Factors Affecting the Hydroxycinnamate ... - Wiley Online Library

JFS M: Food Microbiology and Safety

Factors Affecting the Hydroxycinnamate Decarboxylase/Vinylphenol Reductase Activity of Dekkera/Brettanomyces: Application for Dekkera/Brettanomyces Control in Red Wine Making ABSTRACT: The growth of Dekkera/Brettanomyces yeasts during the ageing of red wines—which can seriously reduce the quality of the final product—is difficult to control. The present study examines the hydroxycinnamate decarboxylase/vinylphenol reductase activity of different strains of Dekkera bruxellensis and Dekkera anomala under a range of growth-limiting conditions with the aim of finding solutions to this problem. The yeasts were cultured in in-house growth media containing different quantities of growth inhibitors such as ethanol, SO 2 , ascorbic acid, benzoic acid and nicostatin, different sugar contents, and at different pHs and temperatures. The reduction of p-coumaric acid and the formation of 4-ethylphenol were periodically monitored by HPLC-PDA. The results of this study allow the optimization of differential media for detecting/culturing these yeasts, and suggest possible ways of controlling these organisms in wineries. Keywords: Dekkera/Brettanomyces, growth inhibitors, hydroxycinnamate decarboxylase/vinylphenol reductase activity, red wines

T

Introduction

he growth of yeasts belonging to the genera Dekkera/ Brettanomyces in wine, especially during ageing, reduces the organoleptic quality of the final product. Unfortunately, this problem is difficult to control. Although slow-growing under oenological conditions, these yeasts sometimes find conditions favorable to them, such as during ageing in barrels when nutrient contents are limited, when SO 2 concentrations are low, when the pH is high, and ˜ when temperatures are above 15 ◦ C (Suárez-Lepe and Inigo 2004; Suárez and others 2007). Under such conditions they grow slowly but surely, using ethanol and residual traces of sugar as their carbon sources. These yeasts also possess hydroxycinnamate decarboxylase (HcDc) (Edlin and others 1998) and vinylphenol reductase (VpR) activity (Dias and others 2003a), allowing them to transform hydroxycinnamic acids into ethylphenols (Figure 1). The sensorial threshold for ethyphenols is low (Suárez and others 2007) and even small amounts can seriously reduce the olfactory quality of wines; descriptors such as “phenolic,” “animal,” “horse sweat,” and “stable” are often used to describe their presence (Chatonnet and others 1992; Chatonnet and others 1993; Rodrigues and others 2001). The formation of volatile phenols is proportional to the size of the Dekkera/Brettanomyces population (Gerbaux and others 2002). The capacity of these yeasts to produce ethylphenols is greater when the ethanol concentration is low (Suárez and others 2007); MS 20080360 Submitted 5/14/2008, Accepted 9/15/2008. Authors Benito, Palomero, Morata, Calderón, and Suárez-Lepe are with Dpto. Tecnolog´ıa de Alimentos, E. T. S. Ingenieros Agrónomos, Univ. Politécnica de Madrid, Ciudad Univ. S/N, Madrid 28040, Spain. Direct inquiries to author Morata (E-mail: [email protected]). R Institute of Food Technologists doi: 10.1111/j.1750-3841.2008.00977.x

