Food Chemistry 129 (2011) 1676–1680

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Food Chemistry

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Fungicide effects on ammonium and amino acids of Monastrell grapes

José Oliva a,⇑, Teresa Garde-Cerdán b, Ana M. Martínez-Gil b, M. Rosario Salinas b, Alberto Barba a

a Dpto. Química Agrícola, Geología y Edafología, Universidad de Murcia, Campus de Espinardo s/n, 30100 Murcia, Spainb Cátedra de Química Agrícola, E.T.S.I. Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain

a r t i c l e i n f o

Article history:Received 22 February 2011Received in revised form 17 June 2011Accepted 18 June 2011Available online 23 June 2011

Keywords:Amino acidsAmmoniumGrapeFungicidesWineVineyard

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.06.030

⇑ Corresponding author. Tel.: +34 868887482; fax:E-mail address: josoliva@um.es (J. Oliva).

a b s t r a c t

The influence of six fungicides (famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfenand trifloxystrobin) on the amino acids and ammonium composition of grapes (var. Monastrell) are stud-ied. The treatments were performed under critical agricultural practices (CAP), 6 h before grape collec-tion. The analytical determination of amino acids and ammonium were made using HPLC with aphotodiode array detector (DAD), after derivatisation of the sample with diethyl (ethoxymethylene)mal-onate (DEEMM). The application of fungicides to the vine decreased concentrations of nitrogenous com-pounds in grapes. Furthermore, the qualitative and quantitative effects on the amino acids in the grapesdepended on the type of fungicide used. The fungicides which affected the highest number of amino acidswere famoxadone and fenhexamid, while quinoxyfen affected the lowest number of amino acids. Grapestreated with famoxadone contained the lowest concentration of total amino acids.

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1. Introduction

Ammonium, free amino acids and low molecular weight poly-peptides represent assimilable nitrogen, essential for the develop-ment and metabolism of microorganisms. They are important ingrapes of yeast and bacteria for successful alcoholic and/or malo-lactic fermentations (Bely, Sablayrolles, & Barre, 1990; Soufleros,Bouloumpasi, & Tsarchopoulos, 2003).

Nitrogen contents can be manipulated, intentionally or other-wise, by numerous viticultural and oenological factors. Thus, thenitrogen concentration and the amino acid profiles in grapes mayvary, depending on the nature of soil, cultivar, rootstock, nitrogenfertilisation, vineyard health, maturation stages, cultivation sys-tems, etc. (Bell & Henschke, 2005; Garde-Cerdán et al., 2011; Gump,Zoecklein, Fugelsang, & Whiton, 2002; Huang & Ough, 1989;Lamikanra & Kassa, 1999; Lee & Schreiner, 2010). Amino acids influ-ence the wine’s organoleptic properties, as they are transformed intoalcohols, aldehydes, esters, acids, etc. (Guitart, Orte, Ferreira, Pena, &Cacho, 1999; Vilanova et al., 2007). These compounds also serve as acriterion of authenticity or adulteration control (Asensio & Valdés,2002). Finally, if the quality and quantity of amino acids is low, itcould cause a decrease in yeast population and the possibility ofcompetition with other microorganisms may result in unwantedchanges in the wines (Mendes-Ferreira, Mendes-Faia, & Leao,

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2004). In contrast, a high content of amino acids and ammoniumcan lead to a high yeast growth, which could affect wine aroma.Moreover, residual nitrogen in the wine medium could cause prob-lems of microbiological instability (Moreno-Arribas & Polo, 2009).

To achieve proper vineyard health, it is essential to apply syn-thetic pesticides, since in modern agriculture without chemicalcontrol it is difficult to ensure a regular, substantial and qualityproduct, although organic farming is constantly expanding.Although there is extensive literature on the effect of differentagronomic factors on the final concentration of nitrogen com-pounds, no literature data have been found dealing with the influ-ence of pesticide treatments on the profile and concentration offree amino acids and ammonium. There are different chromato-graphic methods for the analysis of amino acids. The originalmethod of post-column ninhydrin derivatisation, followed byion-exchange chromatography and UV detection using amino acidanalysers, has been superseded by faster, more sensitive and versa-tile LC methods using pre-column derivatisation (Peace & Gliani,2005).

The aim of this work was to study the influence of the use ofseveral new fungicides widely used in the vineyard on the aminoacids and ammonium composition of grapes. Individual treatmentsat the recommended doses were performed with the selected fun-gicides under critical agricultural practices (CAP); samples werealso taken from an untreated control plot. Quality controls wereperformed in each analysis sequence to check the robustness ofthe method.

