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Higher alcohols, diacetyl, acetoin and 2,3-butanediol biosynthesis in grapes undergoing carbonic maceration
Food Research International 39 (2006) 112–116 www.elsevier.com/locate/foodres
Higher alcohols, diacetyl, acetoin and 2,3-butanediol biosynthesis in grapes undergoing carbonic maceration Dan Yi Yang, Yukio Kakuda ¤, Ronald E. Subden Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 28 November 2004; accepted 20 June 2005
Abstract Two grape varieties, Baco Noir and Gamay, were subjected to carbonic maceration (CM) conditions for 3, 6, 9, 12 and 15 days and the levels of higher alcohols, diacetyl, acetoin and 2,3-butanediol were determined. Diacetyl was not present in any of the CM juices. Acetoin and 2,3-butanediol were produced in the grapes and their amounts increased as the time of CM increased. On the 15th day of CM, acetoin was 12.6 § 0.9 mg/l in Baco Noir juice and 3.2 § 0.1 mg/l in Gamay juice, and 2,3-butanediol was 68.2 § 3.9 mg/l in Baco Noir juice and 19.9 § 1.2 mg/l in Gamay juice. 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 2-methyl-1-butanol appeared in trace amounts (the highest among them was 3.3 § 0.0 mg/l) in the CM Baco Noir juices and were not detected in the CM Gamay juices. Based on these results, the sensory contribution of higher alcohols from grape CM would be inconsequential when compared to that from yeast metabolism. 2005 Elsevier Ltd. All rights reserved. Keywords: Carbonic maceration; Higher alcohols; Fusel oils; Diacetyl; Acetoin; 2,3-Butanediol
1. Introduction Carbonic maceration (CM) is a pre-yeast fermentation treatment of intact grapes, where the grape bunches are kept in CO2 for up to two weeks or longer before crushing and starting yeast fermentation. The anaerobic treatment causes intracellular fermentation, giving distinct characteristics to wine. CM wines have a distinctive aroma, are early maturing and can be enjoyed shortly after yeast fermentation is completed, while other ordinary wines need to wait for at least a year to be consumed (Jackson, 2000). In practice, the grapes are harvested carefully and the intact grape clusters are put into a closed container to keep O2 from entering. Inside the container, O2 is consumed by the respiring grapes, and CM gradually starts
*
Corresponding author. Tel.: +1 519 824 4120; fax: +1 519 824 6631. E-mail address: [email protected] (Y. Kakuda).
0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.06.007
as O2 drops and CO2 builds up. The onset of CM fermentation is rapid if air is Xushed out with CO2 (Sandler & Pinder, 2003) or if there is some berry breakage and the autochthonous yeasts start to ferment the available sugars. During CM, some sugar and malic acid in grape cells are metabolized, producing many compounds such as ethanol and CO2. As a result of the drop in malic acid content, CM wine has a smoother, less sour taste (Flanzy, Flanzy, & Benard, 1987). From 0.5 to 2.2 v/v% ethanol is formed in the grapes during carbonic maceration (Ribereau-Gayon, Peynaud, Ribereau-Gayon, & Sudraud, 1976). Intracellular fermentation will eventually stop because ethanol causes cell membrane damage resulting in the death of the cells (Terrier & Romieu, 1998; Tesnière et al., 1994). The compounds responsible for the aroma of CM wines have not been completely identiWed. Nevertheless, some aspects of the Xavour may be ascribed to ethyl cinnamate and benzaldehyde (Flanzy et al., 1987). Higher
D.Y. Yang et al. / Food Research International 39 (2006) 112–116
concentrations of ethyl decanoate, eugenol, methyl and ethyl vanillates, ethyl and vinyl guaiacols, and ethyl and vinyl phenols were found in CM wines (Ducruet, 1984). Terpenoids liberated from grapes may also contribute to the CM wine Xavour (Bitteur, Tesniere, Fauconnet, Bayonove, & Flanzy, 1996). Although some research has been conducted to compare the composition of CM wines with that of standard wines, there has not been a great deal of research on the composition of juices from CM grapes. It is unclear if the compounds which are found in wine, such as higher alcohols, diacetyl, acetoin and 2,3-butanediol also appear in the juice of whole grapes experiencing CM. The main purpose of this study was to determine the levels of these compounds in grapes undergoing CM.
113
3, 6, 9, 12 and 15 days to provide grape juice for analysis. Fig. 1 summarizes the experimental scheme used to study CM in grapes. 2.3. Collection of juice About 1% of the grapes were broken or bruised. These bottles were harvested Wrst and the aVected berry was trimmed oV, and the bunches were washed carefully with deionized water to avoid contamination of the juice with possible yeast fermentation products from the surface of the grapes. A piece of cheesecloth was wrapped around the grapes and the juice was expressed by hand crushing. Juice samples were collected on day 0, 3, 6, 9, 12 and 15 of CM and frozen at ¡80 °C until analysis. 2.4. CM wines
2. Materials and methods 2.1. Grape variety The grapes used in this study were Baco Noir and Gamay from the Niagara region of Canada. Baco Noir is a Vitis vinifera £ Vitis riparia hybrid widely grown in Canada due to its extreme winter hardiness. Gamay is a V. vinifera (Pinot) £ V. vinifera (Gouais) varietal that is used for the production of Beaujolais Nouveau, the most celebrated of CM wines.
