928 J. Agric. Food Chem. 1980, 28, 928-934 Kepner, R. E., Webb, A. D., Maggiora, L., Am. J. Enol. Viticult. Nordmann, J., Mattioda, G. D., Fr. M. Patent 7593 (1970). 20, 25 (1969). Oser, B. L., Ford, R. A., Food Technol. 32(2), 60 (1978). Kepner, R. E., Webb, A. D., Muller, C. J., Am. J. Enol. Viticult. Piloty, M., Baltes, W., Z. Lebensm.-Untem-Forsch. 168, 374 23, 103 (1972). ( 1979). Leppanen, O., Ronkainen, P., Koivisto, T., Denslow, J., J. Znst. Prochazka, M., Palecek, M., Collect. Czech. Chem. Commun. 35, Brew. 85, 278 (1979). 1399 (1970). Liu, M. T. H., Banjoko, O., Yamamoto, Y., Moritani, I., Tetra- Schreier, P., CRC Crit. Reu. Food Sci. Nutr. 12, 59 (1979). hedron 31, 1645 (1975). Schreier, P., Drawert, F., Winkler, F., J. Agric. Food Chem. 27, Makoto, M., Hoshino, M., Jikeikai Med. J. 23, 189 (1976). 365 (1979). Mattioda, G. D., Ger. Offen. Patent 1903076 (1969). Schreier, P., Drawert, F., Abraham, K. O., Lebensm.-Wiss. Maw, G. A,, Coyne, C. M., Arch. Biochem. 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Volatile Ester Hydrolysis or Formation during Storage of Model Solutions and Wines David D. Ramey and Cornelius S. Ough* The effects of temperature, ethanol concentration, and pH on the rate of hydrolysis of common volatile esters of wine (ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, isobutyl acetate, isoamyl acetate, hexyl acetate, and 2-phenylethyl acetate) were investigated in model situations. The pseu- do-first-order rate constants for temperature and pH effects were calculated as well as the second-order rate constants for [H+]effect on model solution ester hydrolysis. The effect of acid catalysis of species other than H+ was calculated and found to be minor. Ethanol concentration differences in amounts of 10-1470 v/v had little effect on the rates. Activation energies and thermodynamic activation constants were calculated for the esters. Several wines were also analyzed for changes in ester concentration with time at several different temperatures. Volatile esters are introduced into wine primarily by weakly acidic solution such as wine, since the reaction yeast during fermentation. Although small quantities of mechanism differs. esters are present in grapes prior to fermentation, the The probable mechanism of hydrolysis of esters in wine amounts are negligible compared to those introduced en- or solutions modeled after wine is that of Bamford and zymatically by the yeast (Schreier et al., 1976; Stevens et Tipper (1972) and Isaacs (1974) and is given below. The al., 1969; Usseglio-Tomasset and Bosia, 1978; Van Wyk et reaction is entirely reversible, one direction resulting in al., 1967; Webb, 1973). Ethyl esters of straight-chain, ester hydrolysis and the other in esterification of the saturated fatty acids and acetate esters of higher alcohols component acid and alcohol, so that factors affecting the predominate, since these compounds are present in high reaction rate in one direction will affect the reverse reaction concentrations in the fermenting medium and within the similarily. The catalyst may be either a free hydrogen ion yeast cell (Majaama, 1978; Nelson and Wheeler, 1939; or an undissociated proton of an organic acid. Nordstrom, 1963, 1964a). These esters have pleasant, The first studies of esterification rates in model solutions fruitlike aromas which are particularly pronounced in new approximating wine were completed by RibBreau-Gayon wines. and Peynaud (1936). Polyprotic acids such as tartaric, While the enzymatic mechanism of formation of esters malic, and succinic esterified more rapidly than the mo- and the factors affecting the quantities synthesized have noprotic acids acetic, propanoic, and butanoic. Generally, been studied extensively in wine, beer, and synthetic the more complicated the acid within each category, that media, little work has been done on their fates following is, the higher its molecular weight, the more slowly it was fermentation or on the rates at which they are hydrolyzed esterified. Using the equilibrium constant of 4 calculated or formed. Studies have been made of the rates of hy- for esterification by Berthelot (Berthelot and Saint-Gilles, drolysis of various esters in strongly acidic media (>85% 1962-1963) [K, = [ester] [H,O]/([acid][alcohol])], they H,SO,), but this is of limited applicability to a buffered, calculated the theoretical limit of esterification and found that none of the acids studied were esterified to that limit. Nordstrom (1963, 1964a,b) provided a kinetic analysis Department of Viticulture and Enology, University of of ester formation by yeast in synthetic media but em- California, Davis, California 95616. ployed enzyme kinetics. 0021-8561/80/1428-0928$01 .OO/O 0 1980 American Chemical Society Ester Hydrolysis or Formation in Wines J. Agric. Food Chem., Vol. 28, No. 5, 1980 929 Wines. The red wine was a Pinot noir from Oakville, CA. At grape crushing, 75 mg/L SOz was added and the must was fermented at 21 "C (70 OF) by using Saccharo- t-C-OR' myces cerevisiae strain Montrachet yeast. It was pressed at 5" brix and, after finishing fermentation, was cold sta- bilized for 2 weeks at 0 "C, when it was filtered into 750- HH mL screw-cap bottles and sealed with paraffin. The pH was 3.36, the titratable acidity 0.76 g/100 mL, and the ethanol 13.6% v/v. The white wine was a Chardonnay from Oakville, CA. At grape crushing, 75 mg/L SO2 was added, the grapes were pressed, and the juice settled overnight at 0 "C. The clear juice was fermented to dryness at 16 "C (60 OF) with the same strain of yeast (Montrachet) and cold stabilized for 2 weeks at 0 "C and then filtered and bottled. The pH was 2.94, the titratable acidity 1.09 g/100 mL, and the r ethanol 11.2%. Both red and white wines were analyzed immediately after bottling for ester concentrations and then stored in temperature-controlled water baths. Distillation/Extraction. An apparatus similar to that Spirov and Goranov (197'1) analyzed white table wines described by Likens and Nickerson (1964) and Schultz et after 8 months of storage and found increased concen- al. (1977) was used to simultaneously distill the samples trations of ethyl formate, isobutyl acetate, hexyl acetate, and extract the esters. Samples were adjusted to the same ethyl octanoate, and ethyl lactate. alcohol concentration prior to analysis by addition of 100% Simpson (1978) subjected Australian white table wines ethanol [as Killian and Ough (1979)l since variations in to bottled storage at 15 and 50 "C. Both 15 and 50 "C alcoholic strength alter the vapor-liquid partition coeffi- treatments showed increases in ethyl hexanoate, ethyl cient of the volatile solutes and hence the efficiency of their octanoate, ethyl decanoate, ethyl furoate, and diethyl extraction. succinate concentrations by headspace analysis. Hexyl Three hundred and fifty milliliters of model solution acetate concentration decreased. (750 mL of wine) was used. Boiling chips and internal Marais (1978) examined the effects of pH and temper- standards were added. A 250-mL solvent flask was filled ature on South African Colombard wine over a 16-week with 60 mL of redistilled pentane and connected to the period. Isoamyl, hexyl, and 2-phenylethyl acetates all distillation/extraction apparatus. Distilled water and decreased in concentration with time, more so at higher redistilled pentane were used to fill the return tubes at the temperature and at lower pH. Ethyl hexanoate, ethyl bottom of the apparatus. octanoate, and ethyl decanoate concentrations decreased Samples were refluxed in the distillation/extraction only slightly, if at all. Diethyl succinate concentration apparatus for exactly 30 min. The pentane was then increased more rapidly at lower pH and higher tempera- transferred to a 250-mL separatory funnel, and 20 mL of ture. 1 N aqueous NaCl was added to extract any ethanol. The This research was designed to investigate the effects of salt solution was extracted twice more with 25-mL portions temperature, pH, and ethanol concentration on the rates of pentane, and these were combined with the extract. of hydrolysis of the common odorous esters synthesized Anhydrous Na2S04was added to remove any water, and by yeast. The hypothesis was that these esters are intro- the extract was stored overnight at -8 "C. duced enzymatically into wine in excess of their equilib- Concentrations. The extract was transferred into a rium concentrations and that they are gradually hydro- 250-mL round-bottom flask, and the Na2S04was rinsed lyzed until they approach equilibrium with their compo- twice with small portions of pentane. The pentane extract nent acids and alcohols. was distilled through a 40-cm Vigreux-type fractionating MATERIALS AND METHODS column until only 5 mL of extract remained. The residue Model Solutions.