Food Chemistry 170 (2015) 401–406
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Influence of the use of fungicides on the volatile composition of Monastrell red wines obtained from inoculated fermentation J. Oliva a, A.M. Martínez-Gil b, C. Lorenzo b, M.A. Cámara a, M.R. Salinas b, A. Barba a, T. Garde-Cerdán c,⇑ a
Dpto. Química Agrícola, Geología y Edafología, Universidad de Murcia, Campus de Espinardo s/n, 30100 Murcia, Spain Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain c Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja-CSIC-Universidad de La Rioja), Ctra. Mendavia-Logroño NA 134, Km. 90, 26071 Logroño, La Rioja, Spain b
a r t i c l e
i n f o
Article history: Received 8 May 2014 Accepted 12 August 2014 Available online 30 August 2014 Keywords: Aroma Wine Fungicides Grape Vitis vinifera Alcoholic fermentation
a b s t r a c t The influence of six fungicides (famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfen and trifloxystrobin) on the volatile composition of red wines obtained from inoculated fermentation was studied. Although treatments were carried out under critical agricultural practices (CAP), the residues in the wines were below their maximum residue limit (MRL). Ethyl decanoate was the compound most influenced by these fungicides, while diethyl succinate, decanoic acid, b-ionone, and citronellol concentration were not changed with any of the treatments. The treatment of grapes with trifloxystrobin induced changes in only one volatile compound, and the variation in volatile composition of wines from grapes treated with fenhexamid, fluquinconazole and quinoxyfen compared to control wines was almost negligible invaluable. The treatment with famoxadone influenced more volatile compounds than the other ones, except for wine from grapes treated with kresoxim-methyl, which was the only wine that showed a big change in its aromatic composition. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The use of fungicides for the control of pest in the vineyard shows the risk of residues of these compounds in grapes, and their transfer to wines elaborated with these grapes, implying a health hazard. For this reason, their maximum residue limits (MRLs) are controlled by the current legislation. Moreover, these residues can affect the yeast involved in the fermentative process. Hence, their effect on yeast population and fermentation have been studied by several authors (Calhelha, Andrade, Ferreira, & Estevinho, 2006; Comitini & Ciani, 2008; Cus & Raspor, 2008; González-Rodríguez et al., 2011a; Noguerol-Pato, Torrado-Agrasar, González-Barreiro, Cancho-Grande, & Simal-Gándara, 2014; Oliva, Navarro, Barba, Navarro, & Salinas, 1999; Zara et al., 2011). Some suggest that depending on the type of fungicides the effect will be different, as they show specificity for certain species (Cadez, Zupan, & Raspor, 2010). Thus, some types of fungicides can slow down fermentation (Cabras et al., 1999; Navarro, García, Navarro, Oliva, & Barba, 1997), and in extreme cases they may even stop it (González-Álvarez, González-Barreiro,
⇑ Corresponding author. Tel.: +34 941291383; fax: +34 941291392. E-mail address: [email protected] (T. Garde-Cerdán). http://dx.doi.org/10.1016/j.foodchem.2014.08.056 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Cancho-Grande, & Simal-Gándara, 2012), while other types of fungicides do not affect it (Cabras & Angioni, 2000; Oliva et al., 2007; Ubeda, Briones, & Izquierdo, 1996). The aroma of a wine is one of the most important characteristics in defining its quality. Since several wine volatile compounds are produced during fermentation, the potential impact of fungicide residues on their biosynthesis, and so on the total wine aroma, is a matter of a great concern. In fact, new phytosanitary products used to control fungal diseases should be completely inactive against fermentative microflora (González-Álvarez et al., 2012). To our knowledge, there are few data about the influence of new-generation fungicides on aroma biosynthesis (Noguerol-Pato et al., 2014), on volatile composition of white wines (García et al., 2004; González-Rodríguez, Noguerol-Pato, González-Barreiro, Cancho-Grande, & Simal-Gándara, 2011b; González-Álvarez et al., 2012), and red wines (Noguerol-Pato, González-Rodríguez, González-Barreiro, Cancho-Grande, & Simal-Gándara, 2011; Oliva, Zalacain, Payá, Salinas, & Barba, 2008). For these reasons, the aim of this work was to study the influence of the use of several fungicides widely used in the vineyard on the volatile composition of red wines obtained from inoculated fermentation. Individual treatments at the recommended doses were performed with the selected fungicides under critical agricultural practices (CAP).
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2. Materials and methods 2.1. Chemicals Analytical standards of the fungicides (purity P 95%) were purchased from Dr. Ehrenstorfer (Augsburg, Germany); stock standard solutions of 1 mg/l were prepared by accurately weighing individual analytical standards in volumetric flaks and dissolving and diluting them to volume with acetonitrile. The active ingredient used (Table 1) at the doses recommended by the manufacturer in the experiments were: Famoxadone [3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione] with effective preventive effects and broad fungicidal spectrum. Fluquinconazole [3-(2,4-dichlorophenyl)-6-fluoro-2-(1H-1,2,4triazol-1-yl)quinazolin-4(3H)-one] with protective, eradicative and systemic properties; it is used to control Uncinula necator. Kresoxim-methyl [methyl (E)-methoxyimino[2-(o-tolyloxymethyl)phenyl] acetate] is an oximinoacetate (strobilurin type) with protective, curative, eradicative and long residual disease control: it is used to control U. necator in grapes. Quinoxyfen (5,7-dichloro-4-quinolyl-4-fluorophenyl ether); it is a mobile, protective fungicide; it is used in grapes for the control of powdery mildew (U. necator). Fenhexamid (2,3-dichloro-4-hidroxy-1-methylcyclohexanecarboxanilide) with protective action and is not translocated; it is used to control Botrytis cinerea in grapes. Trifloxystrobin [methyl(E)-methoxyimino-{(E)-a-[1-(a,a,a-trifluoro-m-tolyl)ethyl ideneaaminooxy]-0-tolyl}acetate] is the main active ingredient for treating downy and powdery mildews that can be present in grapes and wines. 2.2. Plant materials Red grapes Vitis vinifera var. Monastrell were harvested in an experimental plot in Jumilla, Murcia (SE Spain). The nutritional state and physiological conditions of the grape were suitable to give quality wines. 2.3. Fungicide treatments and sampling Seven experimental plots of 225 m2 were selected (one control and six for the individual treatments with the fungicides under study). All treatments were performed under critical agricultural practices (CAP), i.e., six hours before grape collection. Despite this, the final results in wines for the commercially products used were famoxadone 0.12 mg/l, trifloxystrobin < 0.05 mg/l; fluquinconazole < 0.05 mg/l, quinoxyfen < 0.01 mg/l; fenhexamid 0.89 mg/l, and kresoxim-methyl 0.08 mg/l, all of these under their MRL (Table 1). Table 1 also shows the application doses. The experimental plots had not received previous treatment with the fungicides studied and at the time of application, grapes were exempt from any pesticide residues. Grape samples (15 kg) were destemmed and crushed, and then 80 mg/l of SO2 was added. Musts were inoculated with active dry yeast UCLM S377 (Anfiquimica SL) following the protocol of the manufacturer. Samples were introduced in the fermenters and maintained in dynamic maceration for 8 days at controlled
temperature (24–28 °C). After alcoholic fermentation, wines were decanted, clarified and filtered. All the vinifications were carried out in duplicate. Wine samples (1 l) of each of the selected plots were collected and wine samples were frozen at 30 °C until analysis. 2.4. Analysis of volatile compounds by gas chromatography The wine volatile compounds were extracted by stir bar sorptive extraction (SBSE) according to Lorenzo, Garde-Cerdán, Pedroza, Alonso, and Salinas (2009) and analysed by GC–MS. The volatile compounds were extracted from wines by introducing the polydimethylsiloxane (PDMS) coated stir bar (0.5 mm film thickness, 10 mm length, Twister, Gerstel, Mülheim and der Ruhr, Germany) into 10 ml of sample, to which 100 ll of internal standards c-hexalactone and 3-methyl-1-pentanol solution at 1 ll/ ml, both in absolute ethanol (Merck, Darmstadt, Germany) was added. Samples were stirred at 500 rpm at room temperature for 60 min. The stir bar was then removed from the sample, rinsed with distilled water and dried with a cellulose tissue, and later transferred into a thermal desorption tube for GC–MS analysis. In the thermal desorption tube, the volatile compounds were desorbed from the stir bar under the following conditions: oven temperature, 330 °C; desorption time, 4 min; cold trap temperature, 30 °C; helium inlet flow 45 ml/min. The compounds were transferred into the Hewlett–Packard LC 3D GC–MS (Palo Alto, USA) with a fused silica capillary column (BP21 stationary phase 30 m length, 0.25 mm i.d., and 0.25 lm film thickness; SGE, Ringwood, Australia). The chromatographic program was set at 40 °C (held for 5 min), raised to 230 °C at 10 °C/min (held for 15 min). The total time analysis was 36 min. For mass spectrometry analysis, electron impact mode (EI) at 70 eV was used. The mass range varied from 35 to 500 a, and the detector temperature was 150 °C. The analysis of volatile compounds in the wines was done in duplicate, and since the fermentations were done in duplicate, the results shown for these compounds were the mean of 4 analyses. Identification was carried out using the NIST library and by comparison with the mass spectrum and retention index of chromatographic standards designed by us and data found in the bibliography. Quantification was based on five-point calibration curves of respective standards (Aldrich, Gillingham, England) (R2 > 0.94) in a 12% ethanol (v/v) solution at pH 3.6. 2.5. Analysis of fungicide residues Analytical determination of fluquinconazole was performed with GC-ECD, while that of famoxadone was with LC-DAD, after extraction with acetone and ethyl acetate–hexane (1:1 v/v) (Oliva et al., 2007). The other fungicides were determined using GC–MS/MS and LC–MS/MS, after extraction for the modified version of the QuEChERS method (Payá et al., 2007). 2.6. Statistical analysis The statistical elaboration of the data was performed using SPSS Version 17.0 statistical package for Windows (SPSS, Chicago, IL).
Table 1 Fungicide treatments, dose, pre-harvest interval (PHI) and maximum residue limit (MRL). Fungicide
Commercial name
Manufacturers
Dose (kg/ha)
PHI (days)
MRL (EU) (mg/kg)
Famoxadone Fenhexamid Fluquinconazole Kresoxim-methyl Quinoxyfen Trifloxystrobin
Equation Pro GR (22.5%) Teldor WG (50%) Castellan GD (25%) Stroby WG (50%) Arius SC (25%) Flint WG (50%)
Dupont Ibérica Bayer Hispania Argos Schering AgrEvo BASF Dow Agro Science Bayer Cropscience
0.4 1 0.4 0.2 0.3 0.15
28 14 21 35 28 28
2 5 0.5 1 1 5
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(a)
Isobutanol 40
ab
ab
ab
ab
a
a
a
a a
100 0
C
F
(c)
FL
K
Q
FH
TRI
2-Phenylethanol 16
C
FL
bc
a
8
Q
FH
a
ab
Q
FH
TRI
d
20 mg/l
ab
K
n-Hexanol 25
abc
ab
F
(d)
c bc
mg/l
200 a
a
0
12
b
300
bc
20
Isoamyl alcohols 400
mg/l
mg/l
30
10
(b)
c
15 10
4
5
bc
a
a
c
0
0 C
F
FL
K
Q
FH
C
TRI
F
FL
K
TRI
Fig. 1. Higher alcohols concentration in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI). Different letters indicate differences between the samples (p < 0.05).