Juice (300 ml) from the 15 day CM grapes and Saccharomyces bayanus (0.3 g) were put into a sterile 1 l bottle with an air lock and fermented at 22 °C. The wine was aged with the yeast in the bottle for 20 days after the fermentation stopped, and then transferred to a smaller bottle and sealed against air. The wine was stored at 22 °C for 110 days before analysis. Two kinds of wines, CM Baco Noir wine and CM Gamay wine were produced.
2.2. Carbonic maceration
3. Analytical methods
Healthy whole bunches of grapes were picked in the vineyard in October 2003 and put into 21 sterile 1 l glass bottles and sealed with lids and vapour locks. The vapour lock allowed CO2 generated during the CM to vent but prevented air from entering. Care was taken to avoid bruising or damaging the individual grapes. The bottles were divided into 7 groups (each group contained 3 test bottles) and stored in a lab at 22 °C which reXects ambient conditions in Canada. Two groups were analyzed for CO2/O2 concentration after one day and two days of storage. The other Wve groups were stored for
3.1. CO2 and O2 concentrations during the CM
Group Grapes were sealed in 21 bottles and divided into 7 groups. Each group contained 3 test bottles
The concentrations of CO2 and O2 inside the bottles on day 1, 2, 3, 6, 9, 12 and 15 were determined by piercing the lid septum with the needle from a CO2/O2 detector (PAC CHECK™ 650 dual head space analyzer) and sucking out a small amount of the gas inside the bottle for the detector to analyse. Since this changed the air composition inside the bottles, immediately after the gas was sampled, the grapes were crushed and the juice saved for analysis. Day(s) of Storage
1
1
2
2
3
3
4
6
5
9
6
12
7
15
grape bunches (Baco Noir and Gamay)
Fig. 1. Experimental scheme for CM.
Purposes . Monitored CO2/O2 changes on day 1 and day 2
Monitored CO2/O2 and juice pH Obtain grape juice
114
D.Y. Yang et al. / Food Research International 39 (2006) 112–116
3.2. pH of the grape juices The pH of the grape juice experiencing CM was determined with a pH meter (accumet®, BASIC, AB15) after collecting the juice. The collection of the juice is described above. 3.3. Higher alcohols, diacetyl, acetoin and 2,3-butanediol A 500 ml conical Xask was Wlled with 25 ml of juice and distilled on a hot plate. The distillate was cooled with a condenser (30 cm long, 1 cm internal diameter) kept at ¡10 °C with circulating coolant and collected in a 25 ml volumetric Xask. After 18 ml of distillate was collected, the distillation apparatus was connected to an aspirator, and the Xask was put in a water bath to complete the distillation under vacuum. The temperature of the water bath was gradually increased from room temperature to 100 °C and held for 15 min. During distillation, a magnetic stir bar was used to stir the juice inside the Xask. At the end of 15 min, 4 ml of ethanol was added to the Xask and distilled. The Wnal volume of the distillate was adjusted to 25 ml with ethanol and 1 l was injected into a gas chromatograph (GC) to determine higher alcohols, diacetyl, acetoin and part of 2,3-butanediol. The remainder of the 2,3-butanediol was left in the syrup-like residue on the bottom of the Xask. To recover this part, water was added to the Xask to dissolve the residue and the vacuum distillation procedure repeated two more times. The distillate was collected in a 25 ml volumetric Xask and brought to volume with ethanol. A second injection was made and the concentration of 2,3-butanediol in juice was taken as the sum of the concentrations obtained from the Wrst and the second injections. 3.4. Ethanol A 500 ml conical Xask was Wlled with 25 ml of juice, 25 ml of deionized water and distilled on a hot plate. The distillate was cooled with a condenser (30 cm long, 1 cm internal diameter) kept at ¡10 °C with circulating coolant and collected in a 25 ml volumetric Xask. One microliter was injected into the GC to determine ethanol.
by comparison of their retention times with those of commercial standards. 3.6. Analysis of CM wines The methods used in the determination of ethanol, higher alcohols, diacetyl, acetoin and 2,3-butanediol in the CM wines were the same as those used in the analysis of the juices.
4. Results and discussion The CO2 and O2 concentrations inside the bottles holding the grapes are shown in Table 1. The O2 dropped to zero on day 2, and CO2 increased steadily from day 1 to day 15. The Wnal concentration on day 15 exceeded 90%, showing that N2 was expelled from the bottles during CM. The disappearance of O2 through any means is critical for a successful CM. Table 2 shows the pH values of the juices from Baco Noir and Gamay crushed on day 0, 3, 6, 9, 12 and 15. The pH values of both juices increased with the increase in time, and on day 15 they were about 0.4 units higher than at the beginning. The increase in the juice pH is characteristic of CM, due to the conversion of malic acid into pyruvate and Wnally to ethanol (Robin, Romieu, & Sauvage, 1989; Sneyd, 1989; Terrier & Romieu, 1998), and can be viewed as an indicator of a successful CM. Table 3 lists the concentrations of ethanol, higher alcohols, acetoin and 2,3-butanediol in the CM juices. Ethanol concentrations in fresh grapes (day 0) were very low, but they gradually increased during CM, and reached 2.0% in Baco Noir and 1.8% in Gamay. Ethanol Table 1 Percentage (v/v) of CO 2 and O2 in the atmosphere in the bottles holding the CM grapes (mean § SD) Grape Baco Noir
3.5. GLC An Agilent 6890 GC with a BP-624 (60 m £ 0.25 mm I.D., 1.4 m Wlm thickness) column and 1-pentanol as the internal standard were used for the analyses. The GC conditions were: inlet temperature (250 °C), injection volume (1 l, splitless mode), detector (FID), detector port temperature (280 °C), carrier gas (N2), Xow rate (285 cm/s), temperature program (50 °C, held for 0.5 min; 120 °C/min to 150 °C, held for 8.35 min; 30 °C/min to 180 °C, held for 8.7 min). The compounds were identiWed
Gamay
Day of CM
CO2 (%)
O2 (%)
0 1
0.034 § 0.002 25.7 § 1.4
20.8 § 0.1 0.61 § 0.05
2
47.1 § 1.6
0
3
52.1 § 2.3
0
6
73.4 § 2.8
0
9
89.0 § 1.8
0
12
94.9 § 0.2
0
15
95.7 § 0.4
0
0 1
0.032 § 0.002 21.6 § 0.6
20.7 § 0.1 0.85 § 0.06
2
36.7 § 1.1
0
3
40.6 § 0.7
0
6
57.9 § 5.8
0
9
70.4 § 4.6
0
12
82.0 § 3.4
0
15
91.5 § 1.3
0
D.Y. Yang et al. / Food Research International 39 (2006) 112–116 Table 2 pH values of juices from grapes experiencing CM (mean § SD) Grape
Day of CM
Baco Noir
Gamay
pH of juices
0 3
3.22 § 0.02 3.27 § 0.02
6
3.37 § 0.02
9
3.43 § 0.03
12
3.52 § 0.02
15
3.63 § 0.03
0 3
3.08 § 0.01 3.14 § 0.05
6
3.19 § 0.04
9
3.28 § 0.06
12
3.36 § 0.02
15
3.48 § 0.03
is the characteristic product of grapes under CM and the product of many other plants experiencing anaerobic metabolism. The production of ethanol is the result of intensiWed glycolysis under anaerobic conditions (Crawford, 1978). Table 3 also lists the concentrations of ethanol, higher alcohols, acetoin and 2,3-butanediol in the CM wines made from the 15 day CM juices. Except for acetoin in CM Baco Noir wine, the concentrations of all the compounds tested were much higher. As seen in Table 3, only a few of the targeted compounds showed up in the juices, and they were not detectable until day 9. The 2,3-butanediol was the most prominent, and acetoin was the second most abundant. The 2-methyl-propanol, 3-methyl-1-butanol, 2-methyl-1butanol and 1-propanol were present at very low levels. The concentrations of these compounds increased with
115
an increase in time, and were higher in Baco Noir than in Gamay. The mechanism involved in the synthesis of acetoin and 2,3-butanediol in grapes undergoing CM is not known. However, some insight can be gleaned from the pathways found in microbes (Gibson, Parker, & Woodward, 1991; Hugenholtz, 1993) that produce 2,3-butanediol and acetoin (Fig. 2). Oxygen deprivation forces glycolysis to generate energy to support cell functions (Vartapetian, 1978), which results in the production of more pyruvate. Higher pyruvate concentration may encourage the production of -acetolactate, as -acetolactate synthase has low aYnity for pyruvate – at least in bacteria (Monnet, Phalip, Schmitt, & Divies, 1994; Snoep, Teixeira de Mattos, Starrenburg, & Hugenholtz, 1992), so only when pyruvate is in excess would -acetolactate be synthesized. -acetolactate could then break down to generate either acetoin or diacetyl. The diacetyl could be reduced to acetoin, and acetoin to 2,3-butanediol by NADH from glycolysis under anaerobic condition. When the grapes are deprived of O2, NADH from glycolysis needs to be oxidized to NAD+ so that the redox balance inside the cells can be maintained. Under most circumstances this is accomplished by reducing acetaldehyde to ethanol – CM fermentation generates 1– 2% ethanol. Some NAD+ could also be regenerated by the reduction of diacetyl and acetoin. However, based on the molar concentrations of ethanol (343 mM in Baco Noir and 303 mM in Gamay), acetoin (0.14 mM in Baco Noir and 0.036 mM in Gamay) and 2,3-butanediol (0.75 mM in Baco Noir and 0.22 mM in Gamay) after 15 days of CM, it appears that the majority of the NAD+ is produced by the reduction of acetaldehyde to ethanol.
Table 3 Concentration of ethanol, higher alcohols, acetoin and 2,3-butanediol found in the juices and wines from CM grapes Grape juice or wine
Baco Noir juice
Day of Concentration of ethanol, higher alcohols, acetoin and 2,3-butanediol (mean § SD) CM Ethanol 1-Butanol 2-Phenyl 1-Propanol 2-Methyl-1- 3-Methyl-1- 2-Methyl-1- Acetoin (%, v/v) (mg/l) ethanol (mg/l) propanol butanol butanol (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) 0 3
0.026 § 0.002 0.19 § 0.01
6
0.63 § 0.04
9
1.11 § 0.04
12
1.76 § 0.05
15
2.00 § 0.06
Baco Noir wine 15 Gamay juice
Gamay wine
10.1 § 0.2
0 3
0.008 § 0.001 0.27 § 0.01
–
44.5 § 2.1
2.7 § 0.4
0.9 § 0.2
0.8 § 0.0
3.3 § 0.0
1.5 § 0.2
48.1 § 1.6
45.0 § 1.5
183 § 4.7
2,3-Butanediol (mg/l)
4.4 § 0.3
16.0 § 1.1
7.9 § 0.5
36.9 § 3.3
0.9 § 0.2
12.6 § 0.9
68.2 § 3.9
55.1 § 1.1
8.9 § 0.4
318 § 5.6
6
0.64 § 0.03
9
0.82 § 0.03
12
1.48 § 0.04
2.2 § 0.1
12.6 § 0.6
15
1.77 § 0.04
3.2 § 0.1
19.9 § 1.2
15
11.3 § 0.3
11.5 § 1.5
518.5 § 8.2
6.0 § 1.2
1.1 § 0.1
32.4 § 0.4
98.0 § 1.7
34.1 § 0.8
163 § 4.0
34.0 § 0.2
116
D.Y. Yang et al. / Food Research International 39 (2006) 112–116 α-acetolactate synthase
2 pyruvates
α-acetolactate
enzymatic or nonenzymatic conversion
α-acetolactate decarboxylase
acetoin
NADH
reductase
reductase NAD+
NAD+
NADH
diacetyl
2,3-butanediol
Fig. 2. Pathways for the synthesis of diacetyl, acetoin and 2,3-butanediol in microbes.
