Effect of Saccharomyces cerevisiae F12 on volatile compounds in wines at three different stages of industrial biological ageing
2008; Wiley; Volume: 14; Issue: 2 Linguagem: Inglês
10.1111/j.1755-0238.2008.00012.x
ISSN1755-0238
AutoresD. MUOZ, Rafael A. Peinado, Manuel Medina, Juan Moreno,
Tópico(s)Horticultural and Viticultural Research
ResumoAustralian Journal of Grape and Wine ResearchVolume 14, Issue 2 p. 71-77 Free Access Effect of Saccharomyces cerevisiae F12 on volatile compounds in wines at three different stages of industrial biological ageing D. MUÑOZ, D. MUÑOZ Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this authorR.A. PEINADO, R.A. PEINADO Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this authorM. MEDINA, M. MEDINA Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this authorJ. MORENO, J. MORENO Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this author D. MUÑOZ, D. MUÑOZ Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this authorR.A. PEINADO, R.A. PEINADO Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this authorM. MEDINA, M. MEDINA Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this authorJ. MORENO, J. MORENO Department of Agricultural Chemistry, University of Córdoba, Campus de Rabanales, Edificio C-3, 14014 Córdoba, SpainSearch for more papers by this author First published: 03 July 2008 https://doi.org/10.1111/j.1755-0238.2008.00012.xCitations: 6 Juan Moreno, fax +34 957 212146, e-mail qe1movij@uco.es AboutFiguresReferencesRelatedInformationPDFSectionsAbstract Introduction Materials and methods Results and discussion AcknowledgementsReferencesCiting LiteraturePDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Background and Aims: Wines subjected to biological ageing for variable lengths of time were inoculated with Saccharomyces cerevisiae F12 and were microaerated in order to reduce their overall ageing time. Methods and Results: Volatile compounds as determined by gas chromatography (GC) and GC–mass spectrometry (MS) were grouped according to their aroma descriptors into nine odourant classes, which exhibited similar changes in wines obtained by traditional ageing, and in others inoculated with S. cerevisiae F12 and microaerated. A tasting panel found the wine previously aged for 2 years and inoculated with S. cerevisiae F12 to be of better quality than the identical wine subjected to no inoculation. Conclusion: Based on the results, the ageing time for wines previously aged under typical winery conditions for 0 and 2 years can be shortened by the inoculation of S. cerevisiae F12 flor yeasts. Significance of the Study: The biological ageing system used allows the production cost of fino wines to be reduced by shortening the ageing process. Introduction Fino wines have been traditionally produced in the southern regions of Spain such as Montilla–Moriles and Jerez. Wine is obtained by using a biological ageing process involving the oxidative metabolism of flor yeasts (Martínez et al. 1997, Mauricio et al. 1997, Cortés et al. 1998, Berlanga et al. 2001, Muñoz et al. 2005). Wine is biologically aged in oak casks, using a dynamic system locally known as criaderas and solera, which was recently described by Berlanga et al. (2004). The ageing of wine with this system relies on the development and maintenance of selected flor yeasts on its surface for at least 4 years in order to obtain a high-quality product. Flor yeasts use ethanol, glycerol, various amino acids and dissolved oxygen as major nutrients during the biological ageing of wine. Depletion of such nutrients through yeast metabolism is partially offset by means of