (5 ml) and the internal standard (25 ␮l) used in the above described liquid–liquid extraction method, were added to a 10 ml vial that was sealed with a 19 mm butyl/PTFE septum and 20 mm open capsule. A headspace autosampler (Hewlett-Packard 7694E) connected to a GC (Hewlett-Packard 6890) was used for analysis. The sampler was equipped with a 3 ml injection loop and a transfer liner directly connected to the injector of the chromatograph. The vials were kept at 75 ◦ C for 60 min. The injection loop was heated up to 90 ◦ C and the transfer liner up to 100 ◦ C. The injection conditions in the GC were: 0.2 min time for vial pressurization; 0.15 min for vial equilibrium; 0.10 min for filling the injection loop; 2 min for sample injection. Helium was used for vial pressurization up to 17 psi and as carrier gas to transport the sample from the headspace with a flow of 17 ml min−1 as measured at the exit of the split. Every wine sample was analyzed at least twice. 2.2.3. Stripping with nitrogen This technique was first developed by Mato et al. [14] for determination of isothermal vapor–liquid equilibria and has been adapted in our laboratory for determination of the aromatic fraction of wine. The principle of this technique is the same as of the dynamic headspace technique. The volatile components are swept along with a carrier gas and condensed in a cold trap. The equipment used is presented in Fig. 1. It consists of a bottle of nitrogen (1) used as the inert carrier gas that sweeps the volatile components from the sample; a large volume vessel (2) to damper the oscillations caused by a non-regular nitrogen flow; a drying sequence (3) for drying the carrier gas; a needle valve (4) (Hoke 1315 G6Y) for nitrogen flow regulation; a 3 m long copper coil (1/8 in.) (5) that is submerged in a thermostated bath (6) to take the carrier gas to the temperature of interest (40 ◦ C); two jacketed columns (7) that contain the wine sample (25 ml) soaked in a highly efficient random packing (stainless steel Dixon rings) where the carrier gas is saturated with the volatile components, and a cold trap (8). The advantage of this technique lies in the improvement of the gas–wine contact provided by the packing placed in the columns that allows better volatile stripping

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Fig. 1. Apparatus for stripping with nitrogen (for details see Section 2.2.3).

than other dynamic headspace methods. The saturation columns are 30 cm long and 1 cm in diameter and they are kept at 40 ◦ C by circulation of the fluid in the thermostatic bath (6) through the jackets of the columns. The system where the saturated gas flows towards the cold trap is formed by a glass tube kept at 40 ◦ C through a heat resistance (Nichrome, 0.4 mm × 0.7 mm, 28.14  min−1 ) in order to avoid condensation. This tube ends in a cold trap immersed in liquid nitrogen to allow condensation of the volatile components and the exit of the non-condensable inert gas. The nitrogen flow was low enough (30 ml min−1 ) for the carrier gas to reach saturation in the columns. Each experiment lasted 45 min, the time needed for collecting enough extract for analysis. The solvent of the aromatic components in this case is ethanol that is swept by the carrier gas together with the volatile components and also trapped in the cold trap. 2.2.4. Chromatographic method A GC (Hewlett-Packard 6890) equipped with a FID was used for quantification of the volatile components extracted from the wine samples according to the three techniques previously described. The column was a 50 m × 0.25 mm carbowax BTR with a 0.33 ␮m thick stationary phase. One microliter of extract sample was injected in the splitless mode. The injector temperature was kept at 220 ◦ C and the FID was kept at 250 ◦ C. The carrier gas was helium at 1.3 ml min−1 . The temperature program was: 40 ◦ C for 8 min, raised to 85 ◦ C at 10 ◦ C min−1 and held for

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1 min, raised to 110 ◦ C at 20 ◦ C min−1 and held for 1 min and finally raised to 200 ◦ C at 3 ◦ C min−1 and held for 40 min. Isolated peaks were identified using chemical standards and mass spectral data. A Hewlett-Packard 5973 mass detector fitted with a Hewlett-Packard 6890 GC was used. The ionization of the samples was achieved at 70 eV under the SCAN mode (1 scan s−1 ). The mass range studied was from 30 to 250 m/z. A Hewlett-Packard Chemstation equipped with the Wiley 275 library was used for component identification. The chromatographic conditions were the same as those described above, except for helium flow that was 1 ml min−1 .

3. Results and discussion A summary of the data obtained by the three methods evaluated is presented in Table 1. This table gives comparative information of the compounds isolated by each technique. It also shows the reproducibility achievable by each procedure calculated as coefficients of variation (CVs). 3.1. Liquid–liquid extraction Some of the disadvantages of this method have been described in the previous sections. Some of them may be avoided by the method developed by Moio et al.

Table 1 Compounds identified by each method and the CV of the concentration value for each compound from five different extractions (n = 5) of the same wine Compounds

Reliability of identificationa

Extraction method Liquid–liquid Occurrenceb

SHS CV (%)

Occurrenceb

Stripping CV (%)

Occurrenceb

CV (%)

Fusel alcohols Isobutyl alcohol Isoamyl alcohol 2-Phenyl-1-ethanol

a a a

+ + +

3.20 1.66 2.50

+ + +

4.66 5.12 6.21

+ + +

>15 >15 >15

C6 alcohols Hexanol trans-3-Hexen-1-ol cis-3-Hexen-1-ol

a b a

+ + +

2.54 2.79 2.80

+ − −

5.79

+ − +

>15

Other alcohols Propanol 1-Butanol 3-Methyl-1-pentanol Heptanol Octanol 2,3-Butanediol Methionol Benzyl alcohol

a a b a b a b a

+ + + + + + + +

9.54 5.94 6.52 5.07 2.48 5.85 4.09 10.35

+ + − − − − − −

4.5 5.13

+ + − − − + + −

>15 >15

Ethyl esters Ethyl butanoate Ethyl hexanoate Ethyl octanoate Ethyl decanoate

a b a a

+ + + +

7.08 3.33 10.20 1.84

+ + + +

5.42 4.88 7.56 2.78

+ + + +

>15 >15 >15 >15

Acetates Ethyl acetate Isobutyl acetate Isoamyl acetate Hexyl acetate Ethyl-2-phenyl acetate

a b b b b

− + + + +

+ − + − −

4.88

+ − + − +

>15

3.95 2.53 2.86 3.84

7.08

>15

>15 >15

>15 >15

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Table 1 (Continued ) Compounds

Reliability of identificationa

Extraction method Liquid–liquid Occurrenceb

SHS CV (%)

Occurrenceb

Stripping CV (%)

Occurrenceb

CV (%)

+ +

>15 >15

Other esters Ethyl lactate Diethyl succinate Ethyl-3-methylbutyl succinate Diethyl-2-OH-glutarate 3-OH-ethyl butanoate 4-OH-ethyl butanoate Ethyl-2-methyl butanoate Ethyl-3-methyl butanoate

b b b b b b b b

+ + + + + + + +

2.29 1.30 7.70 4.58 2.57 2.74 7.08 6.54

+ + − − + − + +

5.61 7.27

Acids Hexanoic acid Octanoic acid Decanoic acid

a b b

+ + +

3.62 2.45 3.12

+ − −

11.57

Terpene alcohols Linalol ␣-Terpineol Citronellol Geraniol

a a a a

+ + + +

5.14 5.70 6.76 6.00

− − −

− − − −

b b b c

+ + + +

2.00 3.70 3.19 3.30

− − − −

+ − − −

Phenols Eugenol Vinyl guaiacol Methoxy eugenol

a b b

+ + +

8.62 5.36 10.01

− − −

− − −

Aldehydes Acetaldehyde Benzaldehyde Furfural

a a a

+ + +

3.06 7.96

+ + −

Miscellaneous Acetic acid 3-Methyl-butyl-acetamide 3-Hydroxy-3-butanone

a b b

+ + +

6.09 5.21 3.97

− − +

Lactones ␥-Butyrolactone ␥-Nonalactone 4-Ethoxy carbonyl-␥-butyrolactone 5,6,7,7a-Tetrahydro-4,4,7atrimethylbenzofuran-2(4H)-one

a

5.12 5.36 6.24

2.23 5.96

5.36

− − − − − − − −

>15

+ + +

>15 >15 >15

+ − +

>15 >15

Identification: (a) mass spectrum in agreement with spectra found in the Wiley 275 library and the retention times are the same than those of the pure substances available in the laboratory estimated on the same column; (b) the pure substance was not available but the mass spectrum and the elution order matched with those found in the literature; (c) identification based in the compound assigned by the library Wiley 275 with P > 90%. b Occurrence: (+) compound identified; (−) not detected.

