Food Chemistry 146 (2014) 234–241

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Influence of new generation fungicides on Saccharomyces cerevisiae growth, grape must fermentation and aroma biosynthesis R. Noguerol-Pato, A. Torrado-Agrasar, C. González-Barreiro, B. Cancho-Grande, J. Simal-Gándara ⇑ Nutrition and Bromatology Group, Faculty of Food Science and Technology, University of Vigo, Ourense Campus, E32004 Ourense, Spain

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Article history: Received 27 May 2013 Received in revised form 6 September 2013 Accepted 9 September 2013 Available online 19 September 2013 Keywords: New fungicides Yeast growth Alcoholic fermentation Wine volatiles

a b s t r a c t The influence of ten new generation fungicides (ametoctradin, benthiavalicarb-isopropyl, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone, and pyraclostrobin) on the fermentative activity of Saccharomyces cerevisiae yeast was initially evaluated in pasteurised red must. The presence of ametoctradin, dimethomorph and mepanipyrim seemed to affect sugars-to-ethanol yield in the stationary phase. The same fermentation experiments were carried out for these three fungicides in ecological red must from Vitis vinifera cv. Tempranillo. When ecological must was unfiltered, the fermentative activity of yeasts was unaffected by the presence of these selected fungicides. However, when ecological must was filtered beforehand, a slight decrease of biomass and ethanol production (in terms of biomass-to-ethanol yield and sugars-to-ethanol yield, respectively), as well as a decrease in fruity aroma, were registered with respect to the control wine. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The yeast Saccharomyces cerevisiae is a unicellular, non-pathogenic and easy-to-manipulate eukaryotic model system, which is highly useful in the study of basic biological mechanisms common to fungi, plants, animals and humans. Yeasts were also used successfully as an optimal model system for assessing the toxicity of xenobiotic compounds and oxidative stress because of their simplicity, rapidity, reproducibility and low cost (Bi Fai & Grant, 2009; Braconi et al., 2006; Cabral, Viegas, Teixeira, & Sá-Correia, 2003; Kitagawa, Momose, & Iwahashi, 2003; Ribeiro et al., 2000). S. cerevisiae is a key microorganism in the vinification process. The correct use of phytosanitary products applied to control grey mould (Botrytis cinerea), powdery mildew (Erysiphe necator, formerly Uncinula necator) and downy mildew (Plasmopara viticola) can generate fungicide residues in grapes which pass through the winemaking process and are still present in the wine (De MeloAbreu et al., 2006; Edder et al., 2009; Fernández, Oliva, Barba, & Cámara, 2005; Garau et al., 2009; González-Rodríguez, CanchoGrande, Torrado-Agrasar, & Simal-Gándara, 2009; González-Rodríguez, Cancho-Grande, & Simal-Gándara, 2009, 2011). Fungicide residues can lead to modifications in the structure of the cellular membranes of the yeast and affect their specific function, causing sluggish and stuck fermentations (Calhelha, Andrade, Ferreira, & Estevinho, 2006; Navarro, Pérez, Navarro, Mena, & Vela, 2007).

⇑ Corresponding author. Tel.: +34 988 387000; fax: +34 988 387001. E-mail address: [email protected] (J. Simal-Gándara). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.058

Few data have been published about the influence of new generation fungicides on the behaviour of alcoholic fermentation of the must by S. cerevisiae. Cabras, Farris, Fiori, and Pusino (2003) showed that the presence of fenhexamid did not affect the amount of alcohol produced by S. cerevisiae; they also showed that fungicide was sorbed by two constituents of yeast cell walls, chitin and glucan, which were tested as potential adsorbents with affinity for fenhexamid. Cus and Raspor (2008) evaluated the toxicity of pyrimethanil on the growth of wine yeasts using in vivo and in vitro experimentation; this fungicide has an effect on the course and successful conclusion of spontaneous wine fermentation that was correlated with the initial concentration of yeasts in the must. González-Rodríguez, Cancho-Grande, Torrado-Agrasar, et al. (2009) evaluated the influence of tebuconazole on the fermentative activity of S. cerevisiae yeast and Oenococcus oeni; no effect on the alcoholic or malolactic fermentation was observed. Sarris, Kotseridis, Linga, Galiotou-Panayotou, and Papanikolaou (2009) assessed the ethanol production, volatile compound biosynthesis and quinoxyfen removal during the growth of a newly isolated S. cerevisiae strain in pasteurised grape musts enriched with industrial sugars. Although quinoxyfen was added to the fermentation medium in significant concentrations, biomass production seemed to be unaffected by its presence; in contrast, ethanol production, in terms of both maximum absolute values achieved (in g/L) and conversion yields (g/g) on sugar consumed, seemed slightly affected by the added residue. González-Rodríguez, González-Barreiro, et al. (2011) assessed the influence of different concentrations of metiram and pyraclostrobin on the fermentative activity of S. cerevisiae yeast; these fungicides were assayed as pure active