J. Oliva et al. / Food Chemistry 129 (2011) 1676–1680 1677

2. Materials and methods

2.1. Chemicals

The chemical standards used to determine amino acids andammonium were purchased from Sigma–Aldrich (Gillingham,UK). Solutions of amino acids and ammonium were prepared with0.1 N HCl. HPLC-grade acetonitrile and methanol were obtainedfrom Merck (Darmstadt, Germany). Internal standards (2-aminoa-dipic acid) and derivatisation reagent (diethyl (ethoxymethyl-ene)malonate) were purchased from Sigma–Aldrich. Analyticalstandards of the fungicides, of purity P95%, came from Dr. Ehren-storfer (Augsburg, Germany); stock standard solutions of 1 mg/lwere prepared, considering standard purity, by accurately weigh-ing individual analytical standards in volumetric flasks and dis-solving and diluting them to volume with acetonitrile.

Table 1 shows the commercial formulations used in the exper-iment, whose active ingredients are: famoxadone (3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione) with effective preventive effects and broad fungicidal spectrum. Fluquin-conazole (3-(2,4-dichlorophenyl)-6-fluoro-2-(1H-1,2,4-triazol-1-yl)quinazolin-4(3H)-one) with protective, eradicative and systemicproperties; it is used to control Uncinula necator, a fungus thatcauses powdery mildew in grape. Kresoxim-methyl (methyl (E)-methoxyimino(2-(o-tolyloxymethyl)phenyl)acetate) is an oxi-minoacetate (strobilurin type) with protective, curative, eradica-tive and long residual disease control; it is used to control U.necator in grapes. Quinoxyfen (5,7-dichloro-4-quinolyl-4-fluoro-phenyl ether) is a protective fungicide. It is also used in grapesfor the control of U. necator. Fenhexamid (20,30-dichloro-40-hydro-xy-1-methylcyclohexanecarboxanilide) has protective action andis not translocated; it is used to control Botrytis cinerea in grapes.Trifloxystrobin (methyl (E)-methoxyimino((E)-a-(1-(a,a,a-tri-fluoro-m-tolyl)ethylideneaminooxy)-o-tolyl)acetate) is the mainactive ingredient for treating downy and powdery mildews, whichcan be present in grapes and wines.

2.2. Plant materials

Red grapes, Vitis vinifera var. Monastrell, were harvested inOctober 2008, in an experimental plot in Jumilla, Murcia (SE Spain).The nutritional state and physiological conditions of the grapewere suitable to give quality wines.

2.3. Fungicide treatments and sampling

Seven experimental plots of 225 m2 were marked out on thefarm (one control and six for the individual treatments with thefungicides under study). All treatments were applied under criticalagricultural practices (CAP), i.e., 6 h before grape collection. Table 1shows the commercially-used products and the application doses.The experimental plots had not received previous treatment withthe fungicides studied and at the time of application grapes wereexempt from any pesticide residues.

Table 1Fungicide treatments, dose, pre-harvest interval (PHI) and MRL.

Fungicide Commercial name Manufacturers

Famoxadone Equation Pro GR (22.5%) DuPont IbéricaFenhexamid Teldor WG (50%) Bayer HispaniaFluquinconazole Castellan GD (25%) Argos Schering AgrEKresoxim-methyl Stroby WG (50%) BASFQuinoxyfen Arius SC (25%) Dow Agro ScienceTrifloxystrobin Flint WG (50%) Bayer Cropscience

Grape samples (2 kg) from each of the selected plots were col-lected and immediately transferred to a laboratory to be frozenat �30 �C until crushing, homogenisation and analysis.

2.4. Analysis of amino acids and ammonium by HPLC

Analyses of amino acids and ammonium of the whole andcrushed grapes were made using the method described byGarde-Cerdán et al. (2009). Derivatisation of amino acids andammonium was carried out by reaction of 1.75 mL of borate buffer(1 M, pH 9), 750 lL of methanol, 1 mL of sample (previously fil-tered), 20 lL of internal standard (2-aminoadipic acid, 1 g/L) and30 lL of derivatising reagent diethyl (ethoxymethylene)malonate(DEEMM). The derivatisation reaction was carried out in a screw-cap test tube over 30 min in an ultrasonic bath. The sample wasthen heated at 70–80 �C for 2 h to allow complete degradation ofexcess DEEMM and reagent by-products.

The analyses were performed on an Agilent 1100 HPLC (PaloAlto, CA), with a photodiode array detector. Chromatographic sep-aration was performed using an ACE HPLC column (C18-HL; Hi-Chrom, Reading, UK) particle size 5 lm (250 mm � 4.6 mm). Themobile phases used were A, 25 mM acetate buffer, pH 5.8, with0.4 g of sodium azide; B, 80:20 (v/v) mixture of acetonitrile andmethanol. Amino acids were eluted under the following condi-tions: 0.9 mL/min flow rate, 10% B for 20 min, then elution withlinear gradients from 10% to 17% B for 10 min, from 17% to 19% Bin 0.01 min, maintained for 0.99 min, from 19% to 19.5% B in0.01 min, from 19.5% to 23% B in 8.5 min, from 23% to 29.4% Bin 20.6 min, from 29.4% to 72% B in 8 min, from 72% to 82% B in5 min, from 82% to 100% B in 4 min, maintained for 3 min, followedby washing and reconditioning the column. A photodiode arraydetector monitored at 280, 269 and 300 nm was used for detection.The volume of sample injected was 50 lL. The analysis of aminoacids and ammonium was done in duplicate, and two sampleswere taken from each treatment, so the results for amino acidsand ammonium were the means of four analyses. The target com-pounds were identified according to the retention times and UV–Vis spectral characteristics of corresponding derivatised standards.Quantification was done using the calibration curves of the respec-tive standards (r2 > 0.98) in 0.1 N HCl, which underwent the sameprocess of derivatisation as the samples.

2.5. Analysis of fungicide residues

Analytical determination of fluquinconazole was performedwith GC-ECD, while that of famoxadone was with LC-DAD, afterextraction with acetone and ethyl acetate–hexane (1:1 v/v) (Oliva,Payá, Cámara, & Barba, 2007). The other fungicides were deter-mined using GC–MS/MS and LC–MS/MS, after extraction for themodified version of the QuEChERS method (Payá et al., 2007).

2.6. Statistical analysis

The data were subjected to statistical treatment. First, a test forhomogeneity of variances (Levenes test), indicative of the type of

Dose (kg/ha) PHI (days) MRL (EU) (mg/kg)

0.4 28 21 14 5

vo 0.4 21 0.50.2 35 10.3 28 10.15 28 5

Table 2Concentration (mg/l) of amino acids and ammonium in grape in the control group and the group treated with different fungicides.

Control Famoxadone(1.72 mg/l)

Fluquinconazole(0.12 mg/l)

Kresoxim-methyl(0.15 mg/l)

Quinoxyfen(0.84 mg/l)

Fenhexamid(2.25 mg/l)

Trifloxystrobin(0.19 mg/l)

Aspartic acid 24.1 ± 0.3 16.7 ± 0.6⁄ 22 ± 1⁄ 19 ± 2⁄ 19 ± 1⁄ 18.6 ± 0.4⁄ 20.5 ± 0.5⁄

Glutamic acid 83 ± 2 58 ± 3⁄ 64 ± 2⁄ 59 ± 4⁄ 63 ± 3⁄ 58 ± 3⁄ 70 ± 5⁄

Serine 50 ± 1 42 ± 1⁄ 43.4 ± 0.6⁄ 46 ± 3⁄ 45 ± 6 39 ± 2⁄ 51 ± 4Histidine 20.9 ± 0.4 20.0 ± 0.5 22 ± 1 22 ± 5 22 ± 3 19.6 ± 0.4⁄ 23.3 ± 0.8⁄

Glycine 5.4 ± 0.4 5.8 ± 0.1 5.1 ± 0.1 5.2 ± 0.3 5.0 ± 0.5 5.4 ± 0.4 5.3 ± 0.3Threonine 46 ± 2 40 ± 1⁄ 44.1 ± 0.8 44 ± 5 43 ± 7 39 ± 2⁄ 46 ± 3Arginine 256 ± 11 192 ± 4⁄ 237 ± 20 235 ± 35 218 ± 37 230 ± 4⁄ 238 ± 3⁄

Alanine 130 ± 3 122 ± 3⁄ 121 ± 4⁄ 126 ± 2 115 ± 6⁄ 122 ± 5 136 ± 10Proline 5.0 ± 0.4 3.5 ± 0.5⁄ 3.5 ± 0.7⁄ 6 ± 4 4 ± 2 3 ± 1⁄ 2.1 ± 0.4⁄

