Contribution of critical doses of iprovalicarb, mepanipyrim and tetraconazole to the generation of volatile compounds from Monastrell-based wines

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGThe individual effects of iprovalicarb, mepanipyrim, and tetraconazole on the volatile composition and aromatic profile of Monastrell-based wines were evaluated. To date, no studies about the effect of these fungicides on Monastrell-based wines are available, and the effect on other grape varieties is also unknown. Fungicides were added separately in the cellar to the grape must at two concentration levels (4 and 10 mg/kg for iprovalicarb and mepanipyrim and 1 and 2.5 mg/kg for tetraconazole). The aromatic composition of the final wines was analysed by gas chromatography using flame ionisation and ion trap mass selective detectors. In the presence of fungicides, the most significant variations were observed for isoamyl acetate and 2-phenylethyl acetate (increasing between 20 and 43% compared with the control wine) and ethyl caprate and caprylate (increasing between 12 and 68%). Consequently, treated wines showed a higher global odourant intensity, with increased fresh fruit notes.Ministerio de Economía y Competitividad | Ref. AGL2015-66491-C2-1-RAgencia Estatal de Investigación | Ref. PID2019-105061RB-C2

Mepanipyrim residues on pasteurized red must influence the volatile derived compounds from Saccharomyces cerevisiae metabolism

The impact of mepanipyrim (Mep) and its corresponding commercial formulation (Mep Form) on Saccharomyces cerevisiae metabolites was assessed, separately, by using laboratory-scale wine fermentation assays on pasteurized red must. The presence of Mep did not alter the fermentation course. With regard to volatiles formed at the intracellular level by fermenting yeast cells, Mep residues affected mainly the acetate and ethyl ester biochemical pathways. In particular, the target acetates showed a notorious increment, >90%, in presence of commercial Mep Form at the higher dose assayed. The addition of Mep and Mep Form, at both tested levels, highly increased ethyl caprylate (between 42 and 63%) and ethyl caprate (between 36 and 60%) contents as the same as their respective fatty acid precursors. No important effects were observed on colour and non-volatile pyranoanthocyanins, probably due to the low anthocyanin content characteristic of pasteurized musts.Xunta de Galicia | Ref. EM2013/004POCTEP | Ref. 0377_IBERPHENOL_6_EMinisterio de Economía y Competitividad | Ref. AGL2015-66491-C2-1-

Impact of fungicides on the metabolism of wine yeasts and their implication on the biosynthesis of aroma volatile compounds

Los principios activos de los tratamientos antifúngicos aplicados durante el cultivo de la uva pueden ser transferidos al mosto y, a pesar de reducirse durante el proceso de vinificación, pueden permanecer a niveles residuales en el vino final. La presencia de estos residuos en el mosto, aunque por debajo de sus Límites Máximos Residuales (LMRs) marcados por la legislación europea para uva de vinificación (Reglamento 396/2005 y posteriores modificaciones), podría llegar a influir en la calidad organoléptica de los vinos. Así, el metabolismo de las levaduras puede sufrir alteraciones como consecuencia de la situación de estrés originada por la exposición a residuos de fungicidas durante la etapa de crecimiento, pudiendo retrasarse el inicio de las fermentaciones o incluso producirse paradas fermentativas (Cabras y col., 1987). Por otro lado, dado que las levaduras desempeñan un papel crucial en la formación de los principales compuestos volátiles responsables de la base aromática de los vinos, y en el caso de las levaduras del género Saccharomyces, llegan incluso participar en la liberación de aromas primarios a través de enzimas glucosidasas (Gamero y col., 2014), los residuos de fungicidas pueden afectar a las rutas de biosíntesis de compuestos aromáticos y, alterar, consecuentemente, el perfil sensorial de los vinos (González-Álvarez y col., 2012). Durante la fermentación, las levaduras emplean los nutrientes presentes en el mosto para su desarrollo y crecimiento. El mosto contiene compuestos aromáticos que se encuentran bien en forma libre (olorosos por si mismos) y otros en forma ligada (no volátiles; precursores del aroma). Dentro de los precursores del aroma, podemos distinguir aquellos compuestos específicos (glicosídicos, cisteínicos y glutatiónicos) que pueden originar volátiles olorosos mediante una o dos fragmentaciones de la molécula, quedando aún reconocible la estructura de su precursor (Salinas y col., 2013) y los compuestos inespecíficos, como es el caso de la mayor fuente nitrogenada del mosto: los aminoácidos, precursores metabólicos de los principales compuestos del vino, además de ácidos grasos y carotenoides. El contenido en nitrógeno, en cualquiera de sus formas químicas, orgánica (aminoácidos, péptidos y proteínas) e inorgánica (ión amonio) es muy variable, dependiendo de distintos factores como la variedad de uva, su grado de maduración, características edafoclimáticas y diversos aspectos tecnológicos del proceso de vinificación (Mas y col., 2013). El nitrógeno desempeña un papel importante en muchas de las funciones y procesos biológicos, como el crecimiento y división celular de las levaduras y la biosíntesis de compuestos aromáticos (alcoholes superiores y ésteres de acetato) a través del metabolismo secundario de las levaduras, concretamente mediante la vía catabólica denominada ruta de Ehrlich (descarboxilación y posterior reducción de los α-cetoácidos obtenidos por transaminación de los aminoácidos) (Ayräpää, 1971). Todo lo expuesto hasta ahora, evidencia la triple interacción entre la composición del sustrato fermentable (mosto), levadura empleada y presencia de residuos de fungicidas sobre la biosíntesis de compuestos aromáticos. Sin embargo, en este momento, los mecanismos moleculares de sobreexpresión/represión genética mediante los cuales los fungicidas provocan dichos cambios, no se conocen con exactitud. El principal objetivo del presente proyecto es proporcionar un mayor conocimiento sobre la influencia de los fungicidas habitualmente empleados en el cultivo de la vid sobre el transcurso del proceso fermentativo y el metabolismo secundario de las levaduras y, por lo tanto, sobre el perfil aromático de los vinos obtenidos.Os principios activos dos tratamentos antifúnxicos aplicados durante o cultivo da uva poden ser transferidos ao mosto e, a pesar de verse reducidos polo proceso de vinificación, poden permanecer a niveis traza no viño final. A presenza destes residuos no mosto, aínda que por baixo dos seus Límites Máximos Residuais (LMRs) marcados pola lexislación europea para uva de vinificación (Regulamento 396/2005 e posteriores modificacións), podería chegar a influír na calidade organoléptica dos viños. Así, o metabolismo dos lévedos pode sufrir alteracións como consecuencia da situación de estrés orixinada pola súa exposición a residuos de funxicidas durante a etapa de crecemento, podéndose retrasar o inