SOME NEUTRAL AROMA COMPONENTS VITIS VINIFERA

SOME NEUTRAL AROMA COMPONENTS VITIS VINIFERA VARIETY CARIGNANE Kent H. Sakato, Mark Hoekman, R. E. Kepner, A. D. Webb, and Carlos J. Muller OF WINES OF Respectively graduate students, Professor of Chemistry and Professor of Enology, Departments of Chemistry and of Viticulture and Enology, University of California, Davis, and Associate Professor of Chemistry, Chemistry Department, Louisiana Tech University, Ruston. Presented at the annual meeting of the American Society of Enologists, Monterey, California, June 22-23, 1975. Accepted for publication March 13, 1975. ABSTRACT A methylene chloride extract of a young, glassonly-aged wine of Vitis vini/era variety 'Carignane' was found to contain the following volatile aroma compounds: 3-methyl-l-butanol, 3-methyl-l-pentanol, ethyl lactate, ethyl 3-hydroxybutyrate, levo-2,3- butanediol monoacetate, diethyl succinate, ethyl 4- hydroxybutyrate, diethyl malate, ethyl 2-hydroxy-3- phenylpropionate, ~-butyrolactone, 4-carboethoxy-4- hydroxybutyric acid ~/-lactone, 4R :5R and 4S :5S 4,5- dihydroxyhexanoic acid ~/-lactone, and 4R:5S and 4S : 5R 4,5-dihydroxyhexanoic acid ~-lactone. A volatile component, identified for the first time from wines by infrared and mass spectrometry and by coincidence of gas chromatographic retention times, is 3- hydroxy-4-phenylbutan-2-one. A number of substances present in trace amounts and the isolated acids were not identified. Vitis vinifera variety 'Carignane' is widely planted in several important wine-producing regions of the world. It is believed to have been grown in the south of France since the 12th century A.D. (13). It is still an important variety in that region and is of considerable importance also in Algeria and in California, where 12,332 hectares (3,472 acres) were growing in 1973. It is a variety noted for its high yield and vigorous growth habits. Although its wine is usually considered to lack distinctive varietal character, its high productivity makes it useful in preparing bulk red table wines of standard quality. In terms of volume of wine produced and consumed, it leads the wines of more distinctive and famous, but less productive, varieties such as 'Cabernet Sauvignon' and 'Pinot noir.' 'Carignane' was chosen by Olmo to be crossed with 'Cabernet Sauvignon,' the pollen parent, resulting in 'Ruby Cabernet' (9). Earlier studies have provided knowledge on the aroma constituents of 'Cabernet Sauvignon' and 'Ruby Cabernet' (11). It therefore seemed of interest to investigate the aroma components of 'Carignane' to ascertain whether any insight could be gained on genetic control of the formation of aroma compounds in these three varieties. 7 EXPERIMENTAL The wine (cellar no. 13,158) was made from grapes grown at the vineyard of the University of California, Davis, by standard cellar practice (destemming and crushing, 75 ppm S2, 2% Montrachef pure-culture starter, fermentation at 2 C, and gentle pressing from the skins at approximately 5 o Brix) in the fall of 197. The wine was stored in glass until aroma extractions were begun, in April of 1971. Aroma extraction: One hundred and ninety-seven liters of Carignane wine were extracted with successive two-liter portions of CH2C12 in a 28-liter stainless-steel drum rotated at 1 rpm about its long axis (5). The solvent was changed initially every 24 hours and, after the 1th extraction, every 48 hours, and the extracts were kept separate. The solvent stripped from the initial extract was reused after collection of the 6th fraction. The drum headspace was flushed with nitrogen at every solvent change. Separation of free acids: Free-acids in each of the wine extracts were separated by extraction with seven 1-ml portions of cold 5% Na2C3 solution. Each aqueous phase was back-extracted with two 1-ml portions of CH2C12 to recover nonacidic com-

ponents. The back extracts were combined with the bulk of the neutral fractions and dried over anhydrous Na2SO~. Concentration of neutral extracts: The bulk of the methylene chloride was stripped from each extract in a rotary evaporator under reduced pressure to give concentrates of about 2 ml in volume. The samples were further dried over anhydrous MgSO~ and then each was concentrated to 3-4 ml by distilling-off the residual CH~oCI., at atmospheric pressure through a short Vigreux column. Preparative separation- Preparative separations were carried out on a Loenco dual-column dual-thermal-conductivity-detector gas chromatograph, model 7, fitted with a 6.35-mm-OD X 3-m stainless-steel column packed with 5% FFAP on 6/8-mesh Chromosorb W. Detector temperature was 245 C, injector 24 C, outlet 215 C. Helium flow rate: 4-45 ml/min. Initially, in order to check chromatographic patterns, 2-~1 samples of each of the 17 extracts were chromatographed on a temperatureprogrammed run from 75 to 25 C at a rate of 4 C per minute. Collections were made in capillary tubes from repeated 2-~1 injections. Collected from each injection were five fractions, corresponding to oven temperatures of 1-125, 125-15, 15-18, 18-195, 195-24, and 24-25 C. Isolation and purification of components: Individual components were collected in capillary tubes from repeated injections of the preliminary fractions onto the FFAP column, onto a 6.35-mm X 3-m s.s. column packed with 1~/o SE 3 on 6/8-mesh Chromosorb W, or onto a 6.35-mm X 6-m s.s. column packed with 1~o Carbowax 2M on 6/8-mesh Gaschrom Q. GC conditions were varied to provide the best individual separations for each column and each fraction. Spectroscopic analysis: Infrared spectra were obtained on a Beckman IR-8 spectrometer fitted with a 5X beam condenser using NaC1 microcells. Spectra of the wine components were compared with knowns purified by GC. Mass spectra were obtained with a CEC spectrometer, model 21-14. On some samples, only mass spectral data were obtained because the amounts of material were so small. Synthesis of known 3-hydroxy-4-phenylbutan-2- one: A Darzens condensation of chloroacetone and benzaldehyde was carried out by the procedure of Kwart and Kird (2): under a dry nitrogen atmosphere,.25 moles of sodium methoxide in 5 ml of CH3H were added dropwise over 1 min to a stirred sollution of 3 ml (.29 moles) of benzaldehyde and 21 ml (.25 moles) chloroacetone dissolved in 3 ml CH3H at C. The product, 3,4-epoxy-4- phenylbutan-2-one, was recovered from the reaction mixture by extraction into ether, and was purified by reduced-pressure distillation and crystallization. AROMA COMPONENTS OF CARIGNANE WINESm71 The epoxide was dissolved in ethanol and hydrogenated at atmospheric pressure and room temperature with 5% Pd on charcoal by the technique of Mitsui et al. (3). Gas chromatography of the product on FFAP and SE 3 columns showed one principal product with IR, MS, and NMR spectra in theoretical agreement with those for the phenylhydroxy ketone. RESULTS AND DISCUSSION Fig. 1 is a gas chromatogram of the concentrated essence from the first methylene chloride extraction of the 'Carignane' wine. Each peak on the chromatogram (FFAP column, temperature program: 75-25 C) is identified by the numbering system used by Webb et al. (11) in work on 'Cabernet Sauvignon' and 'Ruby Cabernet.' Table 1 compares relative amounts of each component isolated from the neutral extracts of 'Carignane,' 'Cabernet Sauvignon,' and 'Ruby Cabernet' wines. It is noted that the extracts, while comparable, do not reflect the concentrations of components actually present in the wines because of differences in partition coefficients and because substances with boiling points lower than that of methylene chloride are lost with the stripped extracting solvent. Table 1 shows that all three wines contain alcohols and esters which are common metabolites in alcoholic fermentations. These are indicated by peak numbers 1, 14, ]6A, 17, 18, 37, 38, 46, 51, and 58. Although their relative amounts vary slightly from wine to wine, they constitute the major portion of the volatiles. The amyl alcohol isomers, in particular, contribute significantly to the odor of 'Cabernet Sauvignon' and 'Ruby Cabernet.' Also contributing to the aroma are 3-(methylthio)-l-propanol (peak 38), which has an aroma reminiscent of freshly cut potatoes; 2-phenylethanol (peak 46), which has a rose-like odor; and l-hexanol (peak 17). Diethyl succinate (peak 37) and diethyl malate (peak 5]) have little odor, probably contributing only to the body of the wine (through their viscosity). Similarly, 2,3-butanediol (peak 31A) and its monoacetate ester (peak 32), identified as components