Diacetyl was not detected in any of the juices, indicating no diacetyl was formed or all diacetyl formed was metabolized to acetoin. Higher alcohols which are common in yeast fermentation, e.g., 1-propanol, 2-methyl-1-propanol, 3-methyl1-butanol, 2-methyl-1-butanol, were negligible in the Baco Noir juices, and not detected in the Gamay juices. Other higher alcohols which were reported to be found in grapes (Schreier, Drawert, & Junker, 1976), e.g., 1-hexanol, 1-heptanol, 1-octanol, benzyl alcohol and 2-phenylethanol, were not detectable in any of the juices. 5. Conclusion Acetoin and/or 2,3-butanediol but not diacetyl began to appear in the juices of grapes undergoing CM for 9 days. Their amounts increased with the increase in time of CM, and on the 15th day of CM, acetoin was 12.6 § 0.9 mg/l in Baco Noir juice and 3.2 § 0.1 mg/l in Gamay juice, and 2,3-butanediol was 68.2 § 3.9 mg/l in Baco Noir juice and 19.9 § 1.2 mg/l in Gamay juice. Several higher alcohols in very low amounts were found in the CM Baco Noir juices (the highest one is 3.3 § 0.0 mg/ l 2-methyl-1-propanol), but no higher alcohols were detected in all the CM Gamay juices. References Bitteur, S., Tesniere, C., Fauconnet, A., Bayonove, C., & Flanzy, C. (1996). Carbonic anaerobiosis of Muscat grape, 2. Changes in the distribution of free and bound terpenols. Sciences des Aliments, 16, 37–48. Crawford, R. M. M. (1978). Metabolic adaptations to anoxia. In D. D. Hook & R. M. M. Crawford (Eds.), Plant life in anaerobic environments (pp. 119–136). Michigan: Ann Arbor Science Publishers, Inc. Ducruet, V. (1984). Comparison of the headspace volatiles of carbonic maceration and traditional wine. Lebensmittel Wissenschaft Technologie, 17(4), 217–221.
Flanzy, C., Flanzy, M., & Benard, P. (1987). La ViniWcation par la Maceration Carbonique. Paris: Institute National de la Recherche Agronomique. Gibson, T. D., Parker, S. M., & Woodward, J. R. (1991). PuriWcation and characterization of diacetyl reductase from chicken liver and Streptococcus lactis and enzymic determination of diacetyl and diketones. Enzyme and Microbial Technology, 13, 171–178. Hugenholtz, J. (1993). Citrate metabolism in lactic acid bacteria. FEMS Microbiology Reviews, 12, 165–178. Jackson, R. S. (2000). Wine science – principles, practice, perception. San Diego: Academic Press. Monnet, C., Phalip, V., Schmitt, P., & Divies, C. (1994). Comparison of -acetolactate synthase and -acetolactate decarboxylase in Lactococcus spp. and Leuconostoc spp. Biotechnological Letters, 16, 257– 262. Ribereau-Gayon, J., Peynaud, E., Ribereau-Gayon, P., & Sudraud, P. (1976). Traite d’oenologie. Sciences et techniques du vin. Tome 3 ViniWcations, transformations du vin. Paris: Dunod pp. 289–314. Robin, J. P., Romieu, C., & Sauvage, F. X. (1989). Anaerobic metabolism of organic and aminoacids in grapes. I . A device for measuring the decarboxylation and ethanol releasing kinetics from a single 14C labelled berry. American Journal of Enology and Viticulture, 40(3), 161–169. Sandler, M., & Pinder, R. (2003). Wine – a scientiWc exploration. London: Taylor & Francis pp. 245–246. Schreier, P., Drawert, F., & Junker, A. (1976). IdentiWcation of volatile constituents from grapes. Journal of Agricultural and Food Chemistry, 24, 331–336. Sneyd, T. N. (1989). Carbonic maceration: an overview. Wine Industry Journal, 11, 281–285. Snoep, J. L., Teixeira de Mattos, M. J., Starrenburg, M. J. C., & Hugenholtz, J. (1992). Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and -acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis. Journal of Bacteriology, 174, 4838–4841. Terrier, N., & Romieu, C. (1998). Inhibition of vacuolar proton pumps by ethanol impairs grape berry compartmentation. Australian Journal of Grape and Wine Research, 4, 39–45. Tesnière, C., Romieu, C., Dugelay, I., Nicol, M. Z., Flanzy, C., & Robin, J. P. (1994). Partial recovery of grape energy metabolism upon aeration following anaerobic stress. Journal of Experimental Botany, 45, 145–151. Vartapetian, B. B. (1978). Introduction-life without oxygen. In D. D. Hook & R. M. M. Crawford (Eds.), Plant life in anaerobic environments (pp. 1–11). Michigan: Ann Arbor Science Publishers, Inc.
