Changes in the aromatic composition of Tempranillo wines during spontaneous malolactic fermentation

Changes in the aromatic composition of Tempranillo wines during spontaneous malolactic fermentation

ARTICLE IN PRESS Journal of Food Composition and Analysis 21 (2008) 724– 730 Contents lists available at ScienceDirect Journal of Food Composition a...

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ARTICLE IN PRESS Journal of Food Composition and Analysis 21 (2008) 724– 730

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca

Original Article

Changes in the aromatic composition of Tempranillo wines during spontaneous malolactic fermentation ˜ as a,, E. Garcı´a Romero a, S. Go´mez Alonso a, M.L.L. Palop Herreros b P.M. Izquierdo Can a b

Instituto de la Vid y del Vino de Castilla-La Mancha (IVICAM), Crta. Toledo-Albacete s/n, 13700 Tomelloso (Ciudad Real), Spain ´gico de la Fabrica de Armas, Avda. Carlos III s/n, 45071 Toledo, Spain Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, Campus Tecnolo

a r t i c l e in f o

a b s t r a c t

Article history: Received 8 September 2006 Received in revised form 12 November 2007 Accepted 27 December 2007

The influence of spontaneous malolactic fermentation on the aromatic composition of 16 industrially elaborated Vitis vinifera cv. Tempranillo (Cencibel) red wines was studied. For this purpose, we determined the concentration of 114 volatile compounds at the end of alcoholic fermentation and after malolactic fermentation. This process produces important increases in the content of succinic acid esters (ethyl monosuccinate, diethyl succinate and ethyl-methyl succinate), lactones (b-ethoxy-gbutirolactone and b-(1-hydroxy-ethyl)-g-butirolactone), terpenes (a-terpineol), norisoprenoids (damascenone and 3-hydroxy-b-damascenone) and volatile phenols (vanillin and siringaldehide), thus revealing the importance of the b-glucosidase activity of lactic bacteria in volatile composition and in the improvement of the organoleptic characteristics of wines. & 2008 Published by Elsevier Inc.

Keywords: Cencibel Lactic bacteria Malolactic fermentation MLF Tempranillo Volatile compounds Aromatic compounds Wine Red wine Castilla-La Mancha Spain Oenococcus oeni Food composition

1. Introduction Malolactic fermentation (MLF) is one of the main stages in the elaboration of red wines. In this process lactic bacteria, present spontaneously in wine or added intentionally after alcoholic fermentation, decarboxilate L-malic acid converting it into L-lactic acid. This process, in addition to reducing acidity, enables greater microbiological stability to be achieved and multiple transformations occur that make important contributions to the organoleptic qualities of wines. One of the main species identified during spontaneous MLF is Oenococcus oeni since it is the most tolerant to adverse wine conditions (Lonvaud-Funel, 1999). Some authors (Maicas et al., 1999; Bartowsky and Henschke, 2004) have studied the biosynthesis of aromatic compounds produced during MLF and their organoleptic consequences (Palacios et al., 2003). All agree that the resulting modifications are highly complex and often involve the reduction of vegetable

 Corresponding author. Tel.: +1 501 663 3550; fax: +1 501 954 8882.

˜ as), E-mail addresses: [email protected] (P.M. Izquierdo Can [email protected] (M.L.L. Palop Herreros). 0889-1575/$ - see front matter & 2008 Published by Elsevier Inc. doi:10.1016/j.jfca.2007.12.005

and herbaceous aromas and the appearance of other fruity, floral, nutty or milky aromas. In contrast, Sauvageot and Vivier (1997) observed that changes occurring in MLF are not very important and reported only slight sensorial differences. It is important to highlight the enormous influence of the lactic bacteria strains or strain participating in the process (Gambaro et al., 2001; Pozo-Bayon et al., 2005; Ugliano and Moio, 2005) and the type of elaboration process (industrial or laboratory) (Delaquies et al., 2000) on the aromatic complexity and composition of wine. In industrial processes, the development of MLF starts immediately after alcoholic fermentation (over yeast lies) and the results can be different to those obtained in the laboratory when the MLF is carried out with clean and/or filtered wines. The most grown red grape variety in the region of Castilla-La Mancha (Spain), as in the rest of Spain, is Vitis vinifera cv. Tempranillo, also called ‘‘Cencibel’’. It is common in wineries in this part of the country that MLF occurs spontaneously with the participation of indigenous bacteria, perfectly adapted to local environmental conditions. Few studies have previously examined this grape variety specifically in connection with changes in the composition of aromatic compounds during MLF, and the ones that do have

ARTICLE IN PRESS ˜as et al. / Journal of Food Composition and Analysis 21 (2008) 724–730 P.M. Izquierdo Can

studied these changes in laboratory conditions and with selected bacteria. Therefore, in this research we decided to study the predominant changes in the composition of the volatile fraction of Cencibel wines occurring during spontaneous MLF, carried out by different indigenous strains, under industrial conditions.