Tyrosine 9.0 ± 0.5 8.5 ± 0.3 8.7 ± 0.4 9.3 ± 0.4 9 ± 1 8.8 ± 0.7 9.0 ± 0.4Ammonium 48.9 ± 0.8 44 ± 2⁄ 48.6 ± 0.7 50 ± 1 47 ± 2 48 ± 2 47 ± 5Valine 21.8 ± 0.6 21.4 ± 0.8 20.4 ± 0.5 20 ± 3 21 ± 2 18 ± 1⁄ 22 ± 2Methionine 5.4 ± 0.4 5.2 ± 0.3 5.8 ± 0.2 6.0 ± 0.6⁄ 5.3 ± 0.2 5.6 ± 0.1 5.0 ± 0.3Isoleucine 23.9 ± 0.7 22.4 ± 0.5⁄ 20.8 ± 0.9⁄ 22 ± 2 23.0 ± 0.4 20.3 ± 0.8⁄ 22 ± 1⁄

Leucine 17 ± 1 16.2 ± 0.5 16.3 ± 0.7 16 ± 3 17 ± 2 15.9 ± 0.7 17.0 ± 0.6Phenylalanine 17.6 ± 0.8 15.0 ± 0.7⁄ 16.7 ± 0.5 16 ± 3 17 ± 1 15 ± 1⁄ 18.0 ± 0.7Lysine 4.8 ± 0.2 3.8 ± 0.1⁄ 5.1 ± 0.9 4.0 ± 0.8 4.2 ± 0.4 4.1 ± 0.4⁄ 4.3 ± 0.1⁄

Total aminoacids

721 ± 20 593 ± 8⁄ 656 ± 34⁄ 656 ± 67 631 ± 74 623 ± 20⁄ 689 ± 31

For each compound the asterisk denotes the existence of significant differences to the control (p 6 0.05). The concentrations are shown with their standard deviations (n = 4).

1678 J. Oliva et al. / Food Chemistry 129 (2011) 1676–1680

analysis to use (parametric or non-parametric), was applied. As afunction of results for all the parameters studied, except for methi-onine and glycine, a non-parametric analysis (Mann–Whitney–Wilcoxon test) was carried out. For the rest, parametric analysisof the variance (one-way ANOVA) was performed by applyingthe test of significant minor difference (SMD). The treatment wasperformed using SPSS 15.0 software application for Windows (SPSSInc., Chicago, IL).

3. Results and discussion

Table 2 shows the concentrations of amino acids and ammo-nium in the control grapes and in those treated with six differentfungicides. It also shows the residual values of the fungicides usedwith the harvested grapes. Arginine and alanine were the mostabundant amino acids in all the samples, representing about 50%of total amino acids. These two amino acids are among the mostabundant amino acids in grapes (Bell & Henschke, 2005; Garde-Cerdán et al., 2009). Ammonium levels ranged from 44 to 50 mg/l (Table 2). The ammonium concentration was in the range for thiscation in grape juices, between 19 and 240 mg/l (Garde-Cerdán, Ar-ias-Gil, Marsellés-Fontanet, Ancín-Azpilicueta, & Martín-Belloso,2007). Arginine and ammonium are the yeasts’ principal sourceof nitrogen during fermentation, and alanine is also a good nitro-gen source (Bell & Henschke, 2005; Moreno-Arribas & Polo,2009). However, there was a decrease in the arginine and ammo-nium concentrations for all treated samples, especially for grapeswith famoxadone residues. This decrease does not affect the yeastcount in the grapes or their levels during fermentation (Oliva,Cayuela, et al., 2007). Therefore, this decrease probably will notcause a delay in fermentation, despite there being a minor nitrogensource for the yeasts.

In the cases where significant differences (p 6 0.05) betweenthe concentration of amino acids and ammonium in the controland samples treated with fungicides were found, it was observedthat their concentration in grapes decreased when the treatmentswere carried out, except for histidine in the sample treated withtrifloxystrobin and methionine in the sample treated with kresox-im-methyl, for which the concentrations increased (Table 2). Theseresults are in agreement with Wang, Pawelzik, Weinert, Zhao, andWolf (2004), who observed that amino acid content was mainly

lower in fungicide-treated wheat grains than in untreated grains.The amino acids most affected by the treatments with the six fun-gicides used in this study were aspartic and glutamic acids, sincethese amino acids showed significant differences (p 6 0.05) for allthe grape samples treated with respect to the control, while gly-cine, tyrosine, and leucine concentrations did not change signifi-cantly (p 6 0.05) with the application of any of the six fungicides(Table 2).

The concentration decrease in some amino acids could be due tothe fungicides studied affecting their biosynthesis, whereas forother amino acids the decrease in their concentration could bedue to the decrease of their precursor amino acids. Thus, the datain Table 2 indicate that the presence of fungicides did not affectthe biosynthesis of glycine from glyceric acid, as there are no sig-nificant differences between control and treated samples. How-ever, all fungicides affected the biosynthesis of glutamic acid byamination of a-ketoglutaric acid. Finally, the low formation ofaspartic acid and proline could be due to a strong decrease of glu-tamic acid, which is its precursor amino acid.