Higher alcohols, diacetyl, acetoin and 2,3-butanediol biosynthesis in grapes undergoing carbonic maceration Dan Yi Yang, Yukio Kakuda ¤, Ronald E. Subden Department of Food Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 Received 28 November 2004; accepted 20 June 2005
Abstract Two grape varieties, Baco Noir and Gamay, were subjected to carbonic maceration (CM) conditions for 3, 6, 9, 12 and 15 days and the levels of higher alcohols, diacetyl, acetoin and 2,3-butanediol were determined. Diacetyl was not present in any of the CM juices. Acetoin and 2,3-butanediol were produced in the grapes and their amounts increased as the time of CM increased. On the 15th day of CM, acetoin was 12.6 § 0.9 mg/l in Baco Noir juice and 3.2 § 0.1 mg/l in Gamay juice, and 2,3-butanediol was 68.2 § 3.9 mg/l in Baco Noir juice and 19.9 § 1.2 mg/l in Gamay juice. 1-propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 2-methyl-1-butanol appeared in trace amounts (the highest among them was 3.3 § 0.0 mg/l) in the CM Baco Noir juices and were not detected in the CM Gamay juices. Based on these results, the sensory contribution of higher alcohols from grape CM would be inconsequential when compared to that from yeast metabolism. 2005 Elsevier Ltd. All rights reserved. Keywords: Carbonic maceration; Higher alcohols; Fusel oils; Diacetyl; Acetoin; 2,3-Butanediol
1. Introduction Carbonic maceration (CM) is a pre-yeast fermentation treatment of intact grapes, where the grape bunches are kept in CO2 for up to two weeks or longer before crushing and starting yeast fermentation. The anaerobic treatment causes intracellular fermentation, giving distinct characteristics to wine. CM wines have a distinctive aroma, are early maturing and can be enjoyed shortly after yeast fermentation is completed, while other ordinary wines need to wait for at least a year to be consumed (Jackson, 2000). In practice, the grapes are harvested carefully and the intact grape clusters are put into a closed container to keep O2 from entering. Inside the container, O2 is consumed by the respiring grapes, and CM gradually starts
*
Corresponding author. Tel.: +1 519 824 4120; fax: +1 519 824 6631. E-mail address: [email protected] (Y. Kakuda).
0963-9969/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.06.007
as O2 drops and CO2 builds up. The onset of CM fermentation is rapid if air is Xushed out with CO2 (Sandler & Pinder, 2003) or if there is some berry breakage and the autochthonous yeasts start to ferment the available sugars. During CM, some sugar and malic acid in grape cells are metabolized, producing many compounds such as ethanol and CO2. As a result of the drop in malic acid content, CM wine has a smoother, less sour taste (Flanzy, Flanzy, & Benard, 1987). From 0.5 to 2.2 v/v% ethanol is formed in the grapes during carbonic maceration (Ribereau-Gayon, Peynaud, Ribereau-Gayon, & Sudraud, 1976). Intracellular fermentation will eventually stop because ethanol causes cell membrane damage resulting in the death of the cells (Terrier & Romieu, 1998; Tesnière et al., 1994). The compounds responsible for the aroma of CM wines have not been completely identiWed. Nevertheless, some aspects of the Xavour may be ascribed to ethyl cinnamate and benzaldehyde (Flanzy et al., 1987). Higher
D.Y. Yang et al. / Food Research International 39 (2006) 112–116
concentrations of ethyl decanoate, eugenol, methyl and ethyl vanillates, ethyl and vinyl guaiacols, and ethyl and vinyl phenols were found in CM wines (Ducruet, 1984). Terpenoids liberated from grapes may also contribute to the CM wine Xavour (Bitteur, Tesniere, Fauconnet, Bayonove, & Flanzy, 1996). Although some research has been conducted to compare the composition of CM wines with that of standard wines, there has not been a great deal of research on the composition of juices from CM grapes. It is unclear if the compounds which are found in wine, such as higher alcohols, diacetyl, acetoin and 2,3-butanediol also appear in the juice of whole grapes experiencing CM. The main purpose of this study was to determine the levels of these compounds in grapes undergoing CM.
113
3, 6, 9, 12 and 15 days to provide grape juice for analysis. Fig. 1 summarizes the experimental scheme used to study CM in grapes. 2.3. Collection of juice About 1% of the grapes were broken or bruised. These bottles were harvested Wrst and the aVected berry was trimmed oV, and the bunches were washed carefully with deionized water to avoid contamination of the juice with possible yeast fermentation products from the surface of the grapes. A piece of cheesecloth was wrapped around the grapes and the juice was expressed by hand crushing. Juice samples were collected on day 0, 3, 6, 9, 12 and 15 of CM and frozen at ¡80 °C until analysis. 2.4. CM wines
2. Materials and methods 2.1. Grape variety The grapes used in this study were Baco Noir and Gamay from the Niagara region of Canada. Baco Noir is a Vitis vinifera £ Vitis riparia hybrid widely grown in Canada due to its extreme winter hardiness. Gamay is a V. vinifera (Pinot) £ V. vinifera (Gouais) varietal that is used for the production of Beaujolais Nouveau, the most celebrated of CM wines.
Juice (300 ml) from the 15 day CM grapes and Saccharomyces bayanus (0.3 g) were put into a sterile 1 l bottle with an air lock and fermented at 22 °C. The wine was aged with the yeast in the bottle for 20 days after the fermentation stopped, and then transferred to a smaller bottle and sealed against air. The wine was stored at 22 °C for 110 days before analysis. Two kinds of wines, CM Baco Noir wine and CM Gamay wine were produced.