2. Materials and methods 2.1. Samples Sixteen Cencibel wines were studied from 9 wineries in Castilla-La Mancha (Spain). The elaboration techniques used were the same as those normally employed in each winery. The sampled deposits were selected at random, the only condition being that MLF had to take place spontaneously. Two samples were taken from each wine: one at the end of alcoholic fermentation, determined by measurements of density and glucose–fructose content; and another once MLF was concluded. The beginning and the end of MLF were determined according to the results of the analyses of the L-malic and L-lactic acid content of the wines. All these analyses were carried out by the research team.

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The extract was concentrated by distillation in a Vigreux column to 2 ml and then kept at 20 1C until analysis. A total of 2 mL of the extract was injected in a BP21 column (SGE) with 50 m  0.32 mm internal diameter and 0.25-mm-thick FFAP phase (polyethylenglycol treated with TPA). The chromatographic conditions were as follows: oven temperature, 43 1C (15 min)– 2 1C/min–125 1C–1 1C/min–150 1C–4 1C/min–200 1C (45 min) and carrier gas helium (1.4 ml/min, split 1/15, splitless time 0.5 min). Separated compounds were identified by their mass spectra and their chromatographic retention times, using commercial products ACS grade from Sigma-Aldrich Chemie (Steinheim, Germany), Fluka (Buchs, Germany), Merck (Darmstard, Germany) and Extrasynthese (Genay, France) as a standard. Quantification was performed by analysing the characteristic m/z fragment for each compound using the internal standard method. Linearity was checked for each compound. For available compounds, response factors vs. internal standard were calculated from an injection of a standard mixture solution each with 8 samples. Results for nonavailable products are shown as the relationship between the area of each compound and that of the internal standard. 2.4. Statistical analysis Statistical analysis (medians and percentiles) of the results was carried out to compare the aromatic composition of wines before and after MLF (SPSS 12.0 software).

2.2. Physicochemical analysis The most common physicochemical parameters in the wine were determined, namely density, glucose–fructose content, alcohol content, pH, total acidity, L-malic acid, L-lactic acid, citric acid and volatile acidity, in accordance with the procedures described in the Official Methods of Wine Analysis (European Union, 1990). 2.3. Analysis of volatile compounds Samples were analysed by GC/MS using a TermoQuest mod. GC2000 gas chromatograph and a TRACE2000 mass detector with quadrupole analyser. All masses were obtained in the electronic impact mode at 70 eV. The selected detector and electron multiplier temperatures were 250 1C and 250 V, respectively. For the major volatile compounds, 200 ml of wine was steam distilled up to a volume of 200 ml, and 1 ml of distilled wine with 4-methyl-2-penthanol as the internal standard was directly injected. The chromatographic conditions were as follows: CPWax 57 CB (Varian, Inc.) 50 m  0.32 mm and 0.2 mm phase thick column, with helium as the carrier gas (1.7 ml/min, split 1/25); injector temperature, 220 1C; and oven temperature, 43 1C (5 min)–4 1C/min–100 1C–20 1C/min–190 1C (1 min). For analysis of the minor volatile compounds, 500 ml wine samples containing 100 ml of 10 g/l 4-nonanol as the internal standard were extracted during 24 h with 250 ml of a 60:40 mixture of pentane–dichloromethane.

3. Results and discussion Microbiological analyses of the wine samples have showed that O. oeni was the predominant species involved in MLF in every wine; moreover, the presence of different predominant O. oeni strains at each cellar studied has been demonstrated (data not shown). Table 1 shows the mean value, standard deviation and the minimum and maximum values obtained for the physicochemical parameters analysed in the 16 wines, before and after MLF. When considering the mean values for all samples, during MLF we observed a decrease in the total acidity of around 1 g/l and a subsequent increase in pH of approximately 0.2 units. We also noted an average increase of 0.13 g/l in volatile acidity and the almost total transformation of malic acid into lactic acid, and the decrease of citric acid. The gas chromatography results of the volatile fraction are shown in Tables 2–4. A total of 114 volatile compounds from different groups—alcohols, acids, esters, aldehydes and cetones, lactones, terpenes, norisoprenoids and phenols—were identified and analysed. 3.1. Alcohols Alcohol concentrations tended to increase, except in the case of 2-phenylethanol, c-2-penten-1-ol and 3-methyl-1-pentanol for

Table 1 Mean value, standard deviation, maximum and minimum values of the physicochemical parameters studied in wine Wine after alcoholic fermentation (n ¼ 16)

Alcohol (%) Total acidity (g/l) pH Volatile acidity (g/l) L-malic acid (g/l) L-lactic acid (g/l) Citric acid (g/l)