rocíos, which involves periodically mixing older wines with newer ones in the criaderas and solera system. This process ensures homogeneity in the end product by reducing the influence of differences between vintages on wine composition (Berlanga et al. 2004). The special ageing system used, which involves storing wines for at least 4 years, requires periodic control analyses, and yeast development and maintenance operations that substantially raise the cost of the resulting fino wines. This has led researchers to develop various methods for shortening the biological ageing time required to obtain fino wines of a high quality (Ough and Amerine 1958, 1972, Saavedra and Garrido 1961, Cortés et al. 1999). Recently, Muñoz et al. (2007) proposed using a periodic microaeration in wines previously inoculated with the flor yeast Saccharomyces cerevisiae (capensis) in order to reduce their ageing time. The aim of this work was to reduce the biological ageing time of fino wines previously aged under winery conditions by the inoculation with the flor yeast S. cerevisiae F12 and periodic microaeration. The resulting wines were compared, and the best among them were selected by a tasting panel of the collaborating winery; also, their volatile composition was compared in terms of odourant classes. Materials and methods Yeast strain and inocula A pure culture of S. cerevisiae F12 (Kurtzman and Fell 1998) was used in all tests. This flor yeast was isolated from the velum formed on the surface of fino wines obtained by biological ageing in the Montilla–Moriles region, southern Spain (Guijo et al. 1986). Cells were grown on a yeast medium (0.3% yeast extract, 0.3% malt extract and 0.5% peptone, pH 6.5) containing 1% glucose as carbon source, and were incubated at 27 ± 2°C with shaking for 48 h. Following incubation, the cells were collected by centrifugation at 3,500 × g; the pellet thus obtained being resuspended in a small volume of sterilised wine and used to inoculate the studied wines to a population concentration of 106 viable cells/mL. Wines Wines biologically aged for 0 (unaged), 2 and 4 years were supplied by a winery in the Montilla–Moriles winemaking region. All wines were sterilised by passage through Supra EK filters prior to testing (Seitz, Waldstetten, Germany). Culture and experimental conditions All tests were conducted in 10-L stainless steel vessels containing 8.7 L of sterilised wine (the surface-to-volume ratio was thus 39.3 cm2/L) that were thermostated at 20 ± 1°C. The wines were subjected to biological ageing with S. cerevisiae F12 under a controlled microaeration. Samples for analysis were collected (i) from the wine prior to inoculation; (ii) once the whole surface was covered with a flor film; and (iii) 14, 28, 42, 56 and 70 days after the formation of the flor film. Following the collection, each wine sample was microaerated for a short time. The microaeration was performed in a 1-L sterilised aeration chamber into which air was inserted through a sterilised filter of 0.45-µm pore size. A peristaltic pump was used to circulate the wine through the aeration chamber until a dissolved oxygen concentration of 4 mg/L was reached in the entire volume of the wine. The wine was removed from the ageing vessel at the bottom and was fed back through a piece of Teflon tubing (Figure 1). This procedure allowed the flor film to be maintained across the wine surface. The dissolved oxygen was measured by using a Crison Oxy-92 oxygen sensor on the transfer line driving the wine from the tank to the aeration chamber. All tests were performed in triplicate. Figure 1Open in figure