Eur Food Res Technol (1999) 209 :257–260 Q Springer-Verlag 1999

ORIGINAL PAPER

Encarna Gómez-Plaza 7 Rocío Gil-MunozJuan Carreno-Espín 7 Jose Antonio Fernández-LópezAdrián Martínez-Cutillas

Investigation on the aroma of wines from seven clones ofMonastrell grapes

Received: 21 April 1998 / Revised version: 19 November 1998

E. Gómez-Plaza (Y) 7 R. Gil-Munoz 7 J. Carreno-EspínA. Martínez-CutillasCentro de Investigación y Desarrollo Agroalimentario,Consejería de Medio Ambiente, Agricultura y Agua,Ctra. La Alberca s/n, E-30150 La Alberca, Murcia, Spaine-mail: Encarna.Gomez 6accesosis.es

J.A. Fernández-LópezDepartamento de Ingeniería Química, Escuela Superior deIngenieros Agrónomos, Universidad de Murcia,Paseo Alfonso XIII, Cartagena, Murcia, Spain

Abstract Analysis of the volatile compounds of winesfrom seven clones of Monastrell grapes was performedusing ultrasound extraction of the compounds. Signifi-cant differences were found for some of the compoundsand these compounds were used in order to character-ize and differentiate among the clones. Linear discrimi-nant analysis allowed some grouping of the clones,showing that differentiation among clones of a neutralgrape variety is possible.

Key words Wine 7 Grape 7 Aroma 7 Volatilecompounds 7 Clones

Introduction

Different clones from one grape variety can differ intheir productive characteristics and their ability to pro-duce wines with different organoleptic characteristics[1]. The evaluation of clones in viticulture is a tedioustask since the grapes and also the wines obtained haveto be evaluated.

Research on the differences in aromatic compoundsin grapes and wines from clones of different varietieshas been done. Ia et al. [1] studied the differences inmonoterpene levels in different clones of Chardonnaygrapes; McCarthy [2] studied the clonal effect on Mus-cat blanc à petit grains terpene concentrations, findingthat differences could be found in bound monoterpene

content but not in free monoterpene content. Otherwork with Chardonnay grapes was done by Versini etal. [3], who also found differences among clones regard-ing the monoterpene contents. Versini et al. [4] foundthat no differences could be determined among flavourcompounds of Riesling clones but some were found inthose for Traminer and Chardonnay.

All these studies were done on aromatic grapes.Monastrell is a neutral variety with an insignificant mo-noterpene content [5] but is of great economic impor-tance since it is the second largest cultivar of red varie-ties in Spain. The characteristics of its wines have alrea-dy been studied [6] and it was found that the most im-portant flavour compounds are those arising from thefermentation process.

Owing to its economic importance, a clonal and san-itary selection of this variety has been accomplished [7]and now the enological characteristics of the differentselected clones are being studied. The aim of this paperis to achieve a possible characterization of the winesfrom the different clones of Monastrell grapes bymeans of several volatile compounds, owing due to thehigh discriminant potential power these variables haveproved to possess in similar cases [8–10].

To differentiate among clones using volatile com-pounds as variables, linear discriminant analysis waschosen, since this technique allows differentiationamong pre-established populations. This type of mathe-matical treatment has been applied previously to wineclassification [9, 11, 12].

Material and methods

Seven clones from Monastrell (Vitis vinifera L.) grapes from theclonal and sanitary selection program held in the Centro de In-vestigación y Desarrollo Agroalimentario (Murcia, Spain) weregrown under identical conditions and harvested at optimum ma-turity. Grapes from each clone were carried to a large-scale ex-perimental winery and divided into three batches to have a tripli-cate vinification of each clone. After malolactic fermentation andcold stabilization the wines were bottled and analysed.