2.2. Carbonic maceration
3. Analytical methods
Healthy whole bunches of grapes were picked in the vineyard in October 2003 and put into 21 sterile 1 l glass bottles and sealed with lids and vapour locks. The vapour lock allowed CO2 generated during the CM to vent but prevented air from entering. Care was taken to avoid bruising or damaging the individual grapes. The bottles were divided into 7 groups (each group contained 3 test bottles) and stored in a lab at 22 °C which reXects ambient conditions in Canada. Two groups were analyzed for CO2/O2 concentration after one day and two days of storage. The other Wve groups were stored for
3.1. CO2 and O2 concentrations during the CM
Group Grapes were sealed in 21 bottles and divided into 7 groups. Each group contained 3 test bottles
The concentrations of CO2 and O2 inside the bottles on day 1, 2, 3, 6, 9, 12 and 15 were determined by piercing the lid septum with the needle from a CO2/O2 detector (PAC CHECK™ 650 dual head space analyzer) and sucking out a small amount of the gas inside the bottle for the detector to analyse. Since this changed the air composition inside the bottles, immediately after the gas was sampled, the grapes were crushed and the juice saved for analysis. Day(s) of Storage
1
1
2
2
3
3
4
6
5
9
6
12
7
15
grape bunches (Baco Noir and Gamay)
Fig. 1. Experimental scheme for CM.
Purposes . Monitored CO2/O2 changes on day 1 and day 2
Monitored CO2/O2 and juice pH Obtain grape juice
114
D.Y. Yang et al. / Food Research International 39 (2006) 112–116
3.2. pH of the grape juices The pH of the grape juice experiencing CM was determined with a pH meter (accumet®, BASIC, AB15) after collecting the juice. The collection of the juice is described above. 3.3. Higher alcohols, diacetyl, acetoin and 2,3-butanediol A 500 ml conical Xask was Wlled with 25 ml of juice and distilled on a hot plate. The distillate was cooled with a condenser (30 cm long, 1 cm internal diameter) kept at ¡10 °C with circulating coolant and collected in a 25 ml volumetric Xask. After 18 ml of distillate was collected, the distillation apparatus was connected to an aspirator, and the Xask was put in a water bath to complete the distillation under vacuum. The temperature of the water bath was gradually increased from room temperature to 100 °C and held for 15 min. During distillation, a magnetic stir bar was used to stir the juice inside the Xask. At the end of 15 min, 4 ml of ethanol was added to the Xask and distilled. The Wnal volume of the distillate was adjusted to 25 ml with ethanol and 1 l was injected into a gas chromatograph (GC) to determine higher alcohols, diacetyl, acetoin and part of 2,3-butanediol. The remainder of the 2,3-butanediol was left in the syrup-like residue on the bottom of the Xask. To recover this part, water was added to the Xask to dissolve the residue and the vacuum distillation procedure repeated two more times. The distillate was collected in a 25 ml volumetric Xask and brought to volume with ethanol. A second injection was made and the concentration of 2,3-butanediol in juice was taken as the sum of the concentrations obtained from the Wrst and the second injections. 3.4. Ethanol A 500 ml conical Xask was Wlled with 25 ml of juice, 25 ml of deionized water and distilled on a hot plate. The distillate was cooled with a condenser (30 cm long, 1 cm internal diameter) kept at ¡10 °C with circulating coolant and collected in a 25 ml volumetric Xask. One microliter was injected into the GC to determine ethanol.
by comparison of their retention times with those of commercial standards. 3.6. Analysis of CM wines The methods used in the determination of ethanol, higher alcohols, diacetyl, acetoin and 2,3-butanediol in the CM wines were the same as those used in the analysis of the juices.
4. Results and discussion The CO2 and O2 concentrations inside the bottles holding the grapes are shown in Table 1. The O2 dropped to zero on day 2, and CO2 increased steadily from day 1 to day 15. The Wnal concentration on day 15 exceeded 90%, showing that N2 was expelled from the bottles during CM. The disappearance of O2 through any means is critical for a successful CM. Table 2 shows the pH values of the juices from Baco Noir and Gamay crushed on day 0, 3, 6, 9, 12 and 15. The pH values of both juices increased with the increase in time, and on day 15 they were about 0.4 units higher than at the beginning. The increase in the juice pH is characteristic of CM, due to the conversion of malic acid into pyruvate and Wnally to ethanol (Robin, Romieu, & Sauvage, 1989; Sneyd, 1989; Terrier & Romieu, 1998), and can be viewed as an indicator of a successful CM. Table 3 lists the concentrations of ethanol, higher alcohols, acetoin and 2,3-butanediol in the CM juices. Ethanol concentrations in fresh grapes (day 0) were very low, but they gradually increased during CM, and reached 2.0% in Baco Noir and 1.8% in Gamay. Ethanol Table 1 Percentage (v/v) of CO 2 and O2 in the atmosphere in the bottles holding the CM grapes (mean § SD) Grape Baco Noir