Wine after malolactic fermentation (n ¼ 16)

Mean

SD

Maximum

Minimum

Mean

SD

Maximum

Minimum

13.5 5.86 3.65 0.29 2.01 0.35 0.28

0.44 1.12 0.16 0.08 0.31 0.20 0.07

14.4 7.70 3.89 0.45 2.42 0.76 0.38

12.9 3.93 3.37 0.18 1.27 0.11 0.09

13.6 5.04 3.81 0.42 0.20 1.65 0.06

0.48 1.01 0.22 0.09 0.09 0.27 0.09

14.6 7.11 4.08 0.53 0.34 1.96 0.26

12.9 3.37 3.47 0.27 0.00 1.07 0.00

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Table 2 Percentile 10, median, interquartile range (IQR) and percentile 90 values of the alcohols and acids studied in wine before and after MLF Wine after AF (n ¼ 16)

Wine after MLF (n ¼ 16)

Variation

P10

Median

IQR

P90

P10

Median

IQR

P90

Median (%)

Alcohols Methanol* Propanol* Isobutanol* 1-Butanol* 1-Pentanol** Active amyl+isoamyl* 1-Octanol** 3-Octanol** c-2-penten-1-ol** 3-Methyl-1-pentanol** 3-Methyl-3-buten-1-ol** 1,9-Nonanediol** 1,10-Decanediol** L-2,3-Butanodiol* Meso-2,3-butanodiol* 1-Hexanol* t-2-Hexen-1-ol** t-3-Hexen-1-ol** c-2-Hexen-1-ol** c-3-Hexen-1-ol** 3-Ethoxy-1-propanol** 2-Methyl-thio-etanol**** 3-Methyl-thio-propanol* 6-Methyl-5-hepten-2-ol** 3-Ethyl-thio-propanol** Furfuryl alcohol** Benzyl alcohol* 2-Phenylethanol*

78.17 30.10 29.64 0.68 80.08 164.7 28.20 6.25 30.35 105.1 100.8 4.38 3.36 0.86 5.08 2.33 1.37 96.32 12.09 0.19 290.8 0.03 0.82 3.98 23.99 14.96 0.13 24.05

118.1 36.94 39.73 1.21 113.6 212.3 40.95 6.62 55.23 170.7 123.6 5.53 10.84 1.30 7.66 3.26 6.45 119.0 15.80 0.29 503.3 0.04 1.24 9.13 31.62 21.11 0.23 30.26

33.94 11.04 9.46 0.53 69.72 50.94 20.48 0.50 19.62 64.64 34.83 1.90 8.02 0.68 4.01 1.10 13.22 26.15 3.79 0.14 360.0 0.02 1.05 10.41 20.85 21.74 0.08 11.19

156.0 55.43 51.02 1.60 183.3 238.9 61.94 7.47 87.56 262.4 145.8 8.96 21.72 2.41 15.58 3.86 27.08 131.8 18.48 0.45 1208 0.06 2.67 30.24 52.33 110.1 0.47 44.07

88.46 31.23 33.26 0.68 80.24 167.1 38.69 6.11 27.08 101.2 105.1 7.05 6.49 0.82 5.95 2.52 3.66 98.28 13.84 0.24 306.8 0.03 0.92 8.46 22.28 25.79 0.16 24.65

123.0 38.01 40.96 1.20 126.8 220.2 45.79 6.62 48.67 165.4 127.9 8.31 16.34 1.53 9.24 3.43 12.49 121.0 16.75 0.34 560.0 0.04 1.41 17.00 33.42 41.33 0.27 28.85

15.82 9.02 8.32 0.59 111.5 48.16 20.41 0.52 32.24 70.69 33.36 1.68 9.25 0.90 7.68 1.42 13.58 32.94 4.08 0.13 258.2 0.01 0.94 19.39 20.71 26.34 0.11 9.95

140.6 52.04 45.45 1.77 205.5 238.8 72.56 7.33 95.33 256.4 167.4 14.39 34.01 2.45 16.49 4.23 37.66 162.3 23.98 0.48 1175 0.06 2.59 45.88 55.73 194.9 0.57 40.77

4.1 2.9 3.1 0.7 11.6 3.7 11.8 0.1 11.9 3.1 3.5 50.4 50.8 17.9 20.6 5.2 93.8 1.7 6.0 17.1 11.3 8.6 14.0 86.1 5.7 95.8 15.1 4.7

Acids Propionic acid** Butyric acid* Isobutyric acid* Valeric acid** Isovaleric acid* Hexanoic acid* Octanoic acid* Decanoic acid* t-3-Hexenoic acid** t-2-Hexenoic acid** Benzoic acid** Phenylacetic acid****