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compounds (single and in combination) and as a commercial formulation which contains both fungicides (55% metiram + 5% pyraclostrobin). The presence of pyraclostrobin pure standard in the culture medium, at the highest concentration evaluated (10 mg/ L), increased the biomass and ethanol production rate. No effect on the alcoholic fermentation was observed for metiram pure standard, due to its low solubility in the synthetic medium. However, a total inhibition of yeast growth was observed in the presence of P40 mg/L of the commercial formulation. The objective of the present work was to evaluate whether the presence of fungicide substances of new generation – ametoctradin, benthiavalicarb, boscalid, dimethomorph, cyazofamid, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and/or pyraclostrobin – can negatively affect yeast growth and alcoholic fermentation of the must by S. cerevisiae. For this purpose, in a screening step, S. cerevisiae yeast growth and alcoholic fermentation course in a pasteurised red must was assessed for testing the toxicity of the ten fungicides. For those fungicides capable of causing significant effects on the sugar consumption and biomass courses as well as on the ethanol production, the same experiments were repeated in unfiltered and filtered ecological musts from Vitis vinifera cv. Tempranillo. 2. Materials and methods 2.1. Chemicals Ametoctradin, benthiavalicarb-isopropyl, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone, and pyraclostrobin pure standards were Pestanal grade purchased from Sigma–Aldrich (Steinheim, Germany). The main chemical properties and mode of action of the target fungicides are shown in Table 1 (FRAC Code List., 2012). Ingredients and culture media (peptone, yeast extract and agar) were obtained from Cultimed (Barcelona, Spain) and Liofilchem (Roseto degli Abruzzi, Italy). Glucose PA-ACS, ethanol 96% PA-ACS, glycerol PA-ACS-ISO and sulphuric acid 95–98% PRS-CODEX were obtained from Panreac (Barcelona, Spain). Fructose, tartaric acid and acetic acid were obtained from Sigma–Aldrich. 2.2. Small apparatus Centrifugation was performed in a Rotina 35R centrifuge (Hettich Zentrifugen, Tuttlingen, Germany). In vitro fermentation assays without agitation were performed in a refrigerated incubator Unimax 2010 (Heidolph, Schwabach, Germany). Agitated incubations were done in a thermostated orbital shaker (Optic Ivymen System, Barcelona, Spain). For absorbance measurements a 6505 UV/Vis spectrophotometer (Jenway, Dunmow, UK) was used. 2.3. In vitro assays: inoculation and fermentation 2.3.1. Yeast inoculation The commercial dry yeast used was Uvaferm YSEO VRB (S. cerevisiae var. cerevisiae, strain VRB) from Lallemand (Montreal, Canada). Firstly, the yeast was rehydrated, as described by the manufacturer, and inoculated on YPD-agar plates (10 g/L glucose, 10 g/L fructose, 20 g/L peptone, 20 g/L yeast extract and 20 g/L agar), which were incubated without agitation at 30 °C for 24 h and kept at 5 °C until use. Inoculum was prepared by transferring cells from a sole colony in the plates to YPD liquid broth (without agar) placed in Erlenmeyer flasks, and incubated in an orbital shaker (150 rpm, 30 °C, 48 h). The exponentially growing cells were separated from the supernatant by centrifugation (5000 rpm/ 15 min), washed twice and suspended in the fungicide-enriched