viewerPowerPoint System used for the microaeration of the wine. Selection of wine samples Expert tasters from the collaborating winery were asked to select the particular sample of wine, among those collected during the controlled biological ageing tests, that best met the in-house quality standards for commercial fino wines: a pale colour and a dry, pungent aroma with hazelnut and distinctive 'flor yeast' notes. The profile of volatile compounds in the selected wine samples was determined using gas chromatography–mass spectrometry (GC–MS). Analytical methods The number of viable cells was determined by counting under a light microscope in a Thoma chamber, following staining with methylene blue (European Brewery Convention 1977). Ethanol was quantified with the method of Crowell and Ough (1979), and also titratable acidity, pH and volatile acidity by using the European Union recommended methods (European Economic Community 1990). The absorbances at 280, 420 and 520 nm were measured on a Beckman DU-640 UV spectrophotometer. Major volatile compounds and polyols were quantified on a Model 6890 gas chromatograph from Agilent Technologies (Palo Alto, California, USA) furnished with a CP-WAX 57 CB capillary column (60-m long × 0.25-mm i.d., 0.4-µm film thickness), using the method of Peinado et al. (2004). Minor volatile compounds were determined in a capillary column, using GC–MS after a continuous extraction of 100-mL wine samples with 100 mL of freon-11 for 24 h (Rapp et al. 1976). The samples were previously adjusted to pH 3.5 and were supplemented with 5 mL of a 30 mg/L solution of 2-octanol as internal standard. The freon extracts containing the volatile compounds were concentrated to 0.2 mL in a Kuderna–Danish microconcentrator, and 1.5-µL aliquots were injected into an HP-6890 gas chromatograph from Agilent Technologies equipped with an HP MS 5972A mass detector. An HP-Innowax fused silica capillary column (60-m long × 0.32-mm i.d., 0.25-µm film thickness) was used for this purpose. The temperature program was as follows: an initial temperature of 40°C, held for 10 minutes, ramped to 180°C at 1°C/min, and held for 35 minutes. Helium at a constant flow rate of 0.9 mL/min was used as carrier gas, and a 30 : 1 split ratio was employed at the injection port. The mass detector was used at 1,612 V in the scan mode; the mass range examined spanned values from 39 to 300 amu. Spectral libraries were supplied by Wiley (Chichester, England), and pure chemical compounds were obtained from Merck (Whitehouse Station, New Jersey, USA), Sigma–Aldrich (Buchs SG, Switzerland), Riedel de Haën and Fluka (Seelze, Germany). The retention times for the volatile compounds were used to identify, confirm and prepare standard solutions of the studied volatile compounds. Each compound was quantified from its response factor, which was determined by using standard solutions of known concentration subjected to the same treatment as the samples, and the target ions and qualifier ions were selected for each compound by the Hewlett–Packard Chemstation (Palo Alto, California, USA). Determination of odour perception thresholds The odour perception threshold is defined as the lowest concentration of a substance capable of producing a sensation detectable by at least 50% of the members of a tasting panel (Cutzach et al. 2000). Solutions containing increasing concentrations of each volatile compound concerned were used for this purpose. Starting from the least concentrated solution, the panel members were asked to identify the first solution departing from the stimulus perceived with the control. The control is a 1/10 