618.2 1.14 1.41 8.12 0.74 2.07 1.69 0.44 7.25 24.63 99.29 2.48

853.6 1.78 2.08 11.06 0.99 4.55 3.68 0.61 9.67 39.66 157.3 5.48

439.5 0.47 0.56 3.73 0.35 2.37 1.66 0.45 4.09 32.33 82.08 3.69

1269 2.13 2.55 16.21 1.39 5.44 4.57 1.22 16.39 79.13 376.7 10.53

647.1 1.10 1.64 8.01 0.78 2.48 2.51 0.65 8.45 39.21 191.6 4.74

947.5 1.93 2.33 11.51 0.99 4.52 4.12 1.11 12.00 54.56 269.9 7.78

355.0 0.74 0.62 6.28 0.33 2.83 2.67 0.53 4.19 26.34 130.2 5.46

1360 2.64 2.96 18.50 1.47 6.34 6.33 1.78 18.39 121.0 628.7 13.03

11.0 8.7 11.8 4.1 0.3 0.8 12.1 80.7 24.1 37.6 71.6 42.0

*mg/l; **mg/l; ***area compound/area IS; ****area compound/area IS  1000.

which an insignificant decrease was observed. Maicas et al. (1999) reported the important influence of the LAB strain participating in MLF on the evolution of alcohol concentrations, and affirmed that the decreases observed when certain strains were used may have been due to the physical adsorption of the bacteria. Large increase in median concentrations before and after MLF, higher than 50%, were found for t-2-hexen-1-ol, 1,9-nonanediol, 1,10-decanediol and furfuryl alcohol. Median growths higher than 10% were also observed for 1-pentanol, 1-octanol, L-2,3-butanodiol, meso-2,3-butanodiol, c-3-hexen-1-ol, 3-ethoxy-1-propanol, 3-methyl-thio-propanol and benzyl alcohol. Ugliano and Moio (2005) reported similar results for 1-pentanol, 1-hexanol and 3-etoxy-1-propanol, but reported an increase for 3-methyl-1-pentanol, which slightly decreased in this study. Maicas et al. (1999) also obtained similar results for benzyl alcohol, but never before have increases been reported for t-2-hexen-1-ol, 1,9 nonenodiol and 1,10-decanediol during MLF.

concentrations in wines, their presence makes a significant contribution to wine aroma because of their low perception threshold and harsh aromas (Rodrı´guez et al., 1990). Changes in acids concentration during MLF depend on the strains participating in the process. Thus, while Ugliano and Moio (2005) report increases in octanoic acid, which is responsible for lactic notes in wines and decanoic acid during MLF, Pozo-Bayon et al. (2005) showed significant decreases in decanoic acid content and increases in benzoic and phenylacetic acid content. Results of this study corroborate a considerable increase in the concentration of octanoic, decanoic, benzoic and phenylacetic acids. On the other hand, acids as t-3-hexenoic and t-2-hexenoic also displayed a growth of more than 20%. The concentration of only 2 of the 12 organic acids analysed, isovaleric and hexanoic acids, remained practically unaffected by MLF. Growth seemed to be higher in those acids with more carbon atoms. 3.3. Esters

3.2. Acids Short-chain organic acids (C3–C10) are produced by yeasts during alcoholic fermentation, and despite being present in low

Ester synthesis and hydrolysis during MLF are due to the esterase activity of lactic bacteria. There is disagreement regarding the influence of MLF on the final ester content in wine.

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Table 3 Percentile 10, median, interquartile range (IQR) and percentile 90 values of the esters, aldehydes, cetones and lactones studied in wine before and after MLF Wine after AF (n ¼ 16)

Wine after MLF (n ¼ 16)

Variation

P10

Median

IQR

P90

P10

Median

IQR

P90

Median (%)

2.10 1.44 36.56 2.81 116.6 1.60 33.79 72.63 278.6 0.62 0.68 0.10 6.42 6.79 1.97 0.19 0.76 1.91 0.47 3.23 0.87 1.50 2.65 2.09 0.48 1.64 0.92 0.66 2.02

5.15 3.19 62.59 8.17 172.4 4.69 106.3 219.1 492.2 1.11 1.18 0.15 18.85 11.31 3.08 0.49 1.34 4.25 1.24 7.43 1.52 3.49 5.27 4.47 0.96 2.37 1.66 1.52 2.99

9.63 1.93 15.22 3.43 120.6 4.07 70.58 200.5 199.7 0.77 0.55 0.14 26.11 13.68 1.05 1.70 1.62 2.89 6.00 10.88 0.94 5.41 2.96 2.20 0.51 0.97 1.04 0.85 2.38

57.58 14.36 75.00 15.89 309.7 12.33 267.3 507.9 735.2 1.91 2.13 0.31 45.17 37.11 3.86 5.37 9.45 48.27 17.58 32.89 2.52 10.93 10.16 6.99 1.45 3.75 2.81 2.37 6.11

52.91 4.94 41.43 3.07 126.9 0.90 10.23 38.39 297.7 0.60 0.61 0.10 16.57 5.41 2.34 1.52 2.73 14.59 16.63 14.18 1.72 6.10 3.47 2.47 0.57 2.12 0.82 1.49 3.61

60.09 11.99 57.35 8.42 180.9 5.06 121.3 209.6 484.3 0.93 0.83 0.15 21.59 9.88 3.10 6.03 8.73 31.04 21.95 31.03 2.53 10.55 4.38 3.57 0.93 3.38 1.71 1.83 5.87

39.39 6.44 21.47 5.62 96.80 4.73 119.1 307.9 281.4 0.71 0.68 0.14 13.36 8.84 1.07 3.95 5.42 27.27 6.56 24.11 0.87 11.43 2.27 1.71 0.59 2.75 0.67 0.51 3.60

122.1 28.21 77.97 15.69 310.0 11.16 231.0 512.5 706.6 1.60 1.74 0.31 60.65 21.75 4.68 9.07 12.97 59.92 30.07 46.63 3.31 27.16 12.62 6.47 1.41 6.28 2.67 2.71 12.10

1066 275.8 8.4 3.0 4.9 7.9 14.1 4.4 1.6 16.2 29.9 3.4 14.5 12.6 0.7 1126 550.7 630.7 1670. 317.8 66.9 202.0 16.8 20.1 2.2 42.7 3.1 20.2 96.0

Aldehydes and ketones Ethanal* 2,3-Butanedione* 3-Hydroxy-2-butanone* 3-Hydroxy-2-pentanone**** 1-Hydroxy-2-propanone** 3-Acetyloxy-2-butanone**** Furfural**

7.37 1.26 1.00 23.01 27.35 1.01 10.71

9.65 2.10 1.87 43.55 57.84 1.70 13.50

3.77 0.92 2.89 21.84 32.11 0.85 9.94

14.60 2.92 5.18 87.58 98.24 2.53 30.30

1.51 2.32 0.38 4.35 27.10 1.00 12.80

2.46 3.04 3.99 18.98 42.20 1.60 21.06

1.85 0.90 8.97 46.58 24.36 0.47 12.47

4.08 4.84 21.12 84.58 78.03 2.29 52.09

74.5 44.5 113.5 56.4 27.0 5.8 56.0

Lactones g-Butirolactone* g-Valerolactone** g-Caprolactone** d-Decalactone** Pantolactone** b-Ethoxy-g-butyrolactone**** b-(1-hydroxy-ethyl)-g-butyrolactone**** d-Dodecalactone** Furaneol**

3.64 11.38 9.07 9.23 38.60 2.58 3.73 5.77 11.52

4.71 16.22 13.88 12.37 63.41 4.28 4.99 8.07 18.90

2.19 10.99 5.59 4.52 39.92 4.10 2.30 3.58 10.50

8.92 61.72 19.38 18.72 116.7 41.65 39.76 10.06 39.29

3.88 17.68 9.59 14.15 50.72 8.25 15.47 9.09 19.35

7.78 27.06 14.11 25.05 85.19 27.36 27.04 10.00 25.13

5.39 15.17 8.24 25.95 64.59 60.31 27.61 2.18 9.13

10.75 53.97 23.29 25.95 145.3 92.05 64.74 12.48 43.11

65.1 66.8 1.6 102.6 34.3 539.4 441.8 24.0 33.0

Esters Ethyl lactate* 2-Phenylethyl lactate**** Ethyl acetate* Butyl acetate** Isobutyl acetate** Isoamyl acetate* Hexyl acetate** 2-Phenylethyl acetate** Ethyl butirate** Ethyl hexanoate* Ethyl octanoate* Ethyl decanoate* Ethyl hexadecanoate** Ethyl dodecanoate** Dimethyl succinate** Diethyl succinate* Ethyl-methyl succinate**** Methyl monosuccinate**** Ethyl monosuccinate*** 2-Phenylethyl succinate**** Diethyl glutarate** Diethyl malate**** Isoamyl butanoate** Methyl octanoate** Ethyl 3-hydroxy-butirate* Ethyl 2-furoate** Ethyl 4-hydroxy-butanoate*** Ethyl 3-hydroxy-decanoate**** 2-Hydroxy-ethyl-phenylpropanoate****

*mg/l; **mg/l; ***area compound/area IS; ****area compound/area IS  1000.

Thus, whereas some authors state that the metabolism of lactic bacteria may induce significant increases in the concentrations of different esters originating from alcoholic fermentation (Delaquies et al., 2000), others affirm that their concentrations diminish significantly with the subsequent reduction in fruity attributes (Du Plessis et al., 2002). The present study revealed median concentration increases after MLF higher than 65%, and up to 1670%, for 10 of the 29 esters analysed. Those displaying the greatest increases include ethyl monosuccinate, diethyl succinate, ethyl lactate, methyl monosuccinate ethyl-methyl succinate, 2-phenylethyl succinate, 2-phenylethyl lactate and diethyl malate. The increase in concentration of ethyl lactate is linked to the production of lactic acid and depends on the metabolism of malic acid. This compound is one of the most important subproducts in the metabolism of lactic bacteria. Its concentration may increase

more than fourfold after MLF, depending on the strain of lactic bacteria involved (Izquierdo et al., 2005). Ethyl lactate provides milky notes to wines and is responsible for the sensation of the volume characteristic of wines after MLF (Davis et al., 1985). Although the change of 2-phenylethyl lactate during MLF has not been studied in as much depth, it is clear that its behaviour is due to reasons outlined above and that its formation may be responsible for the observed decrease in 2-phenylethanol. Dicarboxylic acid esters, compounds resulting from esterification of the corresponding acid, displayed great increases during MLF. These results coincide with those published by other authors (Ugliano and Moio, 2005; Pe´rez et al., 2003), although the increases obtained by the latter were not as great as those reported here. The concentration of some short-chain esters such as ethyl acetate, butyl acetate, isobutyl acetate, isoamyl acetate and

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Table 4 Percentile 10, median, interquartile range (IQR) and percentile 90 values of the phenols, terpenes and norisoprenoids studied in wine before and after MLF Wine After AF (n ¼ 16)

Phenols 4-Ethyl-phenol** 4-Vinylphenol**** guaiacol** 4-Ethyl-guaiacol** 4-Vinyl-guaiacol** Phenol** 2,4-Diterbutyl-phenol** Syringol** Eugenol** t-Isoeugenol** Methoxyeugenol** Vanillin** Methyl vanillate** Acetovanillone** Propiovanillone**** Zingerone** Acetosyringone** Tirosol*** Maltol** Tryptophol acetate**** Syringaldehyde** Terpenes a-Terpineol** Citronellol** Nerol** Geraniol** Linalol** Hydroxylinalol**** Hydroxycitronelol**** Norisoprenoids Damascenone**** 3-Hydroxy-b-damascone**** 3-Oxo-a-ionol**** 3-Oxo-7,8-dihydro-a-ionol****

Wine After MLF (n ¼ 16)

Variation

P10

Median

IQR

P90

P10

Median

IQR

P90

Median (%)

0.27 1.41 3.64 0.09 110.8 2.61 1.14 4.20 2.84 0.25 0.91 1.31 1.28 22.42 1.09 3.56 1.74 0.10 8.30 3.04 0.52

0.60 3.93 4.87 0.21 201.5 3.43 1.91 6.54 3.56 0.42 2.03 3.27 2.67 29.27 1.64 5.60 2.90 0.18 9.88 6.50 1.09

0.58 4.05 2.11 0.22 116.8 1.32 0.97 4.72 0.97 0.36 1.20 1.80 1.67 17.56 0.86 3.29 1.55 0.20 5.83 5.09 0.76

1.11 13.58 8.36 0.52 356.0 6.81 3.43 11.68 5.68 1.02 3.73 4.79 4.51 55.20 2.60 8.94 4.97 0.40 21.82 14.46 1.59

0.30 4.81 4.75 0.12 131.1 2.92 1.39 8.07 3.20 0.39 1.91 3.49 2.04 33.84 2.12 0.74 3.35 0.16 12.88 2.25 1.14

0.94 14.32 5.63 0.35 263.5 3.87 2.81 11.01 4.43 0.75 3.23 4.88 3.29 43.61 2.60 4.66 4.70 0.29 14.70 11.58 2.82

0.68 7.61 3.75 0.30 338.0 3.70 4.13 3.85 0.73 0.56 1.87 2.08 1.65 15.21 0.78 5.16 1.44 0.35 2.60 11.86 3.29

1.59 22.63 9.78 0.75 629.8 12.86 9.16 14.56 5.95 1.13 5.91 10.40 5.55 59.60 4.06 8.38 6.23 0.66 24.38 22.05 18.06

57.2 264.0 15.7 64.5 30.7 12.9 47.4 68.4 24.6 80.6 59.3 49.5 23.2 49.0 58.7 -16.8 61.7 56.7 48.7 78.0 158.2