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must in sufficient amount to reach an initial concentration of 2  107 cell/mL. Water cell suspensions were also prepared for measuring yeast concentration by means of turbidity. For that, calibration curves relating the absorbance (measured in a spectrophotometer at 750 nm) with the biomass of different suspensions of concentrated inoculums were developed; this biomass could be expressed as cell/mL (using a Neubauer chamber) and as g/L (determining dry weights). 2.3.2. Alcoholic fermentation assays All the fungicides were tested in fermentation assays performed in both a commercial red pasteurised must enriched with industrial sugars (Greip, Spain) and an ecological Tempranillo red must, which was or was not previously subjected to a filtering step, to check the effect of the fungicide in absence and presence of the natural solids in suspension. For each fungicide tested, the musts were separately supplemented with a 1% volume of an ethanolic fungicide solution prepared at a concentration enough to provide in the must the maximum residue levels (MRLs) established by European legislation in wine grapes (European Commission, 2008). Supplementation was as follows: Experiment 1, ametoctradin at 5.05 mg/L (MRL of 5 mg/kg); Experiment 2, benthiavalicarbisopropyl at 0.22 mg/L (MRL of 0.2 mg/kg); Experiment 3, boscalid at 4.92 mg/L (MRL of 5 mg/kg); Experiment 4, cyazofamid at 0.49 mg/L (MRL of 0.5 mg/kg); Experiment 5, dimethomorph at 3.09 mg/L (MRL of 3.0 mg/kg); Experiment 6, fenhexamid at 5.02 mg/L (MRL of 5.0 mg/kg); Experiment 7, kresoxim-methyl at 0.98 mg/L (MRL of 1.0 mg/kg); Experiment 8, mepanipyrim at 3.04 mg/L (MRL of 3.0 mg/kg); Experiment 9, metrafenone at 5.11 mg/L (MRL of 5.0 mg/kg); and Experiment 10, pyraclostrobin at 2.0 mg/L (MRL of 2.0 mg/kg). A control experiment, Experiment 11, was also included as a control experiment by adding the same volume of a fungicide-free ethanolic solution. Fermentations were done in semi-aerobic conditions in semicovered 10-mL glass tubes filled with 3 mL of the fungicide enriched musts, inoculated as described before and incubated for 14 days, without agitation, in a thermostatically controlled chamber at 20 °C. Twenty tubes were used for each pesticide tested, considering 10 sampling times and duplicated experiments. 2.3.3. Samplings and analyses Two glass tubes from each experiment were aseptically collected at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 10 days after inoculation. The aliquot was centrifuged at 4000 rpm for 10 min. The supernatants were stored at –20 °C for glucose, fructose and ethanol analyses, and yeast cells were washed twice and re-suspended in water to measure the biomass concentration (as turbidity in a spectrometer at 750 nm). Glucose, fructose and ethanol were measured with a Agilent 1100 high-performance liquid chromatograph equipped with a refractive index detector (Agilent, Santa Clara, CA) and a Teknokroma ICSep ICE-ION-300 column (7.8  300 mm) (Barcelona, Spain), eluted with 0.0085 M H2SO4 at 35 °C and a constant flow-rate of 0.4 mL/min for 40 min with 5 lL sample injection volume. Under these conditions, the retention times were 16.10 min for glucose, 17.48 min for fructose and 34.62 min for ethanol. An aliquot of volatile compounds biosynthesised during the alcoholic fermentation (2 mL) was placed in 5-mL polypropylene screw-capped centrifuge tube and 500 lL of dichloromethane were added. The tube was vigorously homogenised in an ultrasonic bath for 1 min. After phase partitioning (2800g, 10 min) in a centrifuge, the organic phase was dispensed into a vial containing an internal standard (2-octanol at 2 lg/mL). Two microlitres of the organic extract were injected into a Trace GC Thermo Finnigan gas chromatograph (Rodano, Italy) equipped with a PolarisQ ion trap mass selective detector (ITMS), using the instrumental conditions de-

