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Keywords:

  • Dekkera/Brettanomyces;
  • growth inhibitors;
  • hydroxycinnamate decarboxylase/vinylphenol reductase activity;
  • red wines

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

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, SO2, 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

The 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 SO2 concentrations are low, when the pH is high, and when temperatures are above 15 °C (Suárez-Lepe and Iñigo 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).

image

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

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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); 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 SO2. The addition of this compound is legal and effective, and it can inhibit the growth of Dekkera/Brettanomyces when the concentration of free SO2 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 (Martorell and others 2002; Monje and others 2002), or stir bar sorptive extraction (Díez and others 2004; Marín 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

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).

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).

Table 1—.  Yeast strains used.
SpeciesStrainOrigin
Dekkera bruxellensisD37IFI-CSIC
D36 
D35 
D34 
6802ETSIA-UPM
R3 
7801 
JR3 
HA11 
Dekkera anomalaDA1IFI-CSIC
DA2 
DA3 

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 SO2, 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 SO2 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 SO2 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.

Table 2—.  Media used with Dekkera bruxellensis D37 and concentrations/values for inhibitory factors.
Inhibitory factorGlucose (g/L)Yeast extract (g/L)Peptone (g/L)p-coumaric acid (mg/L)Ethanol (% v/v)Nitrogen base (g/L)pH
  1. V = variable.

Ethanol (0%, 5%, 10%, 15%, 20%, and 25% v/v)1011120V3.6
pH (1.74, 2.17, 2.62, 3.21, 3.61, and 4.18)10111201V
Glucose (0, 150, 600, 2000, and 5000 mg/L)V12066.73.6
Temperature (0, 10, 20, 30, 40, and 50 °C)101112013.6
Sorbic acid (0, 250, 500, 900, and 1100 mg/L)101112053.6
Benzoic acid (0, 50, 100, 150, 200, and 250 mg/L)101112053.6
Nicostatin (0, 25, 50, and 75 mg/L)101112053.6
Free SO2 (0, 9.6, 20.16, 29.66, and 38.4 mg/L)101112063.5
Table 3—.  Media used with all other Dekkera bruxellensis strains and those of Dekkera anomala and concentrations/values for inhibitory factors.
Inhibitory factorGlucose (g/L)Yeast extract (g/L)Peptone (g/L)p-coumaric acid (mg/L)Ethanol (% v/v)Nitrogen base (g/L)pH
  1. V = variable.

Ethanol1011120153.6
pH101112012.5
Glucose012066.73.6
Temperature (0 and 40 °C)101112013.6
Sorbic acid (750 mg/L)101112053.6
Benzoic acid (200 mg/L)101112053.6
Nicostatin (25 mg/L)101112053.6
Free SO2 (20 mg/L)101112063.5

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 reverse-phase 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 previously filtered (0.45 μm) fermentations were injected into the HPLC. Analyses were performed every 48 h for strain D37 and at 21 d for the remaining strains.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

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

In the absence of all other limiting factors, D. bruxellensis D37 appeared unable to convert p-coumaric acid into 4-ethylphenol (that is, there was no HcDc and/or VpR activity) when the ethanol concentration was >15% (v/v) (Figure 2). A concentration of 10% (v/v) delayed the formation of ethylphenol by more than 3 d compared to results for the 5% (v/v) concentration, while a concentration of 15% delayed it by 7 d. Interestingly, in the absence of ethanol (0%, v/v), 4-ethylphenol production was also delayed (Figure 2) with respect to the 5% (v/v) ethanol fermentation. This may indicate that low ethanol—but not very low—concentrations favor the necessary enzymatic processes of decarboxylation and/or reduction. This might be related to the capacity of Dekkera/Brettanomyces to use ethanol as a carbon source (Kurtzman and Fell 1998; Suárez-Lepe and Iñigo 2004).

image

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).

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The inhibitory action of ethanol might potentiate that of other factors such as SO2, sorbic acid, benzoic acid, DMDC, or antibiotics. Some researchers have described ethanol to be inhibitory in synthetic media at 13% (v/v) (Dias and others 2003a) and 11.4% (v/v) (Medawar and others 2003). However, members of Dekkera/Brettanomyces have been isolated from films in aged sherry wines with an ethanol content of 15% (v/v) (Ibeas and others 1996, 1997). These films may have grown due to the existence of other favorable conditions (such as aerobiosis) or the presence of nutrients or other factors. Nonetheless, an ethanol content of 14% to 14.5% (v/v) might be a factor limiting growth under the conditions used in the production of red table wines (Loureiro and Malfeito-Ferreira 2006).