0.82 6.70 3.39 11.00 2.23 18.33 1.17

2.18 10.20 5.05 14.40 3.35 24.73 2.20

2.48 10.60 2.28 4.50 1.76 20.50 1.57

25.24 21.67 7.39 19.06 6.31 48.23 4.52

2.30 7.68 3.43 11.40 3.06 28.10 1.88

18.45 12.95 4.94 14.37 4.62 46.47 3.36

32.63 7.29 2.51 3.68 2.15 23.36 1.41

49.19 17.74 9.24 3.68 7.32 75.62 4.11

747.2 27.0 -2.2 -0.3 37.8 87.9 52.4

0.64 0.28 2.54 0.25

0.95 0.49 6.98 0.87

0.37 0.53 5.81 0.94

1.40 1.17 15.35 2.46

0.84 0.65 5.12 0.53

1.27 1.00 11.40 1.58

0.67 0.58 7.04 0.97

2.54 1.82 22.42 2.76

33.3 106.0 63.3 81.7

*mg/l; **mg/l; ***area compound/area IS; ****area compound/area IS  1000.

hexyl acetate, some of which contribute to wine aroma providing fruity flavours, showed slight changes with a trend to increase for all of them except for ethyl acetate, which scarcely decreased. These esters are the ones that hydrolyse most quickly in acid conditions and are also strongly affected by the esterases produced by lactic bacteria. Once again, the type of strain involved in this fermentation determines the evolution of these compounds and their concentration may either increase (Laurent et al., 1994) or decrease (Gil et al., 1996). The results obtained in this study enable us to confirm that the increase in volatile acidity does not always produce an increase in acetate concentration. 2-Phenylethyl acetate, which is responsible for the floral/rose hints in wines, also underwent a slight decrease in accordance with the findings reported by Ugliano and Moio (2005). For the other esters analysed, little variation was observed in the median values of the concentrations obtained before and after MLF, although in general they tended to decrease, with the exception of ethyl dodecanoate, ethyl-2-furoate, ethyl-3-hydroxydecanoate and 2-hydroxy-ethyl-phenylpropanoate, which displayed increases between 14% and 96%. 3.4. Aldehydes and ketones The median concentration of 4 of the 7 carbonyl compounds studied decreased after MLF. These decreases were more

important for ethanal, followed by 3-hydroxy-2-pentanone, 1-hydroxy-2-propanone and finally 3-acetyloxy-2-butanone. The decrease in ethanal content, a compound related to wine herbaceous and oxidative notes (Osborne et al., 2000), has been reported before, showing significant differences according to the strain of lactic bacteria used (Izquierdo et al., 2005). As reported by many authors, 2,3-butanedione (diacetyl) and its reduced form, 3-hydroxy-2-butanone (acetoin) are also produced during MLF (Laurent et al., 1994); here, the increases in median of these compounds were 44% and 113%, respectively. These compounds give the wine aromatic complexity, with notes of butter, contributing positively to aroma and organoleptic quality. However, concentrations above 5–7 mg/l of diacetyl are undesirable for wines (Martineau and Henick-Kling, 1995). In this study, concentrations of 2,3-butanedione and 3-hydroxy-2-butanone were under 4.08 and 4.84 mg/l, respectively, in 90% of the analysed samples; hence, probably most of the wines would not be expected to display defects attributable to the presence of these compounds. Furfural median content also rose by more than 55%; this seems to contradict the increase in furfuryl alcohol mentioned previously because this would have been due to the enzymatic reduction of furfural. In general, both compounds appear due to the dehydration and cyclization of sugars and are related to aging in oak wood (Garde-Cerda´n and Ancı´n-Azpilicueta, 2005). There were no mentions of the behaviour of these compounds during MLF in the literature consulted, and hence it would be useful to

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perform additional studies to determine the causes of this increase.

attributed to the action of b-glucosidase enzymes originating from microbiota present in the medium (Ugliano et al., 2003).

3.5. Lactones

3.8. Norisoprenoids

g-Butyrolactone is a compound produced by O. oeni as a byproduct in the metabolism of a-ketoglutarate (Radler, 1986); it is normal for the concentration of this compound to increase during MLF (Maicas et al., 1999). However, the g-butyrolactone median increase is much lower than that observed for other lactones such as b-etoxy-g-butyrolactone and b-(1-hydroxy-ethyl)-g-butyrolactone, which increased by 540% and 440%, respectively. This result is very important due to the fruity and floral aromas attributed to these compounds (Garcı´a et al., 2003). The other lactones analysed also increased, albeit to a lesser extent, with increases ranging between 1% and 100%, adding fruity flavours to the wines.

Norisoprenoids are compounds that contribute fruity, floral or spicy notes; hence, their presence has very beneficial effects for the sensorial quality of wines. In our study, the 4 norisoprenoid compounds analysed displayed increases after MLF (between 33% and 106%). Pozo-Bayon et al. (2005) reported increases in b-ionone during MLF, although it has not been possible to confirm this finding because this compound could not be determined through the extraction procedure employed in this study. The increase of concentrations observed for terpenes, norisoprenoids and volatile phenols such as vanillin may be due to the hydrolysis of their non-volatile precursors. Different studies (Strauss et al., 1986; Gunata et al., 1988) have revealed the existence of the glycosylated form of terpenes, norisoprenoids and volatile phenols, and also, in recent years some researchers have reported that different strains of O. oeni may produce b-glucosidase enzymes (Boio, et al., 2002; Grimaldi et al., 2005).