The highest number of amino acids (11) affected significantly(p 6 0.05) by the application of fungicides occurred in grapes trea-ted with famoxadone and fenhexamid, while the treatment withquinoxyfen only significantly affected (p 6 0.05) the concentrationof three amino acids in grapes (Table 2). Of the six fungicides ap-plied to the grapes, famoxadone, flunquinconazole, and fenhex-amid, affected significantly (p 6 0.05) the total amino acidconcentration in grapes (Table 2); in all three cases the treatedgrapes had lower concentration of total amino acids than the con-trol grapes.

The arginine and proline concentrations have been used inmany varieties for their genetic differentiation (Huang & Ough,1991; Spayd & Andersen-Bagge, 1996; Stines et al., 2000). There-fore, the variation of the concentration of these amino acids by fun-gicide residues could lead to inadequate genetic differentiation.Another indirect consequence of the effect of these pesticide resi-dues in the grapes is that the decrease in the levels of amino acidscould modify the composition of the wine (Garde-Cerdán & Ancín-Azpilicueta, 2008; Miller, Wolff, Bisson, & Ebeler, 2007; Uglianoet al., 2009). Hernández-Orte, Cacho, and Ferreira (2002) showedthat there is a correlation between the decrease in grape aminoacid contents and the profile of the wine aroma. Finally, the de-crease in the grape concentration of certain amino acids, caused

Table 3Amino acids presenting significant differences on comparing treatments pair-wise.

Amino acids Fam–Flu

Fam–Kre

Fam–Qui

Fam–Fen

Fam–Tri

Flu–Kre

Flu–Qui

Flu–Fen

Flu–Tri

Kre–Qui

Kre–Fen

Kre–Tri

Qui–Fen

Qui–Tri

Fen–Tri

Aspartic acid X X X X X X X XGlutamic acid X X X X XSerine X X X X XHistidine X X X XGlycine X X XThreonine X X X XArginine X X X X XAlanine X X X XProline X X XTyrosine XAmmonium X XValine X X X XMethionine X X X X X X XIsoleucine X X X XLeucinePhenylalanine X X X XLysine X XTotal amino

acidsX X X X

Fam: famoxadone; Flu: fluquinconazole; Kre: kresoxim-methyl; Qui: quinoxyfen; Fen: fenhexamid; and Tri: trifloxystrobin.

J. Oliva et al. / Food Chemistry 129 (2011) 1676–1680 1679

by the presence of fungicides, can affect certain beneficial effects ofamino acids in the grapes, such as lower synthesis of protein, lessresistance to stress, decreased cation chelating power, etc. (Feuil-lat, Charpentier, & Mauhean, 1999).

Table 3 shows the amino acids that presented differences intheir concentrations in the grapes with a treatment with one fun-gicide or another, i.e., there are pair-wise comparisons. The aim ofthis comparison is to determine whether the alterations in the lev-els of amino acids depend on the type of fungicide assayed. Thehighest differences were observed when the treatments were withtrifloxystrobin or with famoxadone, as the treatment with one orother of these two fungicides affected the concentration of 11 ami-no acids of the 16 studied (Table 3). Likewise, the treatments of thegrapevines with famoxadone or fluquinconazole or trifloxystrobinor fenhexamid affected the concentration of nine amino acids. Onthe other hand, the lowest differences were observed betweenkresoxim-methyl and fenhexamid (difference in concentration ofone amino acid, serine), and between quinoxyfen and trifloxystrob-in (difference in concentration of one amino acid, alanine) (Table3). This indicates that the alteration of the amino acids fractionwas not due to the presence of fungicides, but depended on thefungicide type (physical and chemical properties, molecular struc-ture, etc.).

4. Conclusions

Application of fungicides to the vine decreased concentrationsof nitrogenous compounds in grapes. Furthermore, the qualitativeand quantitative effects on the amino acids in the grapes dependon the type of fungicide used. The fungicides which affected thehighest number of amino acids were famoxadone and fenhexamid,while quinoxyfen affected the lowest number of amino acids.Grapes treated with famoxadone contained the lowest concentra-tion of total amino acids. In consequence, the application of differ-ent fungicides could affect fermentation and the aromaticcomposition of wines in different ways.

Acknowledgements

Many thanks for the financial support given by Junta de Comun-idades de Castilla-La Mancha through the FPI scholarship forA.M.M.-G. and also to the Ministerio de Educación y Ciencia for theJuan de la Cierva contract for T.G.-C.

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