(a)
(b)
Isoamyl acetate 4
b
2 1
a
a
C
F
a
a
a
a
mg/l
mg/l
3
0 (c)
FL
K
Q
FH
a
a
a
a
mg/l
mg/l
0.5
a
0 C
F
K
Q
FH
0.06 0.05 0.04 0.03 0.02 0.01 0
d c
c
b
b
ab a
C
F
FL
K
Q
F
1.2 1 0.8 0.6 0.4 0.2 0
FH
TRI
c
FL
K
Q
FH
TRI
c
ab
ab
C
F
(f)
Ethyl decanoate
bc
ab
Ethyl octanoate
TRI
mg/l
mg/l
(e)
FL
d
a
C
1.5 a
d bc
(d)
b
1
0.06 0.05 0.04 0.03 0.02 0.01 0
TRI
Ethyl hexanoate 2
2-Phenylethyl acetate
b
a
FL
K
Q
ab
FH
ab
TRI
Diethyl succinate 0.6 0.5 0.4 0.3 0.2 0.1 0
a
a a
C
F
a
FL
a
a
K
Q
a
FH
TRI
Fig. 2. Ester concentrations in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI). Different letters indicate differences between the samples (p < 0.05).
Volatile compound data were processed using variance analysis (ANOVA). Differences between means were compared using the least significant differences (LSD) test at 0.05 probability level. Principal component analysis (PCA) was performed with volatile compound contents in the different samples using InfoStat Professional 2012 version (InfoStat, www.infostat.com.ar).
3. Results and discussion 3.1. Higher alcohols Fig. 1 shows the results of higher alcohols in wines elaborated with grapes from the different treatment carried out in the
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(a)
Octanoic acid 2.5 2
mg/l
(b)
b b
b
0.25 ab
b
ab
mg/l
a
FL
K
Q
FH
C
TRI
b
a
Q
FH
TRI
a a
a
a
C
F
FL
a a
a
Q
FH
0.6 a
a
a
μg/l
a a
K
β -Ionone
0.8
a
FL
1
20 15
F
(d)
Nonanal 25
μg/l
a
0 F
(c)
0.4 0.2
5
0
0 C
F
FL
(e)
K
Q
FH
TRI
Nerolidol 4
(f)
bc ab
c ab
ab
K
TRI
Citronellol 40 30
ab
2
a
μg/l
μg/l
a
0.05 C
1 0
a
a
0.15
0
3
a
0.1
0.5
10
a
0.2
1.5 1
Decanoic acid
a
a
a
a
F
FL
a
a
a
20 10 0
C
F
FL
K
Q
FH
TRI
(g)
C
K
Q
FH
TRI
Farnesol 60
b
μg/l
40 20
a
a
a
a
a
a
Q
FH
TRI
0 C
F
FL
K
Fig. 3. Miscellaneous volatile compounds concentration in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI). Different letters indicate differences between the samples (p < 0.05).
vineyard with the six fungicides studied. Isobutanol concentration was higher in wines from grapes treated with fluquinconazole (FL) and kresoxim-methyl (K) than in the control. However, the other wines did not show significant differences compared to the control (Fig. 1a). In the case of isoamyl alcohols, only the wine from grapes treated with kresoxim-methyl (K) showed a higher concentration than the control (Fig. 1b). 2-Phenylethanol concentration was higher in wines coming from quinoxyfen treatment (Q) than in the others (Fig. 1c). Finally, n-hexanol showed higher concentration in wines from grapes treated with famoxadone (F), kresoxim-methyl (K), and trifloxystrobin (TRI) than in the control wine (Fig. 1d). All the alcohol studied came from alcoholic fermentation, except n-hexanol, formed at any stage preceding alcoholic fermentation (harvest, transport, crushing and pressing of grapes) (González-Álvarez et al., 2012). Therefore, yeast plays an important role in higher alcohols synthesis. As we have written above, depending on the type of fungicide treatment, the yeast population can be affected. Oliva et al. (2007) analysed the influence on grape yeast content and fermentation evolution, of the six fungicides studied in this work. They showed that these six fungicides had
no negative effects on indigenous yeast from the variety and zone or on their fermentative kinetics. In Oliva et al. (2008), in which the only difference with the present study was that yeast was not inoculated, the alcohol evolution was different, showing different behaviours with the same fungicides. So we can say that these fungicides, especially kresoximmethyl (K), showed different effect in spontaneous fermentation than in inoculated fermentation. At concentrations below 300 mg/l, higher alcohols contribute to the desirable complexity of wine aroma (Rapp & Mandery, 1986). Isoamyl alcohols are the most abundant alcohols analysed in these wines (Fig. 1). They have a ‘‘solvent’’ aroma descriptor and at high level concentrations contribute negatively to the aroma quality of wines. This is the case of wines from grapes treated with kresoxim-methyl (Fig. 1b), which showed concentration for these alcohols excessively high, over 300 mg/l. Isobutanol concentration was below its odour threshold in all wines (40 mg/l; Guth, 1997) (Fig. 1a). 2-Phenylethanol is the only fusel alcohol described at a sensory level in pleasant terms (rose aroma); in some wines it was over its odour threshold (7.5 mg/l; Etiévant, 1991) (Fig. 1c). Regarding n-hexanol, whose concentration was over its odour
J. Oliva et al. / Food Chemistry 170 (2015) 401–406
threshold (1.1 mg/l; Etiévant, 1991) in all wines studied (Fig. 1d), it belongs to the group of C6 compounds and is responsible for unpleasant grass flavour in wines. 3.2. Esters Fig. 2 shows the results for esters in wines from grapes treated with the six fungicides studied. The concentration of isoamyl acetate was higher in wines from grapes treated with kresoximmethyl (K) than in the other ones, which did not show significant differences between them (Fig. 2a). In the case of 2-phenylethyl acetate, greater variability among wines was found, and the concentration was higher in wines from grapes treated with kresoxim-methyl (K), and quinoxyfen (Q) than in the others (Fig. 2b). The results for ethyl hexanoate and octanoate were similar to those obtained for isoamyl acetate, with their concentrations in wines from kresoxim-methyl (K) treatment being higher than in the other wines (Fig. 2c–d). The concentration of ethyl decanoate was higher in wines from grapes treated with famoxadone (F), fluquinconazole (FL), and kresoxim-methyl (K) than in the others, which did not show significant differences with the control wine. Only the wine from grapes treated with fenhexamid (FH) showed