3.5. GLC An Agilent 6890 GC with a BP-624 (60 m £ 0.25 mm I.D., 1.4 m Wlm thickness) column and 1-pentanol as the internal standard were used for the analyses. The GC conditions were: inlet temperature (250 °C), injection volume (1 l, splitless mode), detector (FID), detector port temperature (280 °C), carrier gas (N2), Xow rate (285 cm/s), temperature program (50 °C, held for 0.5 min; 120 °C/min to 150 °C, held for 8.35 min; 30 °C/min to 180 °C, held for 8.7 min). The compounds were identiWed
Gamay
Day of CM
CO2 (%)
O2 (%)
0 1
0.034 § 0.002 25.7 § 1.4
20.8 § 0.1 0.61 § 0.05
2
47.1 § 1.6
0
3
52.1 § 2.3
0
6
73.4 § 2.8
0
9
89.0 § 1.8
0
12
94.9 § 0.2
0
15
95.7 § 0.4
0
0 1
0.032 § 0.002 21.6 § 0.6
20.7 § 0.1 0.85 § 0.06
2
36.7 § 1.1
0
3
40.6 § 0.7
0
6
57.9 § 5.8
0
9
70.4 § 4.6
0
12
82.0 § 3.4
0
15
91.5 § 1.3
0
D.Y. Yang et al. / Food Research International 39 (2006) 112–116 Table 2 pH values of juices from grapes experiencing CM (mean § SD) Grape
Day of CM
Baco Noir
Gamay
pH of juices
0 3
3.22 § 0.02 3.27 § 0.02
6
3.37 § 0.02
9
3.43 § 0.03
12
3.52 § 0.02
15
3.63 § 0.03
0 3
3.08 § 0.01 3.14 § 0.05
6
3.19 § 0.04
9
3.28 § 0.06
12
3.36 § 0.02
15
3.48 § 0.03
is the characteristic product of grapes under CM and the product of many other plants experiencing anaerobic metabolism. The production of ethanol is the result of intensiWed glycolysis under anaerobic conditions (Crawford, 1978). Table 3 also lists the concentrations of ethanol, higher alcohols, acetoin and 2,3-butanediol in the CM wines made from the 15 day CM juices. Except for acetoin in CM Baco Noir wine, the concentrations of all the compounds tested were much higher. As seen in Table 3, only a few of the targeted compounds showed up in the juices, and they were not detectable until day 9. The 2,3-butanediol was the most prominent, and acetoin was the second most abundant. The 2-methyl-propanol, 3-methyl-1-butanol, 2-methyl-1butanol and 1-propanol were present at very low levels. The concentrations of these compounds increased with
115
an increase in time, and were higher in Baco Noir than in Gamay. The mechanism involved in the synthesis of acetoin and 2,3-butanediol in grapes undergoing CM is not known. However, some insight can be gleaned from the pathways found in microbes (Gibson, Parker, & Woodward, 1991; Hugenholtz, 1993) that produce 2,3-butanediol and acetoin (Fig. 2). Oxygen deprivation forces glycolysis to generate energy to support cell functions (Vartapetian, 1978), which results in the production of more pyruvate. Higher pyruvate concentration may encourage the production of -acetolactate, as -acetolactate synthase has low aYnity for pyruvate – at least in bacteria (Monnet, Phalip, Schmitt, & Divies, 1994; Snoep, Teixeira de Mattos, Starrenburg, & Hugenholtz, 1992), so only when pyruvate is in excess would -acetolactate be synthesized. -acetolactate could then break down to generate either acetoin or diacetyl. The diacetyl could be reduced to acetoin, and acetoin to 2,3-butanediol by NADH from glycolysis under anaerobic condition. When the grapes are deprived of O2, NADH from glycolysis needs to be oxidized to NAD+ so that the redox balance inside the cells can be maintained. Under most circumstances this is accomplished by reducing acetaldehyde to ethanol – CM fermentation generates 1– 2% ethanol. Some NAD+ could also be regenerated by the reduction of diacetyl and acetoin. However, based on the molar concentrations of ethanol (343 mM in Baco Noir and 303 mM in Gamay), acetoin (0.14 mM in Baco Noir and 0.036 mM in Gamay) and 2,3-butanediol (0.75 mM in Baco Noir and 0.22 mM in Gamay) after 15 days of CM, it appears that the majority of the NAD+ is produced by the reduction of acetaldehyde to ethanol.
Table 3 Concentration of ethanol, higher alcohols, acetoin and 2,3-butanediol found in the juices and wines from CM grapes Grape juice or wine
Baco Noir juice
Day of Concentration of ethanol, higher alcohols, acetoin and 2,3-butanediol (mean § SD) CM Ethanol 1-Butanol 2-Phenyl 1-Propanol 2-Methyl-1- 3-Methyl-1- 2-Methyl-1- Acetoin (%, v/v) (mg/l) ethanol (mg/l) propanol butanol butanol (mg/l) (mg/l) (mg/l) (mg/l) (mg/l) 0 3
0.026 § 0.002 0.19 § 0.01
6
0.63 § 0.04
9
1.11 § 0.04
12
1.76 § 0.05
15
2.00 § 0.06
Baco Noir wine 15 Gamay juice
Gamay wine
10.1 § 0.2
0 3
0.008 § 0.001 0.27 § 0.01
–
44.5 § 2.1
2.7 § 0.4
0.9 § 0.2
0.8 § 0.0
3.3 § 0.0
1.5 § 0.2
48.1 § 1.6
45.0 § 1.5
183 § 4.7
2,3-Butanediol (mg/l)
4.4 § 0.3
16.0 § 1.1
7.9 § 0.5
36.9 § 3.3
0.9 § 0.2
12.6 § 0.9
68.2 § 3.9
55.1 § 1.1
8.9 § 0.4
318 § 5.6
6
0.64 § 0.03
9
0.82 § 0.03
12
1.48 § 0.04
2.2 § 0.1
12.6 § 0.6
15
1.77 § 0.04
3.2 § 0.1
19.9 § 1.2
15
11.3 § 0.3
11.5 § 1.5
518.5 § 8.2
6.0 § 1.2
1.1 § 0.1
32.4 § 0.4
98.0 § 1.7
34.1 § 0.8
163 § 4.0
34.0 § 0.2
116
D.Y. Yang et al. / Food Research International 39 (2006) 112–116 α-acetolactate synthase
2 pyruvates
α-acetolactate
enzymatic or nonenzymatic conversion
α-acetolactate decarboxylase
acetoin
NADH
reductase
reductase NAD+
NAD+
NADH
diacetyl
2,3-butanediol
Fig. 2. Pathways for the synthesis of diacetyl, acetoin and 2,3-butanediol in microbes.