C 2008

Further reproduction without permission is prohibited

above 15.5% (v/v) they are unable to produce these compounds (Ibeas and others 1996, 1997). The temperature at which ageing takes place in barrels also affects the production of these compounds; a temperature of 18 ◦ C is more favorable than one of 13 ◦ C (Couto and others 2005). The additive most commonly used to control the growth of microorganisms in wine is SO 2 . The addition of this compound is legal and effective, and it can inhibit the growth of Dekkera/Brettanomyces when the concentration of free SO 2 is just under 20 mg/L at pH 3.6 to 3.7 (Chatonnet and others 1993). Sorbic acid is unable to stop their growth at the doses legally permitted (Ribéreau-Gayon and others 1975; Chatonnet 2004); in fact, these yeasts can stand up to 950 mg/L of sorbic acid at pH 3.5 (Loureiro and Malfeito-Ferreira 2006). Benzoic acid has been reported to inhibit the growth of these yeasts in soft drinks at concentrations between 100 and 200 mg/L depending on the species (Van Esch 1992), but the use of this agent in wines is not allowed. Dimethyl dicarbonate (DMDC) can also inhibit the growth of Dekkera/Brettanomyces, although even at concentrations close to the legal limit of 400 mg/L it cannot completely prevent the growth of B. anomalus (Suárez and others 2007). The ability of yeasts in the microbiota of wine to generate 4-ethylphenol has been analyzed in different model media containing p-coumaric acid at concentrations higher than those present in wine (Rodrigues and others 2001; Couto and others 2005; Benito and others 2006). Conversion ratios of 90% have been recorded for Dekkera bruxellensis and Dekkera anomala (Dias and others 2003a). Ethyphenols can be detected by subjecting samples prepared by liquid–liquid extraction with organic solvents (Chatonnet and Boidron 1988), headspace solid phase microextraction Vol. 74, Nr. 1, 2009—JOURNAL OF FOOD SCIENCE

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M: Food Microbiology & Safety

´ N, AND J.A. SUA´REZ-LEPE S. BENITO, F. PALOMERO, A. MORATA, F. CALDERO

Factors affecting Dekkera/Brettanomyces . . . (Martorell and others 2002; Monje and others 2002), or stir bar sorptive extraction (D´ıez and others 2004; Mar´ın and others 2005) to gas chromatography. High-performance liquid chromatography (HPLC) is not usually used since this method is insufficiently sensitive for concentrations in the 10 to 100 ppb range. The aim of the present study was to identify molecules and other factors that affect the capacity of different strains of D. bruxellensis and D. anomala to produce 4-ethylphenol, and thus optimize selective/differential media that allow the rapid and reliable identification of these microorganisms in wine, facilitating the control of their growth.

Materials and Methods

Measurement of pH and sterilization conditions The pH of the experimental media was monitored using a Basic 20 pH meter (Crison, Barcelona, Spain). All media were sterilized in an autoclave at 121 ◦ C for 15 min. Thermosensitive growth inhibitors were added after sterilization in a laminar flow cabinet.

Yeast strains examined The yeast strains examined included the model strain Dekkera bruxellensis D37 (from the IFI type collection, CSIC, Madrid, Spain)—a type organism for this species—and the other strains shown in Table 1. Dekkera bruxellensis is the only species to have been isolated from wines to date (Phister and Mills 2003; Dias and others 2003b; Bellon and others 2003; Cocolin and others 2004; Martorell and others 2006).

Reagents Glucose was supplied by J. T. Baker Chemicals B.V. (Denventer, Holland), yeast nitrogen base, bacteriological grade peptone, and yeast extract by Pronadisa (Madrid, Spain), p-coumaric acid by Fluka (Buchs, Switzerland), 4-ethylphenol and 4-vinylphenol by Extrasynthese (Genay, France), nicostatin by Acofarma (Terrasa, Spain), and ethanol, sodium benzoate, potassium sorbate, potassium bisulphite, and orthophosphoric acid by Panreac (Barcelona, Spain).


O

OH

Vinylphenol reductase activity

Hydroxycinnamate decarboxylase activity

OH p-coumaric acid

OH 4-vinylphenol

OH 4-ethylphenol

Figure 1 --- Conversion of p-coumaric acid into 4ethylphenol in wine by Dekkera/Brettanomyces spp. via successive, enzyme-driven decarboxylation and reduction reactions. Table 1 --- Yeast strains used. Species