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Table 1 Chemical group, chemical structure, chemical properties and mode of action of the target fungicides grouped according to their biochemical mode of action (MOA) in the biosynthetic pathways of plant pathogens (FRAC, 2012). Fungicide (CAS number) IUPAC name and chemical group MOA: respiration Ametoctradin (865318-97-4) 5-Ethyl-6-octyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine Triazolo-pyrimidine C15H25N5/MW: 275.39/water solubility (20 °C): 0.15 mg l 1 log Kow (pH 7, 20 °C): 4.4 Control on grapes: downy mildew Target site and code: complex III: cytochrome bc1 (ubiquinone reductase) at Q  (unknown) site (C8) Boscalid (188425-85-6) 2-Chloro-N-(4’-chlorobiphenyl-2-yl)nicotinamide Pyridine-carboxamide C18H12Cl2N2O/MW: 343.21/water solubility (20 °C): 4.6 mg l 1 log Kow (pH 7, 20 °C): 2.96 Control on grapes: powdery mildew and grey mould Target site and code: complex II: succinate-dehydrogenase (C2)

Cyazofamid (120116-88-3) 4-Chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulphonamide Cyano-imidazole C13H13ClN4O2S/MW: 324.78/water solubility (20 °C): 0.114 mg l 1 log Kow (pH 7, 20 °C): 3.2 Control on grapes: downy mildew Target site and code: complex III: cytochrome bc1(ubiquinone reductase) at Qi site (C4)

Kresoxim-methyl (143390-89-0) Methyl (E)-methoxyimino[a-(o-tolyloxy)-o-tolyl]acetate Oximino acetates (strobilurin) C18H19NO4/MW: 313.35/water solubility (20 °C): 2.0 mg l 1 log Kow (pH 7, 20 °C): 3.4 Control on grapes: powdery mildew Target site and code: complex III: cytochrome bc1 (ubiquinol oxidase) at Qo site (cyt b gene) (C3)

Pyraclostrobin (175013-18-0) Methyl {2-[1-(4-chlorophenyl)pyrazol-3-yloxymethyl]phenyl}(methoxy)carbamate Methoxy-carbamates (strobilurin) C19H18CIN3O4/MW: 387.8/water solubility (20 °C): 1.9 mg l 1 log Kow (pH 7, 20 °C): 3.99 Control on grapes: downy mildew and powdery mildew Target site and code: complex III: cytochrome bc1 (ubiquinol oxidase) at Qo site (cyt b gene) (C3)

MOA: cell wall biosynthesis Benthiavalicarb (413615-35-7) [(S)-1-{[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamoyl}-2-methylpropyl]carbamic acid Carbamate C15H18FN3O3S/MW: 339.5/water solubility (20 °C): – log Kow (pH 7, 20 °C): – Control on grapes: downy mildew Target site and code: cellulose synthase (H5) Dimethomorph (110488–70-5) (EZ)-4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine Cinnanic acid amide C21H22ClNO4/MW: 387.86/water solubility (20 °C): 28.95 mg l 1 log Kow (pH 7, 20 °C): 2.68

Chemical structure

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Table 1 (continued) Fungicide (CAS number) IUPAC name and chemical group

Chemical structure

Control on grapes: downy mildew Target site and code: cellulose synthase (H5)

MOA: sterol biosynthesis in membranes Fenhexamid (126833–17-8) 2’,3’-Dichloro-4’-hydroxy-1-methylcyclohexanecarboxanilide Hydroxyanilide C14H17Cl2NO2/MW: 302.20/water solubility (20 °C): 20 mg l 1 log Kow (pH 7, 20 °C): 3.51 Control on grapes: grey mould Target site and code: 3-keto reductase, C4-de-methylation (erg27) (G3) MOA: amino acids and protein synthesis Mepanipyrim (110235-47-7) N-(4-Methyl-6-prop-1-ynylpyrimidin-2-yl)aniline Anilino-pyrimidine C14H13N3/MW: 223.27/water solubility (20 °C): 2.08 mg l 1 log Kow (pH 7, 20 °C): 3.28 Control on grapes: grey mould Target site and code: methionine biosynthesis (proposed) (cgs gene) (D1)