lower concentration than the control wine for this ester (Fig. 2e). Regarding diethyl succinate concentration, no significant difference was observed between wines from the different treatments (Fig. 2f). Oliva et al. (2008) also observed that wines from grapes treated with kresoxim-methyl showed the highest concentration in acetate and ethyl esters in spontaneous fermentations, although with lower differences than in this work. This may be due to the esters’ synthesis being higher in inoculated than in spontaneous fermentations (Garde-Cerdán & Ancín-Azpilicueta, 2006). Like higher alcohols, the esters come from alcoholic fermentation, so it is not surprising that esters formation was greater in wines from grapes treated with kresoxim-methyl (K) than in the others, in the same way as occurred with higher alcohols. In all the wines, the concentration of isoamyl acetate was above its perception threshold (0.03 mg/l; Ferreira, López, & Cacho, 2000), especially in the wines from kresoxim-methyl treatment (K) (Fig. 2a). However, in no case was the threshold level of 2-phenylethyl
405
acetate in wines (0.25 mg/l; Guth, 1997) exceeded (Fig. 2b). Elsewhere, among ethyl esters found in the wines, only the concentration of ethyl hexanoate and octanoate was above its perception thresholds (0.014 and 0.005 mg/l, respectively; Ferreira et al., 2000), especially in the wines from kresoxim-methyl treatment (K) (Fig. 2c–d). Esters are an important factor in wine quality since the concentration of some of them in wine is usually found above the threshold level, adding a floral and fruity aroma (Martínez-Gil et al., 2012; Ribéreau-Gayon, Glories, Maujean, & Dubourdieu, 2006). 3.3. Miscellaneous volatile compounds Fig. 3 shows the results of several volatile compounds in wines from grapes treated with the six fungicides studied. Most of these treatments did not affect the fatty acids concentrations, octanoic and decanoic acid, as their concentrations did not show significant differences in these wines compared to the control wines. Only octanoic acid showed lower concentration in wines from grapes treated with famoxadone (F) (Fig. 3a). The quantity of b-ionone and citronellol in wines was not affected by the treatments (Fig. 3d and f). Finally, the treatment with kresoxim-methyl increased the nonanal concentration; the treatment with fenhexamid slightly increased the nerolidol concentration, and treatment with famoxadone increased farnesol concentration (Fig. 3c, e and g). 3.4. Principal component analysis (PCA) In order to classify the different samples, PCA was performed on data expressing volatile compounds in the control wine and in the wines from the different treatments with the six fungicides (independent variables). The results are shown in Fig. 4. The principal component 1 (PC1) explained 55.6% of the variance and the principal component 2 (PC2) explained 21.8% of the variance (which together accounted for 77.4% of the variance). The PC1 was strongly correlated with isobutanol, isoamyl acetate, isoamyl alcohols, ethyl hexanoate, n-hexanol, nonanal, ethyl octanoate, 2-phenylethyl acetate, b-ionone, and decanoic acid, while the
Fig. 4. Principal component analysis (PCA) carried out with volatile compounds concentration in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI).
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Table 2 Loadings for the two first principal components. PC1 Iso IsoAcet IsoAlc EtHex Hex Non EtOct EtDec DiEtSuc Cit 2-PheAcet 2-Phe Ion Ner C8 C10 Far
0.81 0.95 0.90 0.96 0.95 0.98 0.97 0.65 0.30 0.64 0.70 0.61 0.92 0.26 0.21 0.74 0.20
PC2 0.16 0.21 0.39 0.17 0.28 0.04 0.16 0.65 0.50 0.57 0.50 0.72 0.02 0.32 0.90 0.00 0.83
The most significant values are in bold (loading P 0.70).
PC2 was strongly correlated with 2-phenylethanol, octanoic acid, and farnesol (Table 2). The only wine positively positioned on PC1 was kresoxim-methyl (K) wine (Fig. 4), which showed higher concentration than other wines in several volatile compounds (Figs. 1–3). As for the PC2, on the positive side of this axis were quinoxyfen (Q), control (C), fenhexamid (FH), and trifloxystrobin (TRI) wines, and fluquinconazole (FL), kresoxim-methyl (K), and famoxadone (F) wines were situated on negative side of this axis.
4. Conclusions The fungicide with the greatest effect on wine volatile composition was kresoxim-methyl, as wines from grapes treated with this fungicide showed the highest concentrations of higher alcohols (with the exception of 2-phenylethanol), acetate esters, ethyl hexanoate and decanoate, and nonanal. The most influenced compound by treatments applied was ethyl decanoate, since its concentration in wines changed with four of the six fungicides studied. However, diethyl succinate, decanoic acid, b-ionone, and citronellol concentration were not altered with any of the treatments carried out. In general, all treatments affected at least one compound, although the only wine that showed a great change in its aromatic composition was that from grapes treated with kresoxim-methyl. Acknowledgements T. G.-C. thanks the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)-Gobierno de La Rioja and FEDER of the European Community for her doctoral contract. References Cabras, P., Angioni, A., Garau, V. L., Pirisi, F. M., Farris, G. A., Madau, G., et al. (1999). Pesticides in fermentative processes of wine. Journal of Agricultural Food Chemistry, 47, 3854–3857. Cabras, P., & Angioni, A. (2000). Pesticide residues in grapes, wine and their processing products. Journal of Agricultural Food Chemistry, 48, 967–973. Cadez, N., Zupan, J., & Raspor, P. (2010). The effect of fungicides on yeast communities associated with grape berries. FEMS Yeast Research, 10, 619–630.