Strain

Origin

Dekkera bruxellensis

D37 D36 D35 D34 6802 R3 7801 JR3 HA11 DA1 DA2 DA3

IFI-CSIC

Dekkera anomala

ETSIA-UPM

IFI-CSIC

Growth media For the study of D. bruxellensis D37, 50 mL aliquots of media were placed in 100 mL Erlenmeyer flasks, except in assays involving free SO 2 , in which 60 mL were used. Each flask was inoculated with 100 CFU/mL using 72 h-old synchronized cultures (grown in YEPD medium) of D. bruxellensis D37. All assays were performed in triplicate. All cultures were incubated isothermically at 25 ◦ C, except those used in the SO 2 assay (18 ◦ C) and the variable temperature assays. For all other strains, 10 mL of each medium were placed in 20 mL screw cap test tubes. Each tube was inoculated with 100 CFU/mL using 72 h-old synchronized cultures (YEPD medium). All assays were performed in triplicate. All cultures were incubated isothermically at 25 ◦ C, except those used in the SO 2 assay (18 ◦ C) and the variable temperature assays. Table 2 and 3 show the compositions of the media used and the concentrations/values of inhibitory factors employed in the different fermentations.

HPLC-PDA analysis of p-coumaric acid and 4-ethylphenol The transformation of p-coumaric acid into 4-ethylphenol was monitored using HPLC/photodiode-array (PDA) detection. The phenols in the wines were determined using an Agilent Technologies 1100 (Palo Alto, Calif., U.S.A.) HPLC chromatograph equipped with a quaternary pump, an autosampler, and a PDA detector. Gradients of water/formic acid (90:10, v/v; solvent A) and methanol/formic acid (90:10, v/v; solvent B) were used in a reversephase Nova-pack C18 column (300 × 3.9 mm) as follows: 10% to 50% B, linear (0.8 mL/min) from 0 to 25 min, and 50% to 10% B, linear (0.8 mL/min) from 25 to 30 min. The column was re-equilibrated between 30 and 33 min. Detection was performed by scanning in the 200 to 400 nm range. Quantification was performed by comparison against an external standard at 320, 280, and 260 nm and expressed as a function of the concentration of p-coumaric acid, and 4-ethylphenol. Ten-microliter samples of

Table 2 --- Media used with Dekkera bruxellensis D37 and concentrations/values for inhibitory factors. Inhibitory factor Ethanol (0%, 5%, 10%, 15%, 20%, and 25% v/v) pH (1.74, 2.17, 2.62, 3.21, 3.61, and 4.18) Glucose (0, 150, 600, 2000, and 5000 mg/L) Temperature (0, 10, 20, 30, 40, and 50 ◦ C) Sorbic acid (0, 250, 500, 900, and 1100 mg/L) Benzoic acid (0, 50, 100, 150, 200, and 250 mg/L) Nicostatin (0, 25, 50, and 75 mg/L) Free SO 2 (0, 9.6, 20.16, 29.66, and 38.4 mg/L)

Glucose (g/L)

Yeast extract (g/L)

Peptone (g/L)

p -coumaric acid (mg/L)

Ethanol (% v/v)

Nitrogen base (g/L)

pH

10 10 V 10 10 10 10 10

1 1 --1 1 1 1 1

1 1 --1 1 1 1 1

120 120 120 120 120 120 120 120

V 1 6 1 5 5 5 6

----6.7 -----------

3.6 V 3.6 3.6 3.6 3.6 3.6 3.5

V = variable.