ethanol–water mixture containing no volatile compounds. Our panel consisted of 30 members (males and females of 20–55 years) who were trained experts, but not specifically selected for the test. Odour perception thresholds were determined in accordance with the applicable standard (Asociación Española de Normalización y Certifacición 1997), and the results were consistent with those previously reported by Moreno (2005). Statistical processing The results given are the averages of three independent tests each. The statistical software package Statgraphics Plus v.2, from STSC, Inc. (Rockville, Maryland, USA), was used for one-way analysis of variance (ANOVA) in order to identify the compounds and odourant classes exhibiting significant differences between samples. Results and discussion Thirty days after the inoculation of the studied wines (viz. wines previously aged biologically under winery conditions for 0, 2 and 4 years) with S. cerevisiae F12, a thin (1 mm) brown-cream coloured film was found covering their surface. This flor yeast prevails together with the yeast strain S. cerevisiae G1 in the flor film of the wines biologically aged in the Montilla–Moriles winemaking region. Wine samples were collected for analysis at 14-day intervals from the velum formation to 70 days later. Tasters selected the samples collected 14 days after the film formation as the best for the 0-year-old wine; those corresponding to the film formation for the 2-year-old wine; and those collected 70 days after the film formation for the 4-year-old wine. In a previous paper (Muñoz et al. 2007), wines were inoculated with the flor yeast S. cerevisiae G1, and the taster selected the sample collected 28 days after the film formation as the best for the 0-year-old wines, and those collected 56 days after the film formation for the 2-year-old wine. Winemaking variables During biological ageing, flor yeasts develop an oxidative metabolism, which uses ethanol, glycerol and acetic acid, and which produces acetaldehyde. The values obtained for the winemaking variables (Table 1) are typical of biologically aged wine (Cortés et al. 1998, 1999, Berlanga et al. 2004, Moreno 2005, Muñoz et al. 2005). Worthy of special note are the high concentrations of acetaldehyde, especially in the wine previously aged for 2 years and inoculated with S. cerevisiae F12. Such values may have resulted from the high aldehyde dehydrogenase II activity in this strain (Mauricio et al. 1997). Absorbance measurements at 420 and 520 nm indicate that the controlled microaeration resulted in no browning of the wines (Table 1). Table 1. Winemaking variables quantified in the wines aged in cellar conditions, and in the wines inoculated with Saccharomyces cerevisiae F12 and selected by the tasting panel of the collaborating winery. Compounds Wine ageing in cellar conditions Wine ageing in controlled conditions Young wine 2 years 4 years 0 year + 14 days * 2 years + velum † 4 years + 70 days ‡ Ethanol (% v/v) 16.4 ± 0.3a 15.8 ± 0.3b 15.2 ± 0.1c 15.7 ± 0.3b 14.9 ± 0.3c 13.4 ± 0.3d Glycerol (g/L) 8.3 ± 0.2a 5.2 ± 0.4b 3.8 ± 0.1c 8.4 ± 0.2a 3.5 ± 0.1c 0.4 ± 0.2d Acetaldehyde (mg/L) 100 ± 5a 179 ± 4b 285 ± 9c 750 ± 22d 893 ± 15e 458 ± 34f pH 3.51 ± 0.02a 3.33 ± 0.02b 3.14 ± 0.01c 3.54 ± 0.05a 3.29 ± 0.02b 3.18 ± 0.03c Titratable acidity (meq/L) 40.2 ± 0.3a 52.8 ± 0.4b 65.5 ± 0.5c 38.1 ± 0.5d 52.0 ± 0.4b 50.0 ± 0.9e Volatile acidity (meq/L) 4.8 ± 0.5ab 5.2 ± 0.2a 4.4 ± 0.7b 0.5 ± 0.3c 2.5 ± 0.0d 1.1 ± 0.3c Absorbance 280 nm 7.42 ± 0.08a 8.6 ± 0.6b 8.035 ± 0.003c 7.4 ± 0.1a 8.7 ± 0.2b 8.11 ± 0.09c Absorbance 420 nm 0.17 ± 0.01a 0.190 ± 0.003b 0.162 ± 0.001c 0.157 ± 0.006c 0.160 ± 0.004c 0.13 ± 0.01d Absorbance 520 nm 0.043 ± 0.006a 0.050 ± 0.004b 0.035 ± 0.001c 0.040 ± 0.003ac 0.037 ± 0.006c 0.020 ± 0.004d Different letters indicate significant differences at P ≤ 0.05. * 0 year + 14-day young wine (unaged) subjected to ageing with S. cerevisiae F12 for 14 days. † 2 years + velum: wine previously aged for 2 years in cellar conditions and subjected to ageing with S. cerevisiae F12 until velum formation. ‡ 4 years + 70 days: wine previously aged for 4 years in cellar conditions and subjected to ageing with S. cerevisiae F12 for 70 days. Odour activity values (OAVs) Table 2 lists the OAVs for the 35 volatile compounds studied and the homogeneous groups obtained by ANOVA. The OAV for each compound was obtained as the ratio of its concentration to its odour perception threshold, which is listed in Table 3 together with its odour descriptor. In this way, each volatile compound was identified in terms of its contribution to the wine aroma (OAV) and its odour descriptor. Table 2. Odour activity values of the volatile compounds quantified in the wines aged in cellar conditions, and in the wines inoculated with Saccharomyces cerevisiae F12 and selected by the tasting panel of the collaborating winery. Compounds Wine ageing in cellar conditions Wine ageing in controlled conditions Young wine 2 years 4 years 0 year ± 14 days * 2 years ± velum † 4 years ± 70 days ‡ Acetaldehyde 0.91 ± 0.05a 1.63 ± 0.03b 2.59 ± 0.09c 6.8 ± 0.2d 8.1 ± 0.1e 4.2 ± 0.3f 1,1-Diethoxyethane 13.9 ± 0.7a 22.9 ± 0.6b 36 ± 1c 100 ± 2d 109 ± 2e 52 ± 4f Acetoin 0.11 ± 0.01ab 0.04 ± 0.02a 0.14 ± 0.01b 0.21 ± 0.05c 0.25 ± 0.03c 1.01 ± 0.09d 2,3-Butanediol (levo + meso) 5.4 ± 0.3a 6.5 ± 0.1bc 6.8 ± 0.2b 5.7 ± 0.2a 6.19 ± 0.04c 7.6 ± 0.1d 1-Propanol 0.09 ± 0.01a 0.143 ± 0.001bc 0.139 ± 0.003b 0.09 ± 0.01a 0.153 ± 0.003cd 0.16 ± 0.01d Isobutanol 0.72 ± 0.01a 0.69 ± 0.03ab 0.67 ± 0.01b 0.61 ± 0.03c 0.84 ± 0.04d 0.93 ± 0.03e Isoamyl alcohol 5.85 ± 0.02a 5.8 ± 0.2a 4.56 ± 0.08b 5.2 ± 0.3c 5.8 ± 0.2a 5.1 ± 0.1c 2-Phenethyl alcohol 0.48 ± 0.02a 0.36 ± 0.03cd 0.25 ± 0.01b 0.37 ± 0.03c 0.37 ± 0.01c 0.33 ± 0.02d 1-Butanol 0.02 ± 0.01a 0.043 ± 0.006b 0.06 ± 0.01c 0.02 ± 0.01a 0.043 ± 0.006b 0.09 ± 0.01d 2-Butanol Nd Nd 0.027 ± 0.006a Nd 0.07 ± 0.01b 0.116 ± 0.006c 1-Hexanol 0.89 ± 0.02a 0.50 ± 0.03b 0.64 ± 0.01c 0.76 ± 0.03d 0.60 ± 0.04c 0.47 ± 0.02b 2,3-Butanedione 0.30 ± 0.01a 0.18 ± 0.06b 0.566 ± 0.003c 0.063 ± 0.006d 0.14 ± 0.03b 0.28 ± 0.02a 2,3-Pentanedione Nd 1.50 ± 0.07a 4.6 ± 0.3b 6.5 ± 0.9c 4.02 ± 0.09b 16.5 ± 0.8d Ethyl acetate 4.5 ± 0.1a 5.6 ± 0.6b 3.6 ± 0.1c 3.3 ± 0.2c 4.4 ± 0.2a 2.2 ± 0.3d Isobutyl acetate Nd 0.010 ± 0.002a Nd 0.09 ± 0.01b 0.113 ± 0.006c Nd Isoamyl acetate 5.0 ± 0.6a 2.1 ± 0.2b 1.1 ± 0.4c 1.2 ± 0.2bc 1.7 ± 0.3bc Nd 2-Phenethyl acetate 0.97 ± 0.05a 0.28 ± 0.01b 0.12 ± 0.01c 0.83 ± 0.02d 0.19 ± 0.08c 0.12 ± 0.01c Ethyl propanoate 0.10 ± 0.02a 0.23 ± 0.03ad 0.6 ± 0.1b 1.2 ± 0.2c 0.19 ± 0.08d 0.12 ± 0.01d Ethyl 3-hydroxybutanoate 0.46 ± 0.06a 0.53 ± 0.03a 0.47 ± 0.06a 0.23 ± 0.04b 0.24 ± 0.03b 0.36 ± 0.06a Ethyl butanoate 0.44 ± 0.05a 0.50 ± 0.03a 0.47 ± 0.06a 0.23±0.04b 0.24 ± 0.03b 0.36 ± 0.06a Ethyl octanoate Nd 0.23 ± 0.01a 0.43 ± 0.01b Nd Nd 0.96 ± 0.04c Diethyl malate 0.023 ± 0.006a 0.21 ± 0.04b 0.49 ± 0.02c Nd Nd 0.40 ± 0.02c Isobutanoic acid 0.04 ± 0.02a 0.10 ± 0.02b 0.187 ± 0.006c 0.043 ± 0.006a 0.11 ± 0.01b 0.41 ± 0.04d Butanoic acid 0.53 ± 0.04a 0.7 ± 0.1bd 1.22 ± 0.07c 0.647 ± 0.006ab 0.97 ± 0.03d 2.4 ± 0.3e 2 and 3-Methylbutanoic acids 0.52 ± 0.07a 1.44 ± 0.08b 2.66 ± 0.04c 0.66 ± 0.01a 1.54 ± 0.06b 3.6 ± 0.4d Hexanoic acid 0.26 ± 0.06ab 