MOA: unknown (actin disruption, proposed) Metrafenone (220899-03-6) 3’-Bromo-2,3,4,6’-tetramethoxy-2’,6-dimethylbenzophenone Benzophenone C19H21BrO5/MW: 409.3/water solubility (20 °C): 0.492 mg l log Kow (pH 7, 20 °C): 4.3 Control on grapes: powdery mildew Target site and code: acid disruption (proposed)

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scribed by Noguerol-Pato, González-Barreiro, Cancho-Grande and Simal-Gándara (2011). 2.4. Statistical analysis Statistical analysis was performed with the software package Statgraphics Plus V. 5.1 (Manugistics, Rockville, MD). Significant differences in variables between groups were detected by oneway ANOVA at the 95.0% confidence level. 3. Results and discussion The experimental strategy designed to monitor the interaction of ten new generation fungicides with a S. cerevisiae strain during must fermentation was based on a step-by-step approach: 1. Screening step: During the initial screening for the assessment of the sensitivity of S. cerevisiae strain to several new generation fungicides, separately, a pasteurised red grape must was used. Ten fermentation experiments with one of the fungicides plus one control without fungicides, all in duplicate, with a filtered and pasteurised commercial must were carried out. In order to identify any possible effects, the evolution of glucose and fructose consumption, the biomass production and the ethanol production were registered during these 10-day experiments. Different indices were also used to describe either Saccharomyces growth or must fermentation in both exponential and

stationary phases (see also Table 2). Those fungicides showing significantly higher or lower indices than the control were selected for the following confirmation step. 2. Confirmation step: Run fermentation experiments were done with each of the selected fungicides plus one control without fungicides, all in duplicate, with an ecological Tempranillo red must, before and after filtration to evaluate the effect of suspension of solids in the must. The same measurements were made during the fermentation to calculate again the above indices. 3.1. Screening step: kinetic behaviour of a S. cerevisiae strain grown on pasteurised must 3.1.1. In the exponential phase 3.1.1.1. Glucose and fructose consumption. Assessment of the substrate consumption rate. The consumption of glucose and fructose in the must indicated that the microorganism displayed a higher preference towards glucose consumption with respect to fructose (evolution of individual sugars was not shown). Nevertheless, at the seventh day, both sugars were completely consumed in all experiments. For evaluating the substrate uptake, the sugar consumption rate (rs) was estimated during the exponential growth phase (2– 6 days) in the control experiment (where no fungicide was added) and in the remaining experiments (where each fungicide was added separately to the growth medium at concentrations described previously in the experimental section). As reported by Sarris et al. (2009), most of the fermentations showed comparable

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Table 2 Indices (mean values ± standard deviations) used to describe Saccharomyces cerevisiae exponential and stationary phases.

sugars uptake rates, regardless of the addition of the fungicide to the growth medium (see Table 2). Only in the presence of ametoctradin the sugar was consumed more quickly than in the control. Considering that this fungicide acts as a respiration inhibitor, the higher glucose and fructose consumption rates could be due to the enhancement of the fermentation pathway, which accelerates glycolysis. This is also in accordance with the higher ethanol yield obtained in presence of ametoctradin.

negative effects on the yeast growth and, thus, neither do they affect the fermentative kinetics (they will neither slow down nor stop fermentation). Sarris et al. (2009) evaluated the kinetic behaviour of a newly isolated S. cerevisiae strain (grown on pasteurised grape musts enriched with industrial sugars) after the addition of various concentrations of the fungicide quinoxyfen to the medium and they also concluded that the addition of this fungicide did not seriously inhibit biomass production.

3.1.1.2. Biomass production. Assessment of the maximum specific growth rate. The exponential growth is the period of the highest susceptibility to cell stress (Braconi et al., 2008). For measuring the effect of the fungicides on yeast growth, the maximum specific growth rate (lmax) was evaluated as the slope of the line ln(biomass) = f(t), within the early exponential growth phase. Results in Table 2 showed that the fungicide addition did not appear to affect the specific growth rate except for boscalid (lmax = 1.4, higher than control) and ametoctradin (lmax = 1.16, lower than control). Similar results were published by other authors when they observed that the addition of quinoxyfen (Sarris et al., 2009) and pyrimethanil (Cus & Raspor, 2008) did not seriously inhibit biomass production of S. cerevisiae. Regarding boscalid, the apparent positive effect of the fungicide on yeast growth was also observed previously for metiram and pyraclostrobin (González-Rodríguez, González-Barreiro, et al., 2011). Regarding ametoctradin, the opposite effect on the biomass growth rate observed respect to the control could be also a consequence of the already mentioned inhibition effect of this fungicide on the respiratory pathway in favour of fermentation.