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Table 1 Mean values (standard deviations in parentheses) for the compounds identified in wines from Monastrell clones (mg/l)

Compound Clones

373 118 188 263 21 231 35 CVa

Butyl acetateb 2.74 (0.91) 5.59 (0.94) 3.72 (0.14) 6.94 (0.88) 4.66 (0.91) 3.81 (0.15) 2.95 (0.62) 21Isoamyl alcohol 34.55 (13.05) 47.01 (4.32) 47.75 (4.26) 46.68 (4.80) 45.37 (5.06) 40.46 (11.12) 33.65 (12.20) 11Ethyl hexanoate 0.02 (0.01) 0.04 (0.01) 0.03 (0.01) 0.04 (0.01) 0.02 (0.01) 0.03 (0.01) 0.02 (0.02) 101-Pentanolb 0.01 (0.01) 0.06 (0.05) 0.09 (0.07) 0.01 (0.00) 0.07 (0.06) 0.17 (0.07) 0.04 (0.02) 83-Methylpentanol 0.01 (0.00) 0.01 (0.00) 0.02 (0.00) 0.01 (0.01) 0.01 (0.00) 0.01 (0.00) 0.01 (0.00) 12Ethyl lactateb 7.14 (1.50) 7.30 (0.54) 6.42 (0.44) 8.33 (1.13) 5.72 (1.00) 5.71 (0.68) 6.89 (0.31) 71-Hexanol 0.24 (0.03) 0.31 (0.01) 0.30 (0.02) 0.32 (0.04) 0.29 (0.03) 0.28 (0.04) 0.31 (0.04) 7Ethyl octanoate 1.58 (0.43) 1.85 (0.33) 1.46 (0.93) 1.67 (0.40) 0.83 (0.05) 1.36 (0.53) 1.22 (0.53) 122,3-Butanediol 0.01 (0.01) 0.02 (0.00) 0.02 (0.00) 0.01 (0.01) 0.01 (0.00) 0.01 (0.00) 0.02 (0.00) 151-Octen-4-ol 0.02 (0.00) 0.02 (0.00) 0.02 (0.00) 0.01 (0.01) 0.01 (0.01) 0.02 (0.00) 0.02 (0.00) 142-Methylbutanoic acid 0.24 (0.01) 0.23 (0.02) 0.18 (0.12) 0.26 (0.01) 0.23 (0.03) 0.19 (0.03) 0.19 (0.01) 8Hydroxybutanoneb 0.13 (0.05) 0.07 (0.01) 0.04 (0.03) 0.08 (0.00) 0.04 (0.03) 0.05 (0.00) 0.06 (0.00) 10Butyrolactone 1.70 (0.37) 1.75 (0.06) 1.85 (0.04) 1.54 (0.04) 1.91 (0.64) 1.62 (0.28) 1.73 (0.19) 7Butanoic acid 0.09 (0.01) 0.08 (0.01) 0.07 (0.02) 0.10 (0.02) 0.15 (0.01) 0.06 (0.02) 0.07 (0.01) 123-Methylbutanoic acid 0.30 (0.03) 0.31 (0.03) 0.33 (0.02) 0.31 (0.02) 0.37 (0.1) 0.29 (0.04) 0.28 (0.03) 6Diethyl succinate 1.07 (0.74) 0.55 (0.05) 0.72 (0.05) 0.84 (0.11) 1.11 (0.04) 0.76 (0.13) 0.54 (0.19) 53-Methylthio-l-propanolb 0.06 (0.01) 0.18 (0.02) 0.19 (0.02) 0.16 (0.02) 0.19 (0.04) 0.17 (0.02) 0.18 (0.02) 10Hexanoic acidb 0.25 (0.01) 0.26 (0.02) 0.26 (0.01) 0.32 (0.07) 0.21 (0.00) 0.21 (0.00) 0.22 (0.02) 8Geraniol 0.13 (0.01) 0.12 (0.02) 0.14 (0.00) 0.14 (0.03) 0.12 (0.07) 0.18 (0.01) 0.1 (0.03) 72-Phenylethanol 11.09 (1.35) 11.03 (1.02) 12.28 (0.57) 11.54 (3.84) 13.96 (4.03) 11.24 (0.36) 11.06 (1.50) 64-Ethylguaiacolb 0.02 (0.00) 0.03 (0.02) 0.06 (0.02) 0.02 (0.01) 0.05 (0.02) 0.04 (0.01) 0.05 (0.01) 10Octanoic acid 0.32 (0.05) 0.30 (0.03) 0.31 (0.02) 0.42 (0.10) 0.30 (0.07) 0.27 (0.02) 0.30 (0.01) 84-Ethylphenolb 0.23 (0.03) 0.27 (0.13) 0.39 (0.12) 0.16 (0.06) 0.21 (0.04) 0.26 (0.06) 0.39 (0.05) 7Nonanoic acidb 0.03 (0.02) 0.01 (0.01) 0.01 (0.00) 0.04 (0.01) 0.01 (0.00) 0.01 (0.00) 0.01 (0.00) 142-Propenyl benzeneacetate 0.04 (0.01) 0.04 (0.00) 0.03 (0.02) 0.04 (0.01) 0.05 (0.02) 0.04 (0.00) 0.04 (0.00) 14Decanoic acid 0.04 (0.01) 0.05 (0.01) 0.05 (0.01) 0.04 (0.00) 0.06 (0.02) 0.05 (0.00) 0.05 (0.01) 15Dodecanoic acid 0.02 (0.00) 0.01 (0.01) 0.02 (0.01) 0.02 (0.00) 0.02 (0.01) 0.01 (0.01) 0.01 (0.01) 15Dihydro-b-ionone 0.02 (0.00) 0.04 (0.01) 0.02 (0.01) 0.02 (0.00) 0.06 (0.04) 0.02 (0.01) 0.05 (0.02) 93-Oxo-a-ionol 0.01 (0.00) 0.01 (0.00) 0.01 (0.00) 0.01 (0.00) 0.01 (0.00) 0.01 (0.00) 0.01 (0.00) 72-Phenylethyl isoamylate 0.21 (0.05) 0.10 (0.03) 0.09 (0.04) 0.15 (0.03) 0.15 (0.03) 0.11 (0.04) 0.07 (0.01) 8Sum of estersb 12.80 (4.27) 15.43 (0.49) 12.47 (0.89) 18.01 (1.59) 12.54 (0.89) 11.82 (1.07) 11.19 (0.93)Sum of alcoholsb 46.38 (4.20) 58.76 (2.46) 61.27 (1.62) 59.97 (2.14) 60.29 (3.53) 52.85 (8.12) 45.83 (7.88)Sum of acids 1.29 (0.18) 1.25 (0.02) 1.23 (0.11) 1.51 (0.16) 1.35 (0.21) 1.09 (0.16) 1.13 (0.05)