0.24 ± 0.03a 0.31 ± 0.01bcd 0.34 ± 0.03d 0.28 ± 0.02abc 0.33 ± 0.03cd Octanoic acid 0.16 ± 0.02a 0.09 ± 0.01b 0.07 ± 0.01c 0.16 ± 0.02a Nd 0.031 ± 0.006d 4-Butyrolactone 0.44 ± 0.05a 0.88 ± 0.03b 1.1 ± 0.1c 0.447 ± 0.006a 1.2 ± 0.2cd 1.31 ± 0.07d Pantolactone 0.12 ± 0.04a 0.8 ± 0.1c 1.6 ± 0.1d 0.31 ± 0.02ab 0.5 ± 0.3bc 2.6 ± 0.3e Z-whisky lactone Nd 0.3 ± 0.1a 0.9 ± 0.1b Nd Nd 0.7 ± 0.1c Methionol Nd 4.3 ± 0.4a Nd 3.3 ± 0.4b 4.0 ± 0.2a Nd 4-Ethylguaiacol Nd 1.4 ± 0.2a 3.8 ± 0.3a 3.5 ± 0.5b Nd 3.8 ± 0.6a Furanmethanol Nd 0.017 ± 0.005a 0.033 ± 0.006b Nd Nd 0.037 ± 0.006b 3-Ethoxy-1-propanol Nd 3.3 ± 0.3a 6.0 ± 0.5b 1.3 ± 0.2c Nd 8.0 ± 0.3d Neral 0.20 ± 0.02a 0.23 ± 0.08a Nd 0.44 ± 0.06b 0.18 ± 0.07a 0.19 ± 0.02a Different letters indicate significant differences at P ≤ 0.05. * 0 year + 14-day young wine (unaged) subjected to ageing with S. cerevisiae F12 for 14 days. † 2 years + velum: wine previously aged for 2 years in cellar conditions and subjected to ageing with S. cerevisiae F12 until velum formation. ‡ 4 years + 70 days: wine previously aged for 4 years in cellar conditions and subjected to ageing with S. cerevisiae F12 for 70 days. Nd, not detected. Table 3. Volatile compounds quantified in wines, odour perception thresholds (OPT) in mg/L, odour descriptions and assignment of volatile compounds to odourant classes. Compound OPT Odour descriptor Aroma series* Acetaldehyde 110 Pungent, ripeness apple 1 1,1-Diethoxyethane 1 Liquorice, green fruit 1, 2 Acetoin 150 Buttery, cream 7 2,3-Butanediol 150 Fruity 1 Propanol-1 306 Ripe fruit, alcohol 1, 3 Isobutanol 75 Alcohol, nail polish 3 Isoamyl alcohol 60 Alcohol, nail polish 3 2-Phenylethanol 200 Rose, honey 4 1-Butanol 150 Medicinal 6 2-Butanol 50 Winelike, solvent 3 1-Hexanol 1.1 Herbaceous, grass, woody 5 2,3-Butanedione 3 Buttery, toasted 7, 8 2,3-Pentanodione 0.9 Buttery, toasted 7, 8 Ethyl acetate 12 Pineapple, varnish, balsamic 1, 3 Isobutyl acetate 1.6 Sweet, fruity, apple, banana 1 Isoamyl acetate 0.16 Banana, fruity 1 2-Phenethyl acetate 0.25 Rose, Fruity 4, 1 Ethyl propanoate 1.8 Banana, apple 1 Ethyl-3-hidroxybutanoate 1 Fruity, grape 1 Ethyl butanoate 0.4 Banana, pineapple, strawberry 1 Ethyl octanoate 0.58 Banana, pineapple, pear, floral 4, 1 Diethyl malate 10 Fruity 1 Ethyl lactate 150 Buttery, butterscotch, fruit 1, 7 Isobutanoic acid 30 Rancid butter 7 Butanoic acid 2.2 Cheese, rancid, putrid 7 2 and 3-Methylbutanoic acids 1.5 Rancid 7 Hexanoic acid 8 Rancid, fatty, cheese 7 Octanoic acid 10 Fatty, rancid 7 4-Butyrolactone 20 Caramel, coconut 1, 8 Pantolactone 2.2 Balsamic, smoky, toasted bread 2, 8 Z-whisky lactone 0.07 Coconut 1 Methionol 1.5 Cooked potato, cut hay 5 4-Ethylguaiacol 0.02 Smoky, toasted bread, clove 8, 9 Furanmethanol 15 Floral 4 3-Ethoxy-1-propanol 0.1 Fruity 1 Neral 0.5 Fruity 1 * Odourant classes: 1, fruity; 2, balsamic; 3, solvent; 4, floral; 5, herbaceous; 6, phenolic; 7, fatty; 8, roasty; 9, spicy. Acetaldehyde, 1,1-diethoxyethane, 2,3-butanediol (levo + meso forms), isoamyl alcohols, 2,3-pentanodione, ethyl and isoamyl acetates, butanoic acid, 2- and 3-methylbutanoic acids, γ-butyrolactone, pantolactone, 4-ethylguaiacol, and 3-ethoxy-1-propanol exhibited OAVs greater than 0.8 units in the wines aged under winery conditions (Table 2). Except for acetates and some higher alcohols, the OAVs increased during the biological ageing with S. cerevisiae F12. Acetoin, 1-butanol, 2-butanol, isobutyl acetate, furanmethanol and neral exhibited OAVs 10 times below their odour perception thresholds in the wines aged under winery conditions. The remaining compounds had OAVs ranging from 0.1 to 0.8. 