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Navarro, S., García, B., Navarro, G., Oliva, J., & Barba, A. (1997). Effect of wine-making practices on the concentrations of fenarimol and penconazole in rose wines. Journal of Food Protection, 60, 1120–1124. Noguerol-Pato, R., González-Rodríguez, R. M., González-Barreiro, C., CanchoGrande, B., & Simal-Gándara, J. (2011). Influence of tebuconazole residues on the aroma composition of red wines. Food Chemistry, 124, 1525–1532. Noguerol-Pato, R., Torrado-Agrasar, A., González-Barreiro, C., Cancho-Grande, B., & Simal-Gándara, J. (2014). Influence of new generation fungicides on Saccharomyces cerevisiae growth, grape must fermentation and aroma biosynthesis. Food Chemistry, 146, 234–241. Oliva, J., Navarro, S., Barba, A., Navarro, G., & Salinas, M. R. (1999). Effect of pesticide residues on the aromatic composition of red wines. Journal of Agricultural and Food Chemistry, 47, 2830–2836. Oliva, J., Cayuela, J. M., Payá, P., Martínez-Cacha, A., Cámara, M. A., & Barba, A. (2007). Influence of fungicides on grape yeast content and its evolution in the fermentation. Communications in Agricultural and Applied Biological Sciences, 72, 181–189. Oliva, J., Zalacain, A., Payá, P., Salinas, M. R., & Barba, A. (2008). Effect of the use of recent commercial fungicides [under good and critical agricultural practices] on the aroma composition of Monastrell red wines. Analytica Chimica Acta, 617, 107–118. Payá, P., Anastassiades, M., MacK, D., Sigalova, I., Tasdelen, B., Oliva, J., et al. (2007). Analysis of pesticide residues using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) pesticide multiresidue method in combination with gas and liquid chromatography and tandem mass spectrometric detection. Analytical and Bioanalytical Chemistry, 389, 1697–1714. Rapp, A., & Mandery, H. (1986). Wine aroma. Experientia, 42, 873–884. Ribéreau-Gayon, P., Glories, Y., Maujean, A., & Dubourdieu, D. (2006). Handbook of enology. The chemistry of wine stabilization and treatments. Chichester: John Wiley & Sons. Ubeda, J., Briones, A. I., & Izquierdo, P. M. (1996). Comportamiento in vitro de cepas enológicas de Saccharomyces cerevisiae frente a distintos fungicidas empleados en viticultura. Alimentación, Equipos y Tecnología, 15, 117–120. Zara, S., Caboni, P., Orro, D., Farris, G. A., Pirisi, F., & Angioni, A. (2011). Influence of fenamidone, indoxacarb, pyraclostrobin, and deltamethrin on the population of natural yeast microflora during winemaking of two Sardinian grape cultivars. Journal of Environmental Science and Health B, 46, 491–497.
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Influence of the use of fungicides on the volatile composition of Monastrell red wines obtained from inoculated fermentation J. Oliva a, A.M. Martínez-Gil b, C. Lorenzo b, M.A. Cámara a, M.R. Salinas b, A. Barba a, T. Garde-Cerdán c,⇑ a
Dpto. Química Agrícola, Geología y Edafología, Universidad de Murcia, Campus de Espinardo s/n, 30100 Murcia, Spain Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain c Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja-CSIC-Universidad de La Rioja), Ctra. Mendavia-Logroño NA 134, Km. 90, 26071 Logroño, La Rioja, Spain b
a r t i c l e
i n f o
Article history: Received 8 May 2014 Accepted 12 August 2014 Available online 30 August 2014 Keywords: Aroma Wine Fungicides Grape Vitis vinifera Alcoholic fermentation
a b s t r a c t The influence of six fungicides (famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfen and trifloxystrobin) on the volatile composition of red wines obtained from inoculated fermentation was studied. Although treatments were carried out under critical agricultural practices (CAP), the residues in the wines were below their maximum residue limit (MRL). Ethyl decanoate was the compound most influenced by these fungicides, while diethyl succinate, decanoic acid, b-ionone, and citronellol concentration were not changed with any of the treatments. The treatment of grapes with trifloxystrobin induced changes in only one volatile compound, and the variation in volatile composition of wines from grapes treated with fenhexamid, fluquinconazole and quinoxyfen compared to control wines was almost negligible invaluable. The treatment with famoxadone influenced more volatile compounds than the other ones, except for wine from grapes treated with kresoxim-methyl, which was the only wine that showed a big change in its aromatic composition. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The use of fungicides for the control of pest in the vineyard shows the risk of residues of these compounds in grapes, and their transfer to wines elaborated with these grapes, implying a health hazard. For this reason, their maximum residue limits (MRLs) are controlled by the current legislation. Moreover, these residues can affect the yeast involved in the fermentative process. Hence, their effect on yeast population and fermentation have been studied by several authors (Calhelha, Andrade, Ferreira, & Estevinho, 2006; Comitini & Ciani, 2008; Cus & Raspor, 2008; González-Rodríguez et al., 2011a; Noguerol-Pato, Torrado-Agrasar, González-Barreiro, Cancho-Grande, & Simal-Gándara, 2014; Oliva, Navarro, Barba, Navarro, & Salinas, 1999; Zara et al., 2011). Some suggest that depending on the type of fungicides the effect will be different, as they show specificity for certain species (Cadez, Zupan, & Raspor, 2010). Thus, some types of fungicides can slow down fermentation (Cabras et al., 1999; Navarro, García, Navarro, Oliva, & Barba, 1997), and in extreme cases they may even stop it (González-Álvarez, González-Barreiro,
⇑ Corresponding author. Tel.: +34 941291383; fax: +34 941291392. E-mail address: [email protected] (T. Garde-Cerdán). http://dx.doi.org/10.1016/j.foodchem.2014.08.056 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Cancho-Grande, & Simal-Gándara, 2012), while other types of fungicides do not affect it (Cabras & Angioni, 2000; Oliva et al., 2007; Ubeda, Briones, & Izquierdo, 1996). The aroma of a wine is one of the most important characteristics in defining its quality. Since several wine volatile compounds are produced during fermentation, the potential impact of fungicide residues on their biosynthesis, and so on the total wine aroma, is a matter of a great concern. In fact, new phytosanitary products used to control fungal diseases should be completely inactive against fermentative microflora (González-Álvarez et al., 2012). To our knowledge, there are few data about the influence of new-generation fungicides on aroma biosynthesis (Noguerol-Pato et al., 2014), on volatile composition of white wines (García et al., 2004; González-Rodríguez, Noguerol-Pato, González-Barreiro, Cancho-Grande, & Simal-Gándara, 2011b; González-Álvarez et al., 2012), and red wines (Noguerol-Pato, González-Rodríguez, González-Barreiro, Cancho-Grande, & Simal-Gándara, 2011; Oliva, Zalacain, Payá, Salinas, & Barba, 2008). For these reasons, the aim of this work was to study the influence of the use of several fungicides widely used in the vineyard on the volatile composition of red wines obtained from inoculated fermentation. Individual treatments at the recommended doses were performed with the selected fungicides under critical agricultural practices (CAP).