3.1.2.1. Ethanol production. Ethanol is the main metabolite resulting from the fermentative metabolism of the yeast, and the first indicator of correct vinification. There were no major differences in the ethanol production among the series. The ethanol content reached at the end of the fermentation in the presence of fungicides was identical to the control (73.1 g/L). Only for boscalid, dimethomorph and mepanipyrim were the differences significant, although with different consequences. While boscalid increased the ethanol content by 10% with respect to the control, dimethomorph and mepanipyrim decreased the ethanol content by 8% and 10%, respectively. It is not possible to analyse the effect of each fungicide on ethanol production without considering further possible effects on biomass growth and metabolism. In fact, there were different situations among the fungicides with regard to ethanol and biomass production in comparison to the control: similar ethanol but lower biomass production for ametoctradin and higher biomass production for metrafenone, higher ethanol and biomass production for boscalid, lower ethanol and biomass production for dimethomorph, and lower ethanol and higher biomass production for mepanipyrim. The biomass-to-ethanol yield (defined as the ratio ethanol/biomass concentration) and the sugars-to-ethanol yield (defined as the ratio ethanol concentration/sugars consumption) were calculated at the stationary phase (see Table 2) and analysed.

3.1.2. In the stationary phase In the stationary phase yeast cells are physiologically, biochemically and morphologically distinct from exponentially growing cells (Cabral et al., 2003; Werner-Washburne, Brawn, Johnston, & Singer, 1993). In this stationary phase cells are known to be more resistant than exponential phase cells to a number of types of stress, including heat and oxidative stress and long-term maintenance of viability under nutrient-limited conditions (WernerWashburne et al., 1993). Oliva et al. (2007) concluded that none of six fungicides studied (famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfen and trifloxystrobin) produced

3.1.2.2. Biomass-to-ethanol yield (YE/B). This yield, which reflects the fermentative ability of the yeast, was unaffected by the addition of the fungicide to the culture medium when cyazofamid, fenhexamid, kresoxim-methyl and pyraclostrobin were assayed, while the six remaining fungicides showed a slight but significant effect on this parameter. Among these, four fungicides (benthiavalicarb, boscalid, mepanipyrim and metrafenone) caused a yield decrease

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with regard to control, but ametoctradin and dimethomorph increased significantly the ethanol yield of the biomass. This favourable effect is not surprising in the case of ametoctradin considering that this fungicide had already shown an enhancing effect on the fermentative pathway during the exponential phase, increasing sugars consumption rate and lowering the specific growth rate. In effect, ametoctradin is a mitochondrial respiration inhibitor interfering with the complex III (complex bc1) in the electron transport chain of the microorganism, thus limiting ATP synthesis for the anabolic metabolism (Merk et al., 2011). The positive effect of dimethomorph is more difficult to explain, but its interest is limited since this fungicide gave low ethanol levels, as mentioned before, and generated the lowest biomass concentration (5.9 g/L). The favourable effect of toxic compounds at low concentrations has been also observed previously for other xenobiotics and defined as hormesis (Calabrese, Staudenmayer, Stanek, & Hoffmann, 2006; González-Rodríguez, González-Barreiro, et al., 2011). Murado and Vázquez (2007) pointed that many xenobiotic compounds affect certain receptor enzymes with a relatively low substrate specificity which could modify some endogenous substrates, thus being able to alter metabolic equilibrium. The negative effect of benthiavalicarb, boscalid and mepanipyrim is easier to explain considering that their respective modes of action directly affect biomass generation and, consequently, biomass performance and metabolism, through the inhibition of cell wall biosynthesis, respiration, and methionine biosynthesis, respectively (FRAC, 2012). 3.1.2.3. Sugars-to-ethanol yield (YE/S). The ethanol yield (calculated as the ratio ethanol concentration/sugars consumed at the stationary growth phase) was calculated (see Table 2). Dimethomorph and mepanipyrim presented a lower ethanol yield than the control while ametoctradin presented the highest. 3.2. Confirmation step: kinetic behaviour of a S. cerevisiae strain grown on ecological must Ametoctradin, dimethomorph and mepanipyrim in treated cultures were capable of causing significant differences in biomass and ethanol yields with respect to the control at the stationary