2-Butanol, 2,3-pentanodione, isobutyl acetate, ethyl octanoate, Z-whisky lactone, methionol, 4-ethylguaiacol, furanmethanol and 3-ethoxy-1-propanol exhibited zero OAVs in the wine aged for 0 year (young wine). The high OAVs for Z-whisky lactone, 4-ethylguaiacol and furanmethanol in the wine aged under winery conditions for 4 years were a result of their storage in American oak casks for a long time. Yeasts are hydrolysed during the biological ageing of wine, and dead yeasts depositing at the bottom of casks can contribute to the composition of the resulting wine (Muñoz et al. 2001, Berlanga et al. 2004). The effect of S. cerevisiae F12 can be easily seen by comparing the wines prior to inoculation and after treatment. Thus, a total of 21 compounds exhibited significant differences in concentration in the young wine, 20 compounds in the wine previously aged for 2 years, and 27 compounds in that aged for 4 years. Odourant classes Grouping the volatile aroma compounds with similar odour descriptors into odourant classes is useful for establishing the analytical odour profile of wine. The value for each class is calculated as the combination of the OAVs for the individual compounds in it. We used fruity, balsamic, solvent, floral, herbaceous, phenolic, fatty, roasty and spicy odourant classes (Franco et al. 2004, Peinado et al. 2006, Muñoz et al. 2007) to compare the studied wines. Table 4 shows the overall values for each odourant class. The fruity, balsamic, fatty, phenolic, roasty and spicy classes exhibited significantly increased OAVs as a result of the biological ageing in the winery (0-, 2- and 4-year-old wines). By contrast, the solvent, floral and herbaceous classes had decreased OAVs; however, an increase in the herbaceous class was observed in the wine aged for 2 years, which can be ascribed to the presence of methionol. The increased OAV for the spicy class in the wines aged under winery conditions may have resulted from the extraction of volatile compounds in the oak casks. Table 4. Values of the odourant series in the wines aged in cellar conditions, and in the wines inoculated with Saccharomyces cerevisiae F12 and selected by the tasting panel of the collaborating winery. Compounds Wine ageing in cellar conditions Wine ageing in controlled conditions Young wine 2 years 4 years 0 year ± 14 days * 2 years ± velum † 4 years ± 70 days ‡ Fruity 32.1 ± 0.6a 42 ± 1b 55 ± 1c 117 ± 5d 132 ± 2e 71 ± 2f Balsamic 14.0 ± 0.7a 23.8 ± 0.4b 38 ± 1c 98 ± 5d 109 ± 2e 55 ± 4f Solvent 11.1 ± 0.1a 12.2 ± 0.7b 8.96 ± 0.05c 0.09 ± 0.01d 0.23 ± 0.02d 0.28 ± 0.01d Floral 1.45 ± 0.05a 0.90 ± 0.03b 0.84 ± 0.02b 1.20 ± 0.01c 0.56 ± 0.09e 1.45 ± 0.05a Herbaceous 0.89 ± 0.02a 4.7 ± 0.4b 0.64 ± 0.01a 4.0 ± 0.4c 4.6 ± 0.2b 0.47 ± 0.02a Phenolic 0.02 ± 0.01a 0.043 ± 0.006b 0.057 ± 0.006c 0.020 ± 0.004a 0.043 ± 0.006b 0.09 ± 0.01d Fatty 2.3 ± 0.2a 5.8 ± 0.4b 11.9 ± 0.2c 9.1 ± 0.9d 8.8 ± 0.1d 26.5 ± 0.3e Roasty 0.86 ± 0.04a 4.8 ± 0.3b 11.7 ± 0.1c 10.8 ± 0.5d 5.7 ± 0.4e 24.5 ± 0.3f Spicy Nd 1.4 ± 0.2a 3.8 ± 0.3b 3.5 ± 0.5b Nd 3.8 ± 0.6b Σ odour series 63 ± 1a 95 ± 2b 130 ± 2c 244 ± 11d 261 ± 5e 182 ± 8f Different letters indicate significant differences at P ≤ 0.05. * 0 year + 14-day young wine (unaged) subjected to ageing with S. cerevisiae F12 for 14 days. † 2 years + velum: wine previously aged for 2 years in cellar conditions and subjected to ageing with S. cerevisiae F12 until velum formation. ‡ 4 years + 70 days: wine previously aged for 4 years in cellar conditions and subjected to ageing with S. cerevisiae F12 for 70 days. Nd, not detected. The inoculation of the wines with S. cerevisiae F12 