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[13]. This method has been successfully used for the identification of impact odorants of certain wines by GC–olfactometry [15,16]. However, the compounds isolated have never been identified and the precision of the method has not been studied before. In this method, a minimum solvent/sample volume ratio is used, which increases the concentration without the need for later evaporation, thus avoiding the loss of volatiles, the appearance of solvent impurities and the lack of reproducibility. Furthermore, as the extraction takes place in an oxygen-free atmosphere and at low temperature, the possibility of thermal degradation, oxidation and chemical reaction between the extracted compounds is reduced. Another advantage of this method with respect to the classical liquid–liquid extraction is that the experimentation time is reduced significantly (from at least 24 to 3 h maximum). All these reasons made this method suitable for the straight forward analysis of samples. As can be seen in Table 1, this method was the best of the three procedures tested in terms of the extraction efficiency for the high, medium and low volatility components. Therefore, the esters, alcohols, acids, terpenes, lactones, volatile phenols and aldehydes content of most of the wines can be identified, which are also the most studied and known compounds. This method is sensitive enough to identify trace compounds contained in wine in concentrations of a few ␮g l−1 . However, compounds present in even lower concentrations can only be detected only after a concentration stage. The main disadvantage of this method is the use of an organic solvent (dichloromethane) that is toxic. However, the volume of solvent used is very small if it is compared with other extraction methods. Moreover, as extraction is carried out at low temperature and in a closed vessel, the risk of dichloromethane losses is minimized. The method shows good reproducibility, even for compounds present in low concentrations. Each type of wine studied (white, rose and red) was extracted five times. Each extract was injected twice into the chromatograph. The corresponding relative standard deviations were calculated from 10 data for each type of wine (five replicates × two injections). However, the relative standard deviations shown in Table 1 are the average of the values calculated for each type of wine. This values were <5% and only

in three compounds out of a total of 48 they were >10%, with a maximum value of 10.35 for benzyl alcohol. All the compounds identified, except for one, have previously been described in the literature as wine or grape components. The exception is 5,6,7,7a-tetrahydro-4,4,7a-trimethylbenzofuran-2(4H)-one. This compound appears in all the wines analyzed in remarkable concentrations, therefore, it can play an important role in their flavor. This compound has a chemical structure quite similar to the compound named by Guth [17] as wine lactone: 3a,4,5,7a-tetrahydro3,6-dimethylbenzofuran-2(3H)-one. Guth identified this compound for the first time in Scheurebe and Gewürztraminer wines and it is an important odorant of both varieties; it possesses a “sweet-coconut-like” aroma. Although, both compounds belong to the same chemical family, a comparison of their mass spectra, obtained in the electron impact mode generated at 70 eV shows that both compounds do not give the same ions. The base ion of the compound identified in this work is m/z = 111 (Fig. 2a), while in the case of the wine lactone it is 151 [18] (Fig. 2b). Furthermore, these compounds have been detected in different zones of the chromatogram from polar columns, while wine lactone has been found close to vinyl guaiacol and before decanoic acid (peaks 1 and 2 in Fig. 3); the new compound elutes after decanoic acid. Therefore, these compounds are different. Bonuläder et al. [19] identified for the first time a glucoconjugate precursor of the wine lactone. This compound is the glucose ester of (E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoic acid. It can be expected that the compound identified in this work has a similar precursor. It is likely that this identification has been possible thanks to the fact that the method developed by Moio et al. [13] is not aggressive, what made possible the extraction and detection of compounds with relative low stability without any change in their chemical structure. 3.2. Static headspace The static headspace (SHS) is a suitable method for the normal analysis and quantification of most of the volatile compounds in wine because the preparation of the sample is very simple and the extraction and

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Fig. 2. (a) Mass spectrum (EI) of 5,6,7,7a-tetrahydro-4,4,7a-trimethylbenzofuran-2(3H)-one. (b) Mass spectrum (EI) of wine lactone [16].

analysis is completely automated. Moreover, the cost is quite low and since no organic solvent is used, the danger of toxicity is nil. One of the advantages of this method versus the liquid–liquid extraction is that the analytes are extracted from the sample matrix without the use of an organic solvent, so in the chromatogram the solvent peak does not appear. This is very important for detection of some interesting peaks that elute with the

solvent peak, such as the acetaldehyde and the ethyl acetate. However, the method is only sensitive for detection of highly volatile components or medium volatile ones present in high concentrations, such as 2-phenyl-1-ethanol. This method has a good reproducibility, as can be seen in Table 1. The CVs range from 3 to 8%, being >10% only for hexanoic acid.

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Fig. 3. Chromatogram corresponding to a liquid–liquid extract.