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Factors affecting Dekkera/Brettanomyces . . . previously filtered (0.45 μm) fermentations were injected into the low—concentrations favor the necessary enzymatic processes HPLC. Analyses were performed every 48 h for strain D37 and at of decarboxylation and/or reduction. This might be related to 21 d for the remaining strains. the capacity of Dekkera/Brettanomyces to use ethanol as a car˜ bon source (Kurtzman and Fell 1998; Suárez-Lepe and Inigo 2004). Results and Discussion The inhibitory action of ethanol might potentiate that of other Effect of ethanol concentration on the conversion factors such as SO 2 , sorbic acid, benzoic acid, DMDC, or antibiof p-coumaric acid into 4-ethylphenol otics. Some researchers have described ethanol to be inhibitory In the absence of all other limiting factors, D. bruxellensis D37 in synthetic media at 13% (v/v) (Dias and others 2003a) and appeared unable to convert p-coumaric acid into 4-ethylphenol 11.4% (v/v) (Medawar and others 2003). However, members of (that is, there was no HcDc and/or VpR activity) when the ethanol Dekkera/Brettanomyces have been isolated from films in aged concentration was >15% (v/v) (Figure 2). A concentration of sherry wines with an ethanol content of 15% (v/v) (Ibeas and oth10% (v/v) delayed the formation of ethylphenol by more than ers 1996, 1997). These films may have grown due to the existence 3 d compared to results for the 5% (v/v) concentration, while a of other favorable conditions (such as aerobiosis) or the presence concentration of 15% delayed it by 7 d. Interestingly, in the ab- of nutrients or other factors. Nonetheless, an ethanol content of sence of ethanol (0%, v/v), 4-ethylphenol production was also 14% to 14.5% (v/v) might be a factor limiting growth under the delayed (Figure 2) with respect to the 5% (v/v) ethanol fer- conditions used in the production of red table wines (Loureiro and mentation. This may indicate that low ethanol—but not very Malfeito-Ferreira 2006).

Inhibitory factor Ethanol pH Glucose Temperature (0 and 40 ◦ C) Sorbic acid (750 mg/L) Benzoic acid (200 mg/L) Nicostatin (25 mg/L) Free SO 2 (20 mg/L)

Glucose (g/L)

Yeast extract (g/L)

Peptone (g/L)

p -coumaric acid (mg/L)

Ethanol (% v/v)

Nitrogen base (g/L)

pH

10 10 0 10 10 10 10 10

1 1 --1 1 1 1 1

1 1 --1 1 1 1 1

120 120 120 120 120 120 120 120

15 1 6 1 5 5 5 6

----6.7 -----------

3.6 2.5 3.6 3.6 3.6 3.6 3.6 3.5

V = variable.

p-coumaric acid (mg/l)

140

Figure 2 --- Change in concentrations of p-coumaric acid and 4-ethylphenol during fermentation with Dekkera bruxellensis D37 in the presence of different concentrations of ethanol (% v/v) and in the absence of any other limiting factor (fermentations performed in triplicate).

120 100 80 60 40 20 0 -20

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

8

9

10 11 12

13 14 15 16 17

8

9

10

13

140

4-ethylphenol (mg/l)

120 100 80 60 40 20 0 -20 11

12

14

15

16

17

Time (days)

0

5

10

15

20

25

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Table 3 --- Media used with all other Dekkera bruxellensis strains and those of Dekkera anomala and concentrations/ values for inhibitory factors.

Factors affecting Dekkera/Brettanomyces . . . The remainder of the D. bruxellensis strains studied turned pcoumaric acid into 4-ethylphenol with an efficiency of about 90% in the presence of 14.5% (v/v) ethanol. However, the 3 strains of D. anomala analyzed showed no HcDc/VpR activity. This may explain why D. bruxellensis is the only species to have been isolated from wines (Bellon and others 2003; Dias and others 2003b; Phister and Mills 2003; Cocolin and others 2004; Martorell and others 2006). The D. anomala strains used all originated in the beer industry. Dekkera bruxellensis may therefore be more resistant to alcohol.