The remainder of the D. bruxellensis strains studied turned p-coumaric 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.

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

In the absence of other limiting factors, D. bruxellensis D37 showed no HcDc/VpR activity when the pH was between 1.75 and 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 converted to 4-ethylphenol in approximately 6 d. At pH 2.17, however, there was a 2-d delay in the initiation of p-coumaric acid metabolism. Some researchers recommend the use of pH regulators such as calcium carbonate to prolong the survival of isolated colonies that might suffer from brusque variations in pH caused 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.

image

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).

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pH does not, therefore, appear to be a direct growth limiting factor for D. bruxellensis over the normal range seen in wines. However, low pH has an important effect on the action of SO2, favoring a greater presence of its free molecular form and potentiating the inhibitory effects of sorbic and benzoic acid.

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

For D. bruxellensis D37 the p-coumaric acid content was seen to decrease from the 1st analysis at 48 h, in all repetitions, and at all glucose concentrations (Figure 4). When ethanol was the only carbon source the reduction of p-coumaric acid was detected after 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).

image

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).

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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 SO2 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 SO2 concentration range of 9.6 and 20.16 mg/L. Some researchers report D. bruxellensis to grow at free SO2 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 SO2 concentration of 20 mg/L at pH 3.5. SO2, 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 the pH. It should therefore be checked periodically, especially in wine ageing in wooden barrels, to ensure that protection against Dekkera/Brettanomyces is maintained.

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

In the absence of all other limiting factors, D. bruxellensis D37 showed no HcDc/VpR activity when the sorbic acid concentration 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.

image

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).

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

Although the addition of benzoic acid to wine is not permitted it is a potent inhibitor of yeast growth. In the absence of other growth limiting factors, D. bruxellensis D37 showed no HcDc/VpR activity when the benzoic acid concentration was between 150 and 200 mg/L at a pH of 3.6 (Figure 6). Similar results were reported by Van Esch in 1992 for the concentration range 100 to 200 mg/L depending on the species examined. Although this molecule appears to be an excellent inhibitor, its use is only permitted in musts.

image

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).

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None of the remaining strains could convert p-coumaric acid into 4-ethylphenol at a benzoic acid concentration of 200 mg/L and pH of 3.6. This inhibitor would be ideal were it not for the several disadvantages associated with it (Table 4).

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).
FactorInhibitory effectPreventive effectDisadvantages of preventive concentrationsAdvantages
Ethanol0 to 15% (v/v)> = 15 to 20% (v/v)Too high for table winesWine more stable at higher %v/v ethanol
pH2.17 to 2.621.75 to 2.17Not oenologically viableGreater stability at low pH
Free SO2 (pH = 3.5)0 to 20 mg/L>20 mg/LConcentration gradually fallsEffective against Dekkera/Brettanomyces
pH-dependentWidely used
Sorbic acid (pH = 3.6)0 to 900 mg/L900 to 1100 mg/LLegal limit just 250 mg/LAntifungal agent
Unstable in the presence of lactic acid bacteria 
Benzoic acid (pH = 3.6)0 to 150 mg/L150 to 200 mg/LAffects flavorPreservative
Not authorized for use in wineEffective legal dose for use with musts
Nicostatin 0 to 25 mg/LAction lasts 48 h onlyDisinfectant
SolublePossible use in barrel hygiene
Causes turbidity (especially in white wines)Disappears after 15 d
Temperature15 to 30 °C30 to 40 °CReduces alcohol concentrationEasily controlled if apparatus used is adequate
0 to 15 °CCost 

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 p-coumaric acid into 4-ethylphenol

The optimum temperature for HcDc/VpR activity in D. bruxellensis D37 was 20 to 30 °C; at 15 to 20 °C less activity was seen, and at 30 to 40 and 0 to 15 °C no such activity was seen at all (Figure 7). 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 flash-pasteurization at a temperature of 35 to 40 °C might protect wines from Dekkera/Brettanomyces when ageing in wooden barrels.

image

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).

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

The 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 SO2 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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

This study was funded by the Ministerio de Educación y Ciencia (MEyC) (project AGL2005-06640-C02-01). We thank Director Montserrat Íñiguez 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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
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