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2. Materials and methods 2.1. Chemicals Analytical standards of the fungicides (purity P 95%) were purchased from Dr. Ehrenstorfer (Augsburg, Germany); stock standard solutions of 1 mg/l were prepared by accurately weighing individual analytical standards in volumetric flaks and dissolving and diluting them to volume with acetonitrile. The active ingredient used (Table 1) at the doses recommended by the manufacturer in the experiments were: Famoxadone [3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione] with effective preventive effects and broad fungicidal spectrum. Fluquinconazole [3-(2,4-dichlorophenyl)-6-fluoro-2-(1H-1,2,4triazol-1-yl)quinazolin-4(3H)-one] with protective, eradicative and systemic properties; it is used to control Uncinula necator. Kresoxim-methyl [methyl (E)-methoxyimino[2-(o-tolyloxymethyl)phenyl] acetate] is an oximinoacetate (strobilurin type) with protective, curative, eradicative and long residual disease control: it is used to control U. necator in grapes. Quinoxyfen (5,7-dichloro-4-quinolyl-4-fluorophenyl ether); it is a mobile, protective fungicide; it is used in grapes for the control of powdery mildew (U. necator). Fenhexamid (2,3-dichloro-4-hidroxy-1-methylcyclohexanecarboxanilide) with protective action and is not translocated; it is used to control Botrytis cinerea in grapes. Trifloxystrobin [methyl(E)-methoxyimino-{(E)-a-[1-(a,a,a-trifluoro-m-tolyl)ethyl ideneaaminooxy]-0-tolyl}acetate] is the main active ingredient for treating downy and powdery mildews that can be present in grapes and wines. 2.2. Plant materials Red grapes Vitis vinifera var. Monastrell were harvested in an experimental plot in Jumilla, Murcia (SE Spain). The nutritional state and physiological conditions of the grape were suitable to give quality wines. 2.3. Fungicide treatments and sampling Seven experimental plots of 225 m2 were selected (one control and six for the individual treatments with the fungicides under study). All treatments were performed under critical agricultural practices (CAP), i.e., six hours before grape collection. Despite this, the final results in wines for the commercially products used were famoxadone 0.12 mg/l, trifloxystrobin < 0.05 mg/l; fluquinconazole < 0.05 mg/l, quinoxyfen < 0.01 mg/l; fenhexamid 0.89 mg/l, and kresoxim-methyl 0.08 mg/l, all of these under their MRL (Table 1). Table 1 also shows the application doses. The experimental plots had not received previous treatment with the fungicides studied and at the time of application, grapes were exempt from any pesticide residues. Grape samples (15 kg) were destemmed and crushed, and then 80 mg/l of SO2 was added. Musts were inoculated with active dry yeast UCLM S377 (Anfiquimica SL) following the protocol of the manufacturer. Samples were introduced in the fermenters and maintained in dynamic maceration for 8 days at controlled
temperature (24–28 °C). After alcoholic fermentation, wines were decanted, clarified and filtered. All the vinifications were carried out in duplicate. Wine samples (1 l) of each of the selected plots were collected and wine samples were frozen at 30 °C until analysis. 2.4. Analysis of volatile compounds by gas chromatography The wine volatile compounds were extracted by stir bar sorptive extraction (SBSE) according to Lorenzo, Garde-Cerdán, Pedroza, Alonso, and Salinas (2009) and analysed by GC–MS. The volatile compounds were extracted from wines by introducing the polydimethylsiloxane (PDMS) coated stir bar (0.5 mm film thickness, 10 mm length, Twister, Gerstel, Mülheim and der Ruhr, Germany) into 10 ml of sample, to which 100 ll of internal standards c-hexalactone and 3-methyl-1-pentanol solution at 1 ll/ ml, both in absolute ethanol (Merck, Darmstadt, Germany) was added. Samples were stirred at 500 rpm at room temperature for 60 min. The stir bar was then removed from the sample, rinsed with distilled water and dried with a cellulose tissue, and later transferred into a thermal desorption tube for GC–MS analysis. In the thermal desorption tube, the volatile compounds were desorbed from the stir bar under the following conditions: oven temperature, 330 °C; desorption time, 4 min; cold trap temperature, 30 °C; helium inlet flow 45 ml/min. The compounds were transferred into the Hewlett–Packard LC 3D GC–MS (Palo Alto, USA) with a fused silica capillary column (BP21 stationary phase 30 m length, 0.25 mm i.d., and 0.25 lm film thickness; SGE, Ringwood, Australia). The chromatographic program was set at 40 °C (held for 5 min), raised to 230 °C at 10 °C/min (held for 15 min). The total time analysis was 36 min. For mass spectrometry analysis, electron impact mode (EI) at 70 eV was used. The mass range varied from 35 to 500 a, and the detector temperature was 150 °C. The analysis of volatile compounds in the wines was done in duplicate, and since the fermentations were done in duplicate, the results shown for these compounds were the mean of 4 analyses. Identification was carried out using the NIST library and by comparison with the mass spectrum and retention index of chromatographic standards designed by us and data found in the bibliography. Quantification was based on five-point calibration curves of respective standards (Aldrich, Gillingham, England) (R2 > 0.94) in a 12% ethanol (v/v) solution at pH 3.6. 2.5. Analysis of fungicide residues Analytical determination of fluquinconazole was performed with GC-ECD, while that of famoxadone was with LC-DAD, after extraction with acetone and ethyl acetate–hexane (1:1 v/v) (Oliva et al., 2007). The other fungicides were determined using GC–MS/MS and LC–MS/MS, after extraction for the modified version of the QuEChERS method (Payá et al., 2007). 2.6. Statistical analysis The statistical elaboration of the data was performed using SPSS Version 17.0 statistical package for Windows (SPSS, Chicago, IL).