by their own metabolism (Kurtzman and Fell 1998). However, the present study shows that Dekkera bruxellensis is an acid tolerant species; therefore, the inhibition it experiences in culture media may be due to the toxic action of acetate ions produced during growth and not by any variation in pH. This would also explain the presence of B. naardensis in low pH soft drinks, where it can be the main contaminating agent (Van Esch 1992). The remaining strains all showed HcDc/VpR activity and completely transformed p-coumaric acid into 4-ethylphenol at pH 2.5. pH does not, therefore, appear to be a direct growth limiting factor for D. bruxellensis over the normal range seen in wines. HowEffect of pH on the conversion of p-coumaric ever, low pH has an important effect on the action of SO 2 , favoring acid into 4-ethylphenol a greater presence of its free molecular form and potentiating the In the absence of other limiting factors, D. bruxellensis D37 showed no HcDc/VpR activity when the pH was between 1.75 and inhibitory effects of sorbic and benzoic acid. 2.17 (Figure 3). At pHs equal to or greater than 2.17, no great differences were seen between activities, and all p-coumaric acid was Effect of glucose on the conversion of converted to 4-ethylphenol in approximately 6 d. At pH 2.17, how- p-coumaric acid into 4-ethylphenol ever, there was a 2-d delay in the initiation of p-coumaric acid For D. bruxellensis D37 the p-coumaric acid content was seen metabolism. Some researchers recommend the use of pH regula- to decrease from the 1st analysis at 48 h, in all repetitions, and at tors such as calcium carbonate to prolong the survival of isolated all glucose concentrations (Figure 4). When ethanol was the only colonies that might suffer from brusque variations in pH caused carbon source the reduction of p-coumaric acid was detected after


140

Figure 3 --- Change in concentrations of p-coumaric acid and 4-ethylphenol during fermentation with Dekkera bruxellensis D37 with respect to initial pH and in the absence of any other limiting factor (fermentations performed in triplicate).

120


100

80

60

40

20

0 0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

140 120


100 80 60 40 20 0 -20 Time (days)

1.75 M18

2.17

2.62

3.21


3.61

4.18

Factors affecting Dekkera/Brettanomyces . . . 192 h (Figure 2). The formation of 4-ethylphenol started after 96 h in all repetitions containing glucose; when no glucose was present, its formation was not noticed until 240 h (Figure 4). Greater HcDc/VpR activity was seen in the assays involving concentrations up to 150 mg/L glucose compared to 0 mg/L. The maximum concentration of 4-ethylphenol for the 0 mg/L glucose concentration was reached between 14 and 17 d, explaining the slowness of selective media based on the use of ethanol as the only carbon source (Rodrigues and others 2001; Benito and others 2006). According to some researchers, the fermentation of concentrations of residual sugar close to 300 mg/L is sufficient to form

a quantity of ethylphenols that surpass the sensorial perception threshold (425 μg/L) (Chatonnet and others 1995). The remaining strains were able to use ethanol as the only carbon source and to develop high HcDc/VpR activities; all completely converted the p-coumaric acid present into 4-ethylphenol.

Effect of free SO 2 at pH 3.5 on the conversion of p-coumaric acid into 4-ethylphenol In the absence of other limiting factors, and at a pH of 3.5, the HcDc/VpR activity of D. bruxellensis D37 stopped within the free SO 2 concentration range of 9.6 and 20.16 mg/L. Some researchers

140

Figure 4 --- Change in concentrations of p-coumaric acid and 4-ethylphenol during fermentation with Dekkera bruxellensis D37 in the presence of different concentrations of glucose (mg/L) and in the absence of any other limiting factor (fermentations performed in triplicate).


120 100 80 60 40 20 0 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23

0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16


0 140


120 100 80 60 40 20 0 -20 17 18 19 20 21 22 23

Time (days) 0

0

150 150

600 600

2000 2000

50005000

Table 4 --- Influence of the different inhibitory factor concentrations/values on the HcDc/VpR activity of the Dekkera bruxellensis strains (ranges for all strains taken together). Factor

Disadvantages of preventive concentrations

Inhibitory effect

Preventive effect

Ethanol

0 to 15% (v/v)

> = 15 to 20% (v/v)

Too high for table wines

pH Free SO 2 (pH = 3.5)

2.17 to 2.62 0 to 20 mg/L

1.75 to 2.17 >20 mg/L

Not oenologically viable Concentration gradually falls

Sorbic acid (pH = 3.6)

0 to 900 mg/L

900 to 1100 mg/L

Benzoic acid (pH = 3.6)