a Coefficient of variation of the analytical methodb Difference was significant at the 95% level according to the clone

For the determination of the aroma compounds, triplicateanalyses were done for each wine. The method described by Co-cito et al. [13] was followed. Thus 50 ml of wine in a 200 ml spher-ical flask with 15 ml of dichloromethane were extracted by meansof ultrasound at 20 7C over 10 min. 2-Octanol was used as internalstandard. After separation, the organic layers were dried with an-hydrous sodium sulphate and transferred to a tube. A second anda third extraction were performed with 5 ml of dichloromethaneand the organic layers were collected in the same flask. The ex-tracts were concentrated to a final volume of 0.5 ml under a N2

stream and analysed using a GC-MS system. The concentratedextract was injected into a Hewlett-Packard 5890 gas chromato-graph (Hewlett-Packard, Avondale, Pa.). Separations were per-formed using a HP-20 M column (25 m!0.25 mm, Hewlett-Pack-ard). The oven temperature was programmed from 60 7C to210 7C (4 7C/min). Helium was used as the carrier gas at an aver-age linear velocity of 60 cm/s. The injector was maintained at200 7C and the FID detector at 250 7C. For the identification ofcompounds, a Hewlett-Packard 5890 gas chromatograph coupledwith a Hewlett-Packard 5971 A mass-selective detector was used.The chromatographic separations were performed under thesame conditions described above. The temperature of the detec-tor was 170 7C. The spectra were recorded at an ionization voltageof 70 eV, with a speed of 2.1 scan/s over a mass range of m/z20–260. Data processing was performed with a Hewlett-Packard5895 GC ChemStation.