Table 1 Fungicide treatments, dose, pre-harvest interval (PHI) and maximum residue limit (MRL). Fungicide
Commercial name
Manufacturers
Dose (kg/ha)
PHI (days)
MRL (EU) (mg/kg)
Famoxadone Fenhexamid Fluquinconazole Kresoxim-methyl Quinoxyfen Trifloxystrobin
Equation Pro GR (22.5%) Teldor WG (50%) Castellan GD (25%) Stroby WG (50%) Arius SC (25%) Flint WG (50%)
Dupont Ibérica Bayer Hispania Argos Schering AgrEvo BASF Dow Agro Science Bayer Cropscience
0.4 1 0.4 0.2 0.3 0.15
28 14 21 35 28 28
2 5 0.5 1 1 5
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(a)
Isobutanol 40
ab
ab
ab
ab
a
a
a
a a
100 0
C
F
(c)
FL
K
Q
FH
TRI
2-Phenylethanol 16
C
FL
bc
a
8
Q
FH
a
ab
Q
FH
TRI
d
20 mg/l
ab
K
n-Hexanol 25
abc
ab
F
(d)
c bc
mg/l
200 a
a
0
12
b
300
bc
20
Isoamyl alcohols 400
mg/l
mg/l
30
10
(b)
c
15 10
4
5
bc
a
a
c
0
0 C
F
FL
K
Q
FH
C
TRI
F
FL
K
TRI
Fig. 1. Higher alcohols concentration in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI). Different letters indicate differences between the samples (p < 0.05).
(a)
(b)
Isoamyl acetate 4
b
2 1
a
a
C
F
a
a
a
a
mg/l
mg/l
3
0 (c)
FL
K
Q
FH
a
a
a
a
mg/l
mg/l
0.5
a
0 C
F
K
Q
FH
0.06 0.05 0.04 0.03 0.02 0.01 0
d c
c
b
b
ab a
C
F
FL
K
Q
F
1.2 1 0.8 0.6 0.4 0.2 0
FH
TRI
c
FL
K
Q
FH
TRI
c
ab
ab
C
F
(f)
Ethyl decanoate
bc
ab
Ethyl octanoate
TRI
mg/l
mg/l
(e)
FL
d
a
C
1.5 a
d bc
(d)
b
1
0.06 0.05 0.04 0.03 0.02 0.01 0
TRI
Ethyl hexanoate 2
2-Phenylethyl acetate
b
a
FL
K
Q
ab
FH
ab
TRI
Diethyl succinate 0.6 0.5 0.4 0.3 0.2 0.1 0
a
a a
C
F
a
FL
a
a
K
Q
a
FH
TRI
Fig. 2. Ester concentrations in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI). Different letters indicate differences between the samples (p < 0.05).
Volatile compound data were processed using variance analysis (ANOVA). Differences between means were compared using the least significant differences (LSD) test at 0.05 probability level. Principal component analysis (PCA) was performed with volatile compound contents in the different samples using InfoStat Professional 2012 version (InfoStat, www.infostat.com.ar).
3. Results and discussion 3.1. Higher alcohols Fig. 1 shows the results of higher alcohols in wines elaborated with grapes from the different treatment carried out in the
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(a)
Octanoic acid 2.5 2
mg/l
(b)
b b
b
0.25 ab
b
ab
mg/l
a
FL
K
Q
FH
C
TRI
b
a
Q
FH
TRI
a a
a
a
C
F
FL
a a
a
Q
FH
0.6 a
a
a
μg/l
a a
K
β -Ionone
0.8
a
FL
1
20 15
F
(d)
Nonanal 25
μg/l
a
0 F
(c)
0.4 0.2
5
0
0 C
F
FL
(e)
K
Q
FH
TRI
Nerolidol 4
(f)
bc ab
c ab
ab
K
TRI
Citronellol 40 30
ab
2
a
μg/l
μg/l
a
0.05 C
1 0
a
a
0.15
0
3
a
0.1
0.5
10
a
0.2
1.5 1
Decanoic acid
a
a
a
a
F
FL
a
a
a
20 10 0
C
F
FL
K
Q
FH
TRI
(g)
C
K
Q
FH
TRI
Farnesol 60
b
μg/l
40 20
a
a
a
a
a
a
Q
FH
TRI
0 C
F
FL
K
Fig. 3. Miscellaneous volatile compounds concentration in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI). Different letters indicate differences between the samples (p < 0.05).