0 to 150 mg/L

150 to 200 mg/L

Nicostatin

Temperature

0 to 25 mg/L

15 to 30 ◦ C

pH-dependent Legal limit just 250 mg/L Unstable in the presence of lactic acid bacteria Affects flavor Not authorized for use in wine

30 to 40 ◦ C

Action lasts 48 h only Soluble Causes turbidity (especially in white wines) Reduces alcohol concentration

0 to 15 ◦ C

Cost

Advantages Wine more stable at higher %v/v ethanol Greater stability at low pH Effective against Dekkera/Brettanomyces Widely used Antifungal agent Preservative Effective legal dose for use with musts Disinfectant Possible use in barrel hygiene Disappears after 15 d Easily controlled if apparatus used is adequate


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Factors affecting Dekkera/Brettanomyces . . . report D. bruxellensis to grow at free SO 2 values of >30 mg/L (Froudiére and Laure 1989; Chatonnet and others 1993), and fix its inhibitory threshold at just under 20 mg/L for pHs between 3.6 and 3.7. None of the other Dekkera/Brettanomyces strains showed

any HcDc/VpR activity at a free SO 2 concentration of 20 mg/L at pH 3.5. SO 2 , which is a permitted wine additive, is therefore one of the most effective inhibitors of Dekkera/Brettanomyces; the problem is that over time its concentration falls at a rate dependent on

140

Figure 5 --- Change in concentrations of p-coumaric acid and 4-ethylphenol during fermentation with Dekkera bruxellensis D37 in the presence of different concentrations of sorbic acid (mg/L) and in the absence of any other limiting factor (fermentations performed in triplicate).


120 100 80 60 40 20 0 0 1

2

3 4 5

6 7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

140 1100

900

500

250

0

100 80 60 40 20 0 -20 0

1 2

3 4

5 6

7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Time (days)

1100

900

500

250

0

140

Figure 6 --- Change in concentrations of p-coumaric acid and 4-ethylphenol during fermentation with Dekkera bruxellensis D37 in the presence of different concentrations of benzoic acid (mg/L) and in the absence of any other limiting factor (fermentations performed in triplicate).

p -coumaric acid (mg/l)

120 100 80 60 40 20 0 -20 0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

8

9

10

11 12

13 14

15 16

17

8

9

10

11 12

13 14

15 16

17

140 120 4-ethylphenol (mg/l)



120

100 80 60 40 20 0 -20 Time (days)

0

M20

50

100

150


200

250

Factors affecting Dekkera/Brettanomyces . . . the pH. It should therefore be checked periodically, especially in 200 mg/L at a pH of 3.6 (Figure 6). Similar results were reported by wine ageing in wooden barrels, to ensure that protection against Van Esch in 1992 for the concentration range 100 to 200 mg/L deDekkera/Brettanomyces is maintained. pending on the species examined. Although this molecule appears to be an excellent inhibitor, its use is only permitted in musts. None of the remaining strains could convert p-coumaric acid Effect of sorbic acid on the conversion of into 4-ethylphenol at a benzoic acid concentration of 200 mg/L and p-coumaric acid into 4-ethylphenol In the absence of all other limiting factors, D. bruxellensis D37 pH of 3.6. This inhibitor would be ideal were it not for the several showed no HcDc/VpR activity when the sorbic acid concentra- disadvantages associated with it (Table 4).

Effect of benzoic acid on the conversion of p-coumaric acid into 4-ethylphenol

Effect of nicostatin on the conversion of p-coumaric acid into 4-ethylphenol In the absence of other limiting factors, D. bruxellensis D37 was unable to perform this conversion at nicostatin concentrations of >25 mg/L at a pH of 3.6. It has been reported that concentrations of 10 to 15 mg/L can practically sterilize fungi and yeast in media containing 10% (v/v) ethanol (Ribéreau-Gayon and others 1975). Further, in wine nicostatin is reported to be converted into innocuous molecules (Ribéreau-Gayon and others 1975), facilitating its oenological use. An important disadvantage associated with the use of this inhibitor is the turbidity it causes in the fermentation medium. None of the remaining strains showed any HcDc/VpR activity at 25 mg/L nicostatin either.