Sample components were tentatively identified by mass spec-trum matching with a mass spectral library collection. The tenta-tive MS identifications were verified by comparison of the com-ponent’s experimental retention index with that of reference

standards. The retention index system proposed by Kovats [14]was used.

Statistics were carried out using the STATGRAPHICS Plus2.0 statistical sofware.

Results and discussion

Table 1 gives the results of the volatile compounds ana-lysed. Mean values and standard deviation are re-ported. The coefficient of variation of the analyticalmethod is also reported. In addition, those variablespresenting significant differences among clones(P~0.05) are also stated.

Some aspects concerning the successful analyticaldifferentiation of grape and wine varieties are a com-plete enrichment of the aroma compounds and a com-plete and quantitatively reproducible separation of thearoma compounds. Cocito et al. [13] developed a newtechnique for the extraction of aroma compounds inmust and wines by means of ultrasound, showing goodrecovery, linearity and reproducibility with shorter op-eration times. We have applied this method to our in-vestigations. Three analyses were done on each wine to

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examine the reproducibility of the extraction method.The extraction efficiency and linearity proved to begood. Thus, considering the simplicity and quantitativeaccuracy of the ultrasound extraction, this method canbe considered a valid alternative to long, laborious ex-tractions and this is particularly true for the rapidscreening in clonal selection. We found coefficients ofvariation somewhat higher than those found by Cocitoet al. [13]. The highest value was found for butyl ace-tate, probably owing to its high volatility. For thosecompounds occurring in low concentrations, higher var-iations were found.

Thirty compounds have been quantified in the winesfrom the seven clones of Monastrell grapes. Among thealiphatic alcohols, isoamyl alcohol shows the highestconcentration in all of the samples. Aliphatic alcohols,the major compounds in wines, are formed in aminoacid metabolism. The higher aliphatic alcohols may beformed from related aldehydes by reduction duringyeast fermentation.

Another alcohol present at very high concentrationis 2-phenylethanol. Aromatic alcohols are mainly deri-vatives of the phenylpropanoid metabolism in thegrapes or are formed by the yeast from several grapeprecursors. It has a pleasant fruity aroma [15]. In Noblemuscadine wine [16], 2-phenylethanol is a major com-pound formed during vinification, although it can befound in Muscadine grape skins as it also occurs inMonastrell grapes [5] in free form as well as glycosidi-cally bound. Rankine and Pocock [15] observed thatthe grape variety influenced the amount of 2-phenyle-thanol.

The total sum of the alcohols showed that clones 373and 35 had significantly lower values of alcohols thanclones 263, 118, 188 and 21 (LSD multiple range test,P~0.05).

Among the esters, important quantities of ethyl lac-tate, ethyl octanoate and diethyl succinate were found.These compounds are synthesized during must fermen-tation and they are particularly important in youngwine aroma. They usually occur at levels above theirthresholds. Van der Merwe and Van Wyk [17] showedthat ethyl esters may have a suppressor effect on thearomatic intensity of acetates; however, they contributepositively to generate quality wines. The quantities ofthese esters prior to fermentation are negligible. Theformation of volatile esters is attributable to enzymaticaction of yeast during fermentation [18]. Esters are eas-ily hydrolysed. Ethyl acetate, hexanoate, octanoate anddecanoate all decreased with time. The concentrationof ethyl lactate ranged from 5.71 mg/l for clones 21 and231 to 8.33 mg/l for clone 263. The sum of the estersshowed that clone 263 differed significantly from clones21, 188, 231, 35 and 373.