of 'Carignane,' probably contribute only to the sweetness and viscosity of the wine. Of greater interest are those compounds which appear to be present in only one of the parents ('Cabernet Sauvignon' and 'Carignane'), or which have been identified only in the progeny ('Ruby Cabernet'). Of these, the esters 2-phenylethyl acetate (peak 41), isoamyl caproate (peak 26), ethyl 2-hydroxyisocaproate (peak 31), and isoamyl caprylate (peak 37A) seem to be present only in 'Cabernet Sauvignon' (11), which may indicate that their formation is under genetic control. If that is so, the control is not simply ester formation or no ester formation, however, because ethyl 3-hydroxybutyrate

72--AROMA COMPONENTS OF CARIGNANE WINES 46,4- CARIGNANE ILl Z & D..J >- 7 W 1- ll. >- X O 11:: I 42 35 L,J l- l.l.j Z _I._I a a w w - - 18 z z I- F-- 29 IT Fig. 1. Gas chromatogram of volatile components extracted from 'Carignane' wine. (peak 29) is only questionably present in 'Cabernet Sauvignon' but present in 'Carignane' and 'Ruby Cabernet.' Similarly, the esters ethyl caprylate (peak 24) and ethyl caprate (peak 36) are missing from 'Carignane' but found in 'Cabernet Sauvignon' and 'Ruby Cabernet.' At this point, it seems better to reserve judgment concerning genetic control of the formation of aroma compounds (particularly esters) until more detailed and quantitative data are available. This decision seems even more advisable when one considers that the age of the wine and its mode of preservation (glass or wood) can have pronounced effects on the types and amounts of esters present: very young wines, still containing enzymes, can contain high concentrations of ethyl esters from reaction of ethanol with acyl-s-coa (8), and, after precipitation of all enzymes, slow esterification and transesterifications can occur through hydrogen-ion catalysis even at the low temperatures of wine storage. More acetate could be produced by further oxida- tion of the acetaldehyde resulting from the coupled autoxidation of ethanol by vicinal di- or tri-hydroxyphenols in young wines, according to Wildenradt and Singleton (12). Further, as the pigment and tannin monomers hydrolyze during wine aging, new quantities of acetic and substituted cinnamic acids, as well as glucose, are liberated (1)o Finally, during the later stages of bottle aging, polymerization of the pigment and tannin monomers is an oxidation reaction which requires that some other component of the wine be reduced. Levels of acetate might well be lowered by this mechanism. Table 1 shows that the three wines are quite different in distribution of the four gamma-lactones : y-butyrolactone (peak 35), 4-ethoxy-7-butyrolactone (peak 39), 4,5-dihydroxyhexanoic acid-,/-lactone (peak 53), and 4-carboethoxy-~-butyrolactone (peak 57). Biosynthetic interrelations among these lactones have been proposed by Muller et al (7). If the pathways proposed are those actually followed in biosynthesis of the four lactones in wines, it is evident that

AROMA COMPONENTS OF CARIGNANE WINES--73 Table 1. Comparison of extracts of 'Cabernet Sauvignon,' 'Carignane,' and 'Ruby Cabernet' wines. Peak no. Component 196 1967 'Cabernet 197 'Ruby Sauvignon' - 'Carignane' Cabernet '- 1 14 16A 17 18 2-Methyl-l-propanol 2 & 3-Methyl-l-butanols 3-M ethyl- 1 -pent ano I 1-Hexanol Ethyl lactate -~b --]--t- -'t""{--~r - ++++ ++++ ++++ + +? +++ ++ + ++++ +++ ++++ 24 26 29 31 31A Ethyl caprylate + + - + Isoamyl caproate + - - Ethyl 3-hydroxybutyrate 9 + + + Ethyl 2-hydroxyisocaproate + -? 2,3-Butanediol - + - 32 35 36 37 37A 2,3-Butanediol monoacetate 7-Butyrolactone Ethyl caprate Diethyl succinate Isoamyl caprylate - + -? ++++ ++ + - + ++ +++ ++ + -? 