vineyard with the six fungicides studied. Isobutanol concentration was higher in wines from grapes treated with fluquinconazole (FL) and kresoxim-methyl (K) than in the control. However, the other wines did not show significant differences compared to the control (Fig. 1a). In the case of isoamyl alcohols, only the wine from grapes treated with kresoxim-methyl (K) showed a higher concentration than the control (Fig. 1b). 2-Phenylethanol concentration was higher in wines coming from quinoxyfen treatment (Q) than in the others (Fig. 1c). Finally, n-hexanol showed higher concentration in wines from grapes treated with famoxadone (F), kresoxim-methyl (K), and trifloxystrobin (TRI) than in the control wine (Fig. 1d). All the alcohol studied came from alcoholic fermentation, except n-hexanol, formed at any stage preceding alcoholic fermentation (harvest, transport, crushing and pressing of grapes) (González-Álvarez et al., 2012). Therefore, yeast plays an important role in higher alcohols synthesis. As we have written above, depending on the type of fungicide treatment, the yeast population can be affected. Oliva et al. (2007) analysed the influence on grape yeast content and fermentation evolution, of the six fungicides studied in this work. They showed that these six fungicides had
no negative effects on indigenous yeast from the variety and zone or on their fermentative kinetics. In Oliva et al. (2008), in which the only difference with the present study was that yeast was not inoculated, the alcohol evolution was different, showing different behaviours with the same fungicides. So we can say that these fungicides, especially kresoximmethyl (K), showed different effect in spontaneous fermentation than in inoculated fermentation. At concentrations below 300 mg/l, higher alcohols contribute to the desirable complexity of wine aroma (Rapp & Mandery, 1986). Isoamyl alcohols are the most abundant alcohols analysed in these wines (Fig. 1). They have a ‘‘solvent’’ aroma descriptor and at high level concentrations contribute negatively to the aroma quality of wines. This is the case of wines from grapes treated with kresoxim-methyl (Fig. 1b), which showed concentration for these alcohols excessively high, over 300 mg/l. Isobutanol concentration was below its odour threshold in all wines (40 mg/l; Guth, 1997) (Fig. 1a). 2-Phenylethanol is the only fusel alcohol described at a sensory level in pleasant terms (rose aroma); in some wines it was over its odour threshold (7.5 mg/l; Etiévant, 1991) (Fig. 1c). Regarding n-hexanol, whose concentration was over its odour
J. Oliva et al. / Food Chemistry 170 (2015) 401–406
threshold (1.1 mg/l; Etiévant, 1991) in all wines studied (Fig. 1d), it belongs to the group of C6 compounds and is responsible for unpleasant grass flavour in wines. 3.2. Esters Fig. 2 shows the results for esters in wines from grapes treated with the six fungicides studied. The concentration of isoamyl acetate was higher in wines from grapes treated with kresoximmethyl (K) than in the other ones, which did not show significant differences between them (Fig. 2a). In the case of 2-phenylethyl acetate, greater variability among wines was found, and the concentration was higher in wines from grapes treated with kresoxim-methyl (K), and quinoxyfen (Q) than in the others (Fig. 2b). The results for ethyl hexanoate and octanoate were similar to those obtained for isoamyl acetate, with their concentrations in wines from kresoxim-methyl (K) treatment being higher than in the other wines (Fig. 2c–d). The concentration of ethyl decanoate was higher in wines from grapes treated with famoxadone (F), fluquinconazole (FL), and kresoxim-methyl (K) than in the others, which did not show significant differences with the control wine. Only the wine from grapes treated with fenhexamid (FH) showed lower concentration than the control wine for this ester (Fig. 2e). Regarding diethyl succinate concentration, no significant difference was observed between wines from the different treatments (Fig. 2f). Oliva et al. (2008) also observed that wines from grapes treated with kresoxim-methyl showed the highest concentration in acetate and ethyl esters in spontaneous fermentations, although with lower differences than in this work. This may be due to the esters’ synthesis being higher in inoculated than in spontaneous fermentations (Garde-Cerdán & Ancín-Azpilicueta, 2006). Like higher alcohols, the esters come from alcoholic fermentation, so it is not surprising that esters formation was greater in wines from grapes treated with kresoxim-methyl (K) than in the others, in the same way as occurred with higher alcohols. In all the wines, the concentration of isoamyl acetate was above its perception threshold (0.03 mg/l; Ferreira, López, & Cacho, 2000), especially in the wines from kresoxim-methyl treatment (K) (Fig. 2a). However, in no case was the threshold level of 2-phenylethyl
405
acetate in wines (0.25 mg/l; Guth, 1997) exceeded (Fig. 2b). Elsewhere, among ethyl esters found in the wines, only the concentration of ethyl hexanoate and octanoate was above its perception thresholds (0.014 and 0.005 mg/l, respectively; Ferreira et al., 2000), especially in the wines from kresoxim-methyl treatment (K) (Fig. 2c–d). Esters are an important factor in wine quality since the concentration of some of them in wine is usually found above the threshold level, adding a floral and fruity aroma (Martínez-Gil et al., 2012; Ribéreau-Gayon, Glories, Maujean, & Dubourdieu, 2006). 3.3. Miscellaneous volatile compounds Fig. 3 shows the results of several volatile compounds in wines from grapes treated with the six fungicides studied. Most of these treatments did not affect the fatty acids concentrations, octanoic and decanoic acid, as their concentrations did not show significant differences in these wines compared to the control wines. Only octanoic acid showed lower concentration in wines from grapes treated with famoxadone (F) (Fig. 3a). The quantity of b-ionone and citronellol in wines was not affected by the treatments (Fig. 3d and f). Finally, the treatment with kresoxim-methyl increased the nonanal concentration; the treatment with fenhexamid slightly increased the nerolidol concentration, and treatment with famoxadone increased farnesol concentration (Fig. 3c, e and g). 3.4. Principal component analysis (PCA) In order to classify the different samples, PCA was performed on data expressing volatile compounds in the control wine and in the wines from the different treatments with the six fungicides (independent variables). The results are shown in Fig. 4. The principal component 1 (PC1) explained 55.6% of the variance and the principal component 2 (PC2) explained 21.8% of the variance (which together accounted for 77.4% of the variance). The PC1 was strongly correlated with isobutanol, isoamyl acetate, isoamyl alcohols, ethyl hexanoate, n-hexanol, nonanal, ethyl octanoate, 2-phenylethyl acetate, b-ionone, and decanoic acid, while the
Fig. 4. Principal component analysis (PCA) carried out with volatile compounds concentration in the different Monastrell wines: control (C), famoxadone (F), fluquinconazole (FL), kresoxim-methyl (K), quinoxyfen (Q), fenhexamid (FH), trifloxystrobin (TRI).
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Table 2 Loadings for the two first principal components. PC1 Iso IsoAcet IsoAlc EtHex Hex Non EtOct EtDec DiEtSuc Cit 2-PheAcet 2-Phe Ion Ner C8 C10 Far
0.81 0.95 0.90 0.96 0.95 0.98 0.97 0.65 0.30 0.64 0.70 0.61 0.92 0.26 0.21 0.74 0.20
PC2 0.16 0.21 0.39 0.17 0.28 0.04 0.16 0.65 0.50 0.57 0.50 0.72 0.02 0.32 0.90 0.00 0.83
The most significant values are in bold (loading P 0.70).
PC2 was strongly correlated with 2-phenylethanol, octanoic acid, and farnesol (Table 2). The only wine positively positioned on PC1 was kresoxim-methyl (K) wine (Fig. 4), which showed higher concentration than other wines in several volatile compounds (Figs. 1–3). As for the PC2, on the positive side of this axis were quinoxyfen (Q), control (C), fenhexamid (FH), and trifloxystrobin (TRI) wines, and fluquinconazole (FL), kresoxim-methyl (K), and famoxadone (F) wines were situated on negative side of this axis.
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