3.6. Phenols In the group of volatile phenols, ethyl-phenols are particularly important because they undermine the final quality of wine (Licker et al., 1999). Different studies have been carried out to determine the capacity of certain bacteria to produce volatile phenols (Couto et al., 2006). These authors have described the production of 4-vinylphenol from phenolic acids by some lactic acid bacteria; however, only some strains of Lactobacillus are able to reduce 4-vinylphenol to 4-ethylphenol. The presence of ethylphenols, above a certain threshold level from which wine sensory quality is negatively affected, has been attributed to the growth of Brettanomyces (Cavin et al., 1993; Chatonnet et al., 1995). This research has found a median perceptual increase for 4-vinylphenol above 260% after MLF, with that for 4-ethylphenol being much lower, around 50%. After MLF 4-ethylphenol content remained below 1.59 mg/l in 90% of the samples. These concentrations were lower than 0.68 mg/l, the level from which the 4-ethylphenol content of wine could imply Brett character (Licker et al., 1999). Of the other phenols analysed, very few references were found in the bibliography that relate them to MLF. However, the important increases observed for all of these phenols, except zingerone (Table 4), suggest that the microbiota present in MLF participate actively in their formation, and this aspect should be studied in greater depth. The phenols displaying the greatest increases included, most notably, syringaldehyde, which increased by more than 150%, and syringol, t-isoeugenol, metoxyeugenol, propiovainillone, acetovanillone, acetosyringone and vanillin, which increased between 50% and 100%, respectively. These compounds give wines their spicy and smoked characteristics (Ferreira et al., 1995). 3.7. Terpenes Terpenes are present in free and glycosylated form in grapes; the content of free terpenes often increases during alcoholic fermentation due to the b-glucosidase activity of yeasts (Gil et al., 1996). Coinciding with these findings, we observed a generalized increase in all the terpenes studied, with the exception of nerol and geraniol whose concentration remained constant, and particularly a-terpineol whose median displayed an increase of 740%. This increase may be due to the acid hydrolysis of compounds such as nerol and citronerol over time. However, the decrease in nerol was not correlated with the important increase in a-terpineol. For this reason, the increase observed must be

4. Conclusions The results obtained in this study revealed that in industrial winery conditions, spontaneous MLF produced changes in the concentrations of most of the 114 volatile compounds analysed, mostly alcohols, acids, esters, aldehydes and ketones, phenols, terpenes and norisoprenoids. Some of the results obtained, such as those for terpenes and norisoprenoids, confirmed findings reported elsewhere by other authors. For other compounds such as volatile phenols or furfural, this is the first time that their evolution has been described during MLF. The increase in the concentration of some volatile compounds seems to show the determining contribution of the b-glucosidasic activity of lactic acid bacteria to wine flavour. Since this study was performed with samples taken from different wineries, covering a large geographical area, the results obtained allowed us to identify changes that could be considered as general or characteristic of industrial spontaneous MLF.

Acknowledgements The authors thank the Consejerı´a de Educacio´n y Ciencia de la JCCM for the Project PCC-05-003-2 and the wineries that have taken part in this study: Bodegas Illana de Pozoamargo (Cuenca), Bodegas La Cerca de Casarrubios del Monte (Toledo), Bodegas ˜ edos Mun ˜ oz de Lozano de Villarrobledo (Albacete), Bodegas y Vin ˜ a (Toledo), Cooperativa Provedo de Villarrubia de los Ojos Ocan ˜ as de Tomelloso (Ciudad Real), Cooperativa Virgen de las Vin (Ciudad Real), Dehesa del Carrizal de Retuerta del Bullaque ˜ as (Ciudad Real) (Ciudad Real), Finca Marisa´nchez de Valdepen and Videma S.A.T. de Madrigueras (Albacete). References Bartowsky, E., Henschke, P.A., 2004. The ‘‘buttery’’ attribute of wine-diacetyl—desirability, spoilage and beyond. International Journal of Food and Microbiology 96, 325–352. Boio, E., Lloret, A., Medina, K., Carrau, F., Dellacassa, E., 2002. Effect of b-glycosidase activity of Oenococcus oeni on the glycosylated flavor precursors of tannat wine during malolactic fermentation. Journal of Agricultural and Food Chemistry 50, 2344–2349. Cavin, J.F., Audioc, V., Etie´vant, P.X., Divies, C., 1993. Ability of wine lactic acid bacteria to metabolize phenol carboxylic acids. American Journal of Enology and Viticulture 44, 76–80.

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