38 39 41 42 45 3-(Methylthio)-l-propanol? + + 4-Ethoxy-~,-butyrolactone + + + c 2-Phenylethyl acetate + + + - "~ Ethyl 4-hydroxybutyrate 9 + +? Benzyl alcohol + - + 46 51 53 57 57A 58 2-Phenylethanol Diethyl malate 4,5-Dihyd roxyhexanoic acid-7-1actones 4-Carboethoxy-7-butyrolactone 3-Hyd ro xy-4-p he nyl butan-2-o ne Ethyl 2-hyd roxy-3-phenylpropionate ++++ ++++ ++++ + +++ 9 9 + 9 - +++ + + + + by IR.,, Data from reference (11). b_ - no peak,? = small peak identified by retention time only, + to + ++ + = relative amounts of compounds identified e Data from reference (11) and identification (6). all three varieties must have the enzymes necessary to carry out all of the steps from glutamate to ~/- butyrolactone, and that the blocks occur in 'Cabernet Sauvignon' after the production of 2-oxoglutarate on the route to 4-carboethoxy-7-butyrolactone, and in 'Carignane' after the 4-oxobutyrate on the route to 4-ethoxy-~/-butyrolactone. It is possible, too, that the diastereomeric 4,5-dihydroxyhexanoic acid-7-1actones, first isolated and identified by Muller et al (4) in flor sherries, may be formed by another route in red wines. A thiamine-catalyzed condensation of acetaldehyde with 2-oxoglutarate, followed by a loss of carbon dioxide and reduction of the oxo group (now at position 4) and then closure of the lactone ring seems as likely to occur as the under-the-film reduction of 4-acetyl-7-butyrolactone proposed as the route in flor sherries. 3-Hydroxy-4-phenylbutan-2-one, first isolated but not identified by van Wyk in 1966 from 'White Riesling' wine (14), has been unambiguously characterized as a volatile constituent of 'Carignane' wines. This compound likely represents another instance of thiamine-catalyzed condensations to form vicinal ketols. In this mechanism pyruvate is proposed to condense with 2-phenylacetaldehyde, with loss of carbon dioxide;the 2-phenylacetaldehyde required could arise in turn by decarboxylation of the 2-keto analog of phenylalanine. Schmauder and Gr6ger (1) have shown that the analagous 3-hydroxy-3-phenylpropan-2-one can be formed by adding ~mall amounts of benzaldehyde to a buffered molasses solution actively fermenting with baker's yeast. They, too, postulate that the 2- (a-hydroxyethyl) thiamine pyrophosphate formed from pyruvate during fermentation condenses with the added aldehyde, yielding the ketol. The odor of the 3-hydroxy-4-phenylbutan-2-one isolated from 'Carignane' is bland and indistinct. It probably contributes little to the overall aroma of wine. Am. J. Enol. Viticult., Voh 26, No. 2, 1975

74--AROMA COMPONENTS OF CARIGNANE WINES LITERATURE CITED 1. Anderson, D. W., D. E Gueffroy, A. D. Webb, and R. E. Kepner. Identification of acetic acid as an acylating agent of anthocyanin pigments in grapes. Phytochem. 9:1579-83 (197). 2. Kwart, H., and L. G. Kirk. Steric considerations in base catalyzed condensation; the Darzen's reaction. J. Org. Chem. 22:116-2 (1957). 3. Mitsui, S., Y. Senda, T. Shimodaira, and H. Ichikawa. The selective hydrogenolysis of (~,/~-epoxyketones. Bull. Chem. Soc. Japan 38:1897-19 (1965). 4. Muller, C. J., Linda Maggiora, R. E. Kepner, and A. D. Webb. Identification of two isomers of 4,5-dihydroxyhexanoic acid gamma lactone in California and Spanish flor sherries. Agr. Food Chem. 17:1373-76 (1969). 5. Muller, C. J., R. E. Kepner, and A. D. Webb. Identification of 3(methylthio)-propanol as an aroma constituent in 'Cabernet Sauvignon' and 'Ruby Cabernet' wines. Amer. J. Enol. Viticuit. 22:156-6 (1971). 6. Muller, C. J., R. E. Kepner, and A. D. Webb. Identification of 4-ethoxy-4-hydroxybutyric acid lactone [5-ethoxydihydro- 2(3H)-furanone] as an aroma component of wine from Vitis vinifera var. Ruby Cabernet. Agr. Food Chem. 2:193-5 (1972). 7. Muller, C. J., R. E. Kepner, and A. D. Webb. Lactones in wines m a review. Amer. J. Enol. Viticult. 24:5-9 (1973). 8. NordstrSm, K. Formation of ethyl acetate in fermentation with brewer's yeast. IV. Metabolism of acetyl-coenzyme A. J. Inst. Brew. 69:142-53 (1963). 9. Olmo, H. P. Ruby Cabernet and Emerald Riesling. Univ. Calif. Agr. Expt. Sta. Bull. 74 (1948). 1. Schmauder, H. P., and D. GrSger. Studien zur Acyloinbildung durch Saccharomyces cerevisiae. Pharmazie 23:32-31 (1968). 11. Webb, A. D., R. E. Kepner, and Linda Maggiora. Some volatile components of wines of Vitis vinifera varieties Cabernet Sauvignon and Ruby Cabernet. I. Neutral compounds. Amer. J. Enol. Viticult. 2:16-24 (1969). 12. Wildenradt, H. L., and V. L. Singleton. The production of aldehydes as a result of oxidation of polyphenolic compounds and its relation to wine aging. Amer. J. Enol. Viticult. 25:119-126 (1974). 13. Winkler, A. J. General Viticulture. U. C. Press, Berkeley and Los Angeles. p. 589 (1962). 14. van Wyk, C. J. The aroma constituents of grapes and wines of Vitis vinifera var. White Riesling. Ph.D. dissertation. Univ. Calif. Davis Library (1966).