Fatty acids are also formed during yeast metabolism.Eight fatty acids were detected, from C4 to C12. Hexa-noic acid and octanoic acid occurred more abundantly.The total sum of the acids showed that clone 231 pre-sented the lowest proportion of fatty acids and clones

263 and 21 the highest, but no significant differencescould be found at the 95% significance level.

Important quantities of butyrolactone have been de-tected in these wines. Although some lactones havebeen found in grapes stored in anaerobic conditions un-der CO2 [19], they are mainly formed by the yeastalong the amino acid and keto acid metabolism [20].

Two volatile phenols, 4-ethylguaiacol and 4-ethyl-phenol, have been identified. Beyond a certain concen-tration they may be responsible for sensory faults (phe-nolic or pharmaceutical) in wine aroma [21].

To attempt a differentiation of the different clones,linear discriminant analysis was chosen, since this tech-nique allows differentiation among pre-establishedpopulations. When distinguishing the wines from thedifferent clones the variables that showed significantdifferences (P~0.05) were chosen. Those variables in-cluded butyl acetate, 1-pentanol, ethyl lactate, hydroxy-butanone, 3-methylthio-1-propanol, hexanoic acid, 4-ethylguaiacol, 4-ethylphenol and nonanoic acid. Thevariables sum of the esters and sum of the alcoholswere not included since they are linear combinations ofother variables. Among these variables, 4-ethylguaiacoland 4-ethylphenol may arise from the phenolic acids ofthe hydroxycinnamic series (p-coumaric and ferulicacids) [21]. This fact suggests that differences in thephenolic content of the grapes from the different clonesmay occur. Two esters are included in the variablesused in the discriminant analysis. Butyl acetate hasbeen found in grapes and must prior to fermentation,but ethyl lactate is a fermentation product. The forma-tion of esters along the fermentation proccess is relatedto vinification factors (temperature, yeasts) but also tomust composition [8], so differences in ethyl lactatemay be due to differences in must composition of thedifferent clones. Alvarez et al. [8] also found that estersallowed a good classification of white wines from differ-ent geographical origins.

Six discriminating functions were obtained. The firsttwo of them, statistically significant (P~0.05), explain88% of the variance. The standardized coefficients ofthese two discriminant functions showed that ethyl lac-tate and nonanoic acid had the higher weight in dis-criminating among clones (Table 2). The projection ac-cording to the first and second discriminant functionsfor the different clones is shown in Fig. 1. The classifi-cation functions obtained for each clone using the se-lected variables enabled a 100% correct classification ofthe samples. For this reason, according to Fig. 1, it canbe stated that wines from different clones of Monastrellgrapes can be distinguished according to their aromaticcomposition.

Conclusions

Significant statistical changes in volatile compositionwithin seven different clones from Monastrell grapeshave been detected. Linear discriminant analysis using

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Table 2 Discriminant function standardized coefficients for clone

Compound Discriminant functions

1 2 3 4 5 6

Butyl acetate 0.378 P2.821 P0.895 0.003 0.287 0.5081-Pentanol 0.529 P0.548 P0.752 P0.880 P0.261 0.381Ethyl lactate 2.293 1.503 0.428 P0.347 0.047 P0.351Hydroxybutanone 0.388 P1.925 P0.981 P0.595 1.217 0.3973-Methylthio-1-propanol P1.025 P0.142 P1.321 0.024 0.837 P0.410Hexanoic acid 0.973 0.123 P0.321 0.265 P0.662 0.9924-Ethylguaiacol 1.018 0.226 0.405 P0.005 P0.233 P0.0494-Ethylphenol P0.634 P2.017 P0.073 0.215 P0.159 0.101Nonanoic acid 1.417 1.555 0.208 P0.380 0.104 P1.540

Fig. 1 Plot of discriminant functions

those variables with significant differences amongclones showed that a 100% correct classification couldbe obtained, although, owing to the low number ofsamples in each group no validation was carried out.These results lead us to the conclusion that the volatilecompounds can be a valuable tool for the classificationand discrimination of different clones and that winesmay possess different organoleptic characteristics in re-lation not only to varieties but also to clones.

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