Effect of temperature on the conversion of

Although the addition of benzoic acid to wine is not permit- p-coumaric acid into 4-ethylphenol The optimum temperature for HcDc/VpR activity in D. bruxelted it is a potent inhibitor of yeast growth. In the absence of other growth limiting factors, D. bruxellensis D37 showed no HcDc/VpR lensis D37 was 20 to 30 ◦ C; at 15 to 20 ◦ C less activity was seen, and activity when the benzoic acid concentration was between 150 and at 30 to 40 and 0 to 15 ◦ C no such activity was seen at all (Figure 7).

140

Figure 7 --- Change in concentrations of p-coumaric acid and 4-ethylphenol during fermentation with Dekkera bruxellensis D37 at different incubation temperatures expressed in ◦ C (fermentations performed in triplicate).

120

p -coumaric acid (mg/l)

100 80 60 40 20 0 -20 0

1

2

3

4

5

6

7

8

9

10

11

12

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

14

15

16

140


120 100 80 60 40 20 0 -20 13

16

Time (days) T0

T15

T20

T30

T40

T50


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tion was >1100 mg/L and the pH was 3.6; inhibitory action began at a concentration of 900 mg/L. HcDc/VpR activity is therefore very likely at the maximum dose authorized for use in wines: 200 mg/L (IOC 2006). This result agrees with those reported by other researchers who indicate D. bruxellensis to be among the species most resistant to this inhibitor, with activity occurring up to concentrations of 1000 mg/L (Ribéreau-Gayon and others 1975; Loureiro and Malfeito-Ferreira 2006). This inhibitor has been used in culture media as a selection factor (Chatonnet and others 1992). Figure 5 shows that it clearly delays the conversion of p-coumaric acid into 4-ethylphenol in a dose-dependent fashion. None of the D. anomala strains was able to complete the transformation at a concentration of 750 mg/L sorbic acid at pH 3.6.

Factors affecting Dekkera/Brettanomyces . . . These results show temperature to directly influence the activity of one/both of these enzymes, as reported by other researchers (Couto and others 2005). The other strains studied showed a lack of HcDc/VpR activity at 40 and 0 ◦ C. These results suggest that flashpasteurization at a temperature of 35 to 40 ◦ C might protect wines from Dekkera/Brettanomyces when ageing in wooden barrels.

T

Conclusions


he HcDc/VpR activity of Dekkera bruxellensis D37 was inhibited under the following conditions: ethanol 15% to 20% (v/v), pH 1.75 to 2, free SO 2 9.6 to 20.2 mg/L at pH 3.5, sorbic acid 900 to 1100 mg/L, benzoic acid 150 to 200 mg/L, nicostatin 25 mg/L, and temperatures of 30 to 40 and 0 to 15 ◦ C. Other strains of D. bruxellensis and D. anomala showed similar results or were more sensitive. Although the concentrations of some of these inhibitors surpass the legal limit, synergic effects may be sought between them at lower, legal concentrations. Although HPLC is not normally used for the analysis of small quantities (10 to 100 ppb) of volatile phenolic compounds in wines, it can be successfully used to monitor HcDc/VpR activity in synthetic media with high concentrations of hydroxycinnamic acids. Under such conditions the technique is rapid, shows adequate sensitivity, and no complex preparation of samples is required. The results of the present study may help in the understanding of Dekkera/Brettanomyces HcDc/VpR activity in wine. They may also be of use in the development of microbiological techniques for investigating the presence of these yeasts in wine and the development of corrective/palliative measures.

Acknowledgments This study was funded by the Ministerio de Educación y Ciencia (MEyC) (project AGL2005-06640-C02-01). We thank Director ˜ Montserrat Íniguez and the rest of the team at the Estación Enológica de Haro for their excellent collaboration, Dr. José Barcenilla (IFI, CSIC), and Susana Somolinos and Juan Antonio Sánchez (ETSIA, UPM) for excellent technical assistance.

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