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phase of yeasts grown in pasteurised red musts. So they were selected to run identical fermentation experiments described above with an ecological red must from V. vinifera cv. Tempranillo, before and after filtration, in order to evaluate the effect of suspended solids in the must. The findings to note in the second set of experiments were as follows. 3.2.1. Fermentation activity of yeasts Fermentation experiments in unfiltered Tempranillo must seem to indicate that yeasts were not negatively affected by ametoctradin, dimethomorph and/or mepanipyrim (Table 2). This could be due to the fungicide adsorption on solid particles suspended in must. Adsorption of several fungicides (benalaxyl-M, benalaxyl, cyazofamid, famoxadone, iprovalicarb, mandipropamid, pyraclostrobin and valifenalate) onto pomaces and deposits from musts during the initial steps of winemaking was previously confirmed (González-Rodríguez, Cancho-Grande, & Simal-Gándara, 2009; González-Rodríguez, Cancho-Grande, et al., 2011). When the same experiments were carried out in filtered must, no differences in sugar consumption and biomass production were observed in the exponential growth phase. However, at the stationary phase biomass concentration decreased with respect to the control as follows: control > dimethomorph > mepanipyrim > ametoctradin as well as the ethanol content: control = ametoctradin > dimetomorph > mepanipirym. As a consequence, biomass and/or sugars-to-ethanol yields decreased (see Table 2). 3.2.2. Volatile compounds biosynthesis during yeast growth Fermentation activity of yeasts in filtered musts seemed to be affected by fungicide residues in ecological filtered musts. The main purpose of this section was then to examine how the presence of ametoctradin, dimethomorph and/or mepanipyrim residues in filtered musts could also affect the profile of volatile yeast metabolites that contribute to wine aroma. Alcohols and esters are basically the secondary products of fermentation and they provide a background to the aroma of any wine. The contribution of each compound to the aroma of the resulting four wines was estimated via its odour activity value (OAV), also known as the ‘‘aroma index’’, which was calculated

Table 3 Odour descriptors, odour thresholds and OAVs of volatile compounds biosynthesised by Saccharomyces cerevisiae during growth in control and enriched ecological filtered musts.

Within rows, dark grey means the highest differences respect to OAV control wine; medium grey means differences between 30% and 50%; light grey means differences between 15% and 30% and no grey colour means differences lower than 15%. a Escudero, Campo, Fariña, Cacho, and Ferreira (2007). b Moyano et al. (2002); thresholds were calculated in a 14% ethanol–water mixture (v/v) with the pH adjusted to 3.5. c Culleré, Escudero, Cacho, and Ferreira (2004); thresholds were calculated in a 10% ethanol–water mixture containing 5 g/L tartaric acid with the pH adjusted to 3.2. d Li, Tao, Wang, and Zhang (2008); thresholds were calculated in a 12% ethanol–water mixture.

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Fruity * 10

Floral

Alcohol

0

5

10

15

20

25

30

35

40

45

OAV Control Wine

Wine with ametoctradin residues

Wine with dimethomorph residues

Wine with mepanipirym residues

Fig. 1. OAVs of each odorant series in all wines tested.