Diacetyl was not detected in any of the juices, indicating no diacetyl was formed or all diacetyl formed was metabolized to acetoin. Higher alcohols which are common in yeast fermentation, e.g., 1-propanol, 2-methyl-1-propanol, 3-methyl1-butanol, 2-methyl-1-butanol, were negligible in the Baco Noir juices, and not detected in the Gamay juices. Other higher alcohols which were reported to be found in grapes (Schreier, Drawert, & Junker, 1976), e.g., 1-hexanol, 1-heptanol, 1-octanol, benzyl alcohol and 2-phenylethanol, were not detectable in any of the juices. 5. Conclusion Acetoin and/or 2,3-butanediol but not diacetyl began to appear in the juices of grapes undergoing CM for 9 days. Their amounts increased with the increase in time of CM, and on the 15th day of CM, acetoin was 12.6 § 0.9 mg/l in Baco Noir juice and 3.2 § 0.1 mg/l in Gamay juice, and 2,3-butanediol was 68.2 § 3.9 mg/l in Baco Noir juice and 19.9 § 1.2 mg/l in Gamay juice. Several higher alcohols in very low amounts were found in the CM Baco Noir juices (the highest one is 3.3 § 0.0 mg/ l 2-methyl-1-propanol), but no higher alcohols were detected in all the CM Gamay juices. References Bitteur, S., Tesniere, C., Fauconnet, A., Bayonove, C., & Flanzy, C. (1996). Carbonic anaerobiosis of Muscat grape, 2. Changes in the distribution of free and bound terpenols. Sciences des Aliments, 16, 37–48. Crawford, R. M. M. (1978). Metabolic adaptations to anoxia. In D. D. Hook & R. M. M. Crawford (Eds.), Plant life in anaerobic environments (pp. 119–136). Michigan: Ann Arbor Science Publishers, Inc. Ducruet, V. (1984). Comparison of the headspace volatiles of carbonic maceration and traditional wine. Lebensmittel Wissenschaft Technologie, 17(4), 217–221.
Flanzy, C., Flanzy, M., & Benard, P. (1987). La ViniWcation par la Maceration Carbonique. Paris: Institute National de la Recherche Agronomique. Gibson, T. D., Parker, S. M., & Woodward, J. R. (1991). PuriWcation and characterization of diacetyl reductase from chicken liver and Streptococcus lactis and enzymic determination of diacetyl and diketones. Enzyme and Microbial Technology, 13, 171–178. Hugenholtz, J. (1993). Citrate metabolism in lactic acid bacteria. FEMS Microbiology Reviews, 12, 165–178. Jackson, R. S. (2000). Wine science – principles, practice, perception. San Diego: Academic Press. Monnet, C., Phalip, V., Schmitt, P., & Divies, C. (1994). Comparison of -acetolactate synthase and -acetolactate decarboxylase in Lactococcus spp. and Leuconostoc spp. Biotechnological Letters, 16, 257– 262. Ribereau-Gayon, J., Peynaud, E., Ribereau-Gayon, P., & Sudraud, P. (1976). Traite d’oenologie. Sciences et techniques du vin. Tome 3 ViniWcations, transformations du vin. Paris: Dunod pp. 289–314. Robin, J. P., Romieu, C., & Sauvage, F. X. (1989). Anaerobic metabolism of organic and aminoacids in grapes. I . A device for measuring the decarboxylation and ethanol releasing kinetics from a single 14C labelled berry. American Journal of Enology and Viticulture, 40(3), 161–169. Sandler, M., & Pinder, R. (2003). Wine – a scientiWc exploration. London: Taylor & Francis pp. 245–246. Schreier, P., Drawert, F., & Junker, A. (1976). IdentiWcation of volatile constituents from grapes. Journal of Agricultural and Food Chemistry, 24, 331–336. Sneyd, T. N. (1989). Carbonic maceration: an overview. Wine Industry Journal, 11, 281–285. Snoep, J. L., Teixeira de Mattos, M. J., Starrenburg, M. J. C., & Hugenholtz, J. (1992). Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and -acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis. Journal of Bacteriology, 174, 4838–4841. Terrier, N., & Romieu, C. (1998). Inhibition of vacuolar proton pumps by ethanol impairs grape berry compartmentation. Australian Journal of Grape and Wine Research, 4, 39–45. Tesnière, C., Romieu, C., Dugelay, I., Nicol, M. Z., Flanzy, C., & Robin, J. P. (1994). Partial recovery of grape energy metabolism upon aeration following anaerobic stress. Journal of Experimental Botany, 45, 145–151. Vartapetian, B. B. (1978). Introduction-life without oxygen. In D. D. Hook & R. M. M. Crawford (Eds.), Plant life in anaerobic environments (pp. 1–11). Michigan: Ann Arbor Science Publishers, Inc.