3.3. Stripping with nitrogen This method shows some of the advantages of the static headspace method, such as the non-use of an

organic solvent. Therefore, no peaks due to impurities of the solvent appear in the extract. The possibility of thermal degradation is reduced since the temperature is controlled and kept low (40 ◦ C maximum). The

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oxygen is removed from the system by the nitrogen, thus avoiding the oxidation of the volatiles. Furthermore, no concentration step is needed. The time of extraction is quite short and the sample preparation is minimum, thus the total time of analysis is reduced. As can be seen in Table 1, the new method developed is more efficient than the static headspace method, although it does not allow identification of as many compounds as by liquid–liquid extraction. Only high and medium volatility compounds can be identified. The volume of the extract obtained under the conditions used in this work is quite small, about 500 ␮l. Bigger volumes provoke a dilution effect since more ethanol, which functions as a solvent, is extracted, so the sensitivity of the method would be reduced. On the other hand, due to the fact that part of the sample remains condensed in the walls of the conductor tube, the method has a very poor reproducibility (CV > 15%). From all these facts, it can be concluded that the method developed in our laboratory allows an extract to be obtained that is free of artifacts and foreign substances that were not in the original wine. As no organic solvent is used, this extract can be smelt without solvent interference or temperature problems and it can also be tasted, what can be very useful for the study of impact odorants, completing the information obtained by GC–olfactometry which does not allow retronasal evaluation. However, this method is not suitable for giving quantitative data.

4. Conclusions A new volatile compound, never described before as a wine component, has been identified. The new method developed in our laboratory, after a more detailed study and optimization of some of the parameters affecting the extraction process, could be used as a laboratory technique or even applied at an industrial level to obtain unaltered wine aroma extracts

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representative of the original wine aroma. Furthermore, one of the main advantages of this method is that the extracts are suitable for analysis by sensorial techniques, due to the fact that no organic solvent is employed in the extraction process and this extract is just formed by wine components. References [1] G.P. Blanch, G. Reglero, M. Herraiz, J. Tabera, J. Chromatogr. Sci. 29 (1991) 11. [2] P.X. Etievant, Crit. Rev. Food Sci. Nutr. 36 (7) (1996) 733. [3] J. Villén, F.J.D. Señoráns, G. Reglero, M. Herra´ız, J. Agric. Food Chem. 43 (1995) 717. [4] Y. Zhou, R. Riesen, C.S. Gilpin, J. Agric. Food Chem. 44 (1996) 818. [5] R. Schneider, R. Baumes, C. Bayonove, A. Razungles, J. Agric. Food Chem. 46 (1998) 3230. [6] V. Lavigne, R. Henry, D. Dubourdieu, Sci. des Aliments 18 (1998) 175. [7] T.H. Parliament, Solvent extraction and distillation techniques, in: R. Marsili (Ed.), Techniques for Analyzing Food Aroma, Marcel Dekker, New York, 1997, p. 1. [8] G. Takeoka, Aroma compounds, in: R. Wittkowski, R. Matissek (Eds.), Capillary gas chromatography in Food Control and Research, Lancaster, 1990, p. 109. [9] T.P. Wampler, Analysis of food volatiles using headspace–gas chromatographic techniques, in: R. Marsili (Ed.), Techniques for Analyzing Food Aroma, Marcel Dekker, New York, 1997, p. 27. [10] H.T. Badings, C. de Jong, J. High Resol. Chromatogr. Chromatogr. Commun. 8 (1985) 755. [11] M.R. Salinas, G.L. Alonso, F.J. Esteban-Infantes, J. Agric. Food Chem. 42 (1994) 1328. [12] C. Garc´ıa-Jares, S. Garc´ıa-Mart´ın, R. Cela-Torrijo, J. Agric. Food Chem. 43 (1995) 764. [13] L. Moio, E. Chambellant, I. Lesschaeve, S. Issanchou, P. Schlich, P.X. Etievant, Ital. Food Sci. 3 (1995) 265. [14] F. Mato, F. Fdz-Polanco, F. Sobrón, J. Santos, Ann. Quim. 79 (1983) 121. [15] L. Moio, P.X. Etievant, Am. J. Enol. Vitic. 46 (3) (1995) 393. [16] C. Priser, P.X. Etievant, S. Nicklaus, O. Brun, J. Agric. Food Chem. 45 (1997) 3511. [17] H. Guth, J. Agric. Food Chem. 45 (1997) 3022. [18] H. Guth, J. Agric. Food Chem. 45 (1997) 3027. [19] B. Bonuländer, B. Baderschneider, M. Messerer, P. Winterhalter, J. Agric. Food Chem. 46 (1998) 1474.