as the ratio of its concentration in each wine to its odour threshold. Only those compounds with OAV > 1 were deemed active odorants and variations in OAV were used to assess sensory changes in the wines in the presence of fungicide residues. Those volatile compounds biosynthesised during the fermentation with OAV P 1 in all tested wines are grouped in Table 3, together with sensory descriptors and the perception thresholds taken from the literature. Based on their OAVs, only nine volatile compounds can be considered as active odorants. Two alcohols (isoamyl alcohol and 2-phenylethanol), with OAVs greater than unity, are formed from their amino acid precursors in yeast cells and then transferred to the wine (see Table 3). The first compound contributes more markedly to odour intensity than it does to aroma quality, and the second imparts a floral nuance. Similar 2-phenylethanol OAVs resulted in all wines tested, probably because amino acids were similarly consumed by the yeasts in the presence of fungicide residues (MacDonall, Reeve, Ruddlesden, & White, 1984). The same conclusions were previously established by González-Rodríguez, Noguerol-Pato, González-Barreiro, Cancho-Grande, and Simal-Gándara (2011) and González-Álvarez, González-Barreiro, Cancho-Grande, and Simal-Gándara (2012) when they prepared white wines from Godello grapes treated with new-generation fungicides. For those wines with dimethomorph and/or mepanipyrim residues, isoamyl alcohol OAVs decreased slightly. The principal esters of wine are enzymatically synthesised from alcohols and acids by yeasts during alcoholic fermentation. In the presence of the enzyme alcohol acetyltransferase, acetyl-CoA condenses with higher alcohols to form acetate esters (Peddie, 1990). Acyl-CoA formed by fatty acid synthesis or degradation gives fatty ethyl esters by enzymatic ethanolysis (Lambrechts & Pretorius, 2000). In terms of OAV, ethyl butyrate, ethyl caproate, ethyl caprylate, ethyl caprate, ethyl myristate, isoamyl acetate and 2-phenylethyl acetate most markedly contributed to the aroma of the wines with fruity nuances. The fungicide residues might induce some modifications of yeast metabolism due to the marked decrease of isoamyl acetate and ethyl caprylate contents (Table 3). At the same time, volatile aroma compounds with similar descriptors were grouped into an odorant series. It allowed the sensory profile of wine to be established (Moyano, Zea, Moreno, & Medina, 2002) as can be seen in Fig. 1. We used the following odour series for all wines: fruity (represented by isoamyl acetate,

ethyl butyrate, ethyl caproate and ethyl caprylate, ethyl caprate); floral (2-phenylethanol and 2-phenylethyl acetate); and alcohol (isoamyl alcohol and ethyl myristate). Ametoctradin, dimethomorph and/or mepanipyrim residues reduced drastically the fruity nuances due to the decrease of isoamyl acetate and ethyl caprylate contents. An opposite effect was observed by other authors who described that fruity aroma increased in wines with fungicide residues (González-Álvarez et al., 2012; Oliva, Navarro, Barba, Navarro, & Salinas, 1999; Oliva, Zalacain, Payá, Salinas, & Barba, 2008). The reduction of floral and alcohol nuances with fungicide residues was not so marked. 4. Conclusions The sugars-to-ethanol yield of S. cerevisiae yeasts, at the stationary phase, was altered in pasteurised musts spiked beforehand with ametoctradin, dimethomorph and/or mepanipyrim at their corresponding MRLs. The same results were reached when fermentation experiments were repeated in filtered ecological musts. The assessment of volatile compounds biosynthesised by yeasts revealed that these active substances were also capable of reducing the fruity character of the resulting wines. When the filtration process was omitted, no effect was observed in the fermentative activity, probably as a consequence of the adsorption of fungicide residues onto the solid particles of must. Acknowledgements This work was granted by EU FEDER funds and by the Spanish Ministry of Education and Science grant (AGL2011-30378-C0301). R. Noguerol-Pato acknowledges the grant from the Spanish Researchers Resources Program. References Bi Fai, P., & Grant, A. (2009). A comparative study of Saccharomyces cerevisiae sensitivity against eight yeast species sensitivities to a range of toxicants. Chemosphere, 75, 289–296. Braconi, D., Possenti, S., Laschi, M., Geminiani, M., Lusini, P., Bernardini, G., et al. (2008). Oxidative damage mediated by herbicides on yeast cells. Journal of Agricultural and Food Chemistry, 56, 3836–3845. Braconi, D., Sotgiu, M., Millucci, L., Paffetti, A., Tasso, F., Alisi, C., et al. (2006). Comparative analysis of the effects of locally used herbicides and their active

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