had a strong impact on their odour profiles. Thus, the OAVs for the fruity and balsamic classes increased by 16–90 units, especially in the wines previously aged for 0 and 2 years (Table 4). Muñoz et al. (2007) reported no differences in the fruity and phenolic classes of wines previously aged for 0 and 2 years, and in the balsamic classes of wines previously aged for 2 years and inoculated with S. cerevisiae G1. The OAVs for the fatty and roasty classes also increased by 1–14 units, particularly in the wines aged in the winery for 4 years and then under S. cerevisiae F12 for 70 days. By contrast, the OAV for the solvent class decreased significantly in all wines inoculated with S. cerevisiae F12. The floral character increased only in the wine previously aged for 4 years, while the herbaceous character increased in the unaged wine alone. The OAV for the phenolic class only increased significantly in the wine previously aged for 4 years, whereas that for the spicy class changed significantly in the wines previously aged for 0 and 2 years. Overall, the effects of S. cerevisiae F12 on the volatile compounds were closely related to the age of the initial wine. Thus, the fruity and balsamic classes exhibited the smallest changes in the wine aged for 4 years, which was when the fatty and roasty classes exhibited their largest changes. The opposite was true of the former classes in the wines previously aged under winery conditions for 0 and 2 years. The members of the winery tasting panel were asked to select the best samples among all wines aged under S. cerevisiae F12 relative to the wine aged under winery conditions for 4 years. They found the best samples to be that of the wine previously aged for 4 years and then for a further 70 days under controlled conditions in the presence of the inoculated yeast. In addition, the panel emphasised the improvement in the wine aged under winery conditions for 2 years and subsequently inoculated with S. cerevisiae F12 with respect to the same wine without inoculation. In this case, the results for the odourant classes in the inoculated young wine only revealed the effect of yeast metabolism, whereas those for the wines aged for 2 and 4 years, and inoculated with S. cerevisiae F12, displayed the effects of both yeast metabolism and previous ageing under winery conditions. As stated previously, the OAVs for the roasty and spicy classes exhibited significant differences between the young wine and the wine previously aged under winery conditions. The volatile compounds in these two classes are the result of yeast autolysis (Muñoz et al. 2001) or extraction from cask wood (Moreno 2005), so their importance depends on the ageing time. Finally, the increased OAVs for the fruity and balsamic classes in the wine aged for 4 years and then 70 days with yeast may account for the panel selecting it as the best. Although the tasters' choices may be consistent with analytically determined changes in odourant classes, it is impossible to establish a direct relation between tasters' selection and odourant classes. However, grouping the volatile aroma compounds into odourant classes is useful with a view to comparing wines obtained with different treatments, and also to studying changes in wine during ageing. In addition, the approach used here greatly reduces the number of variables involved in such studies, and allows changes during the ageing process to be discussed in terms of several aroma descriptors while retaining their relative importance in accordance with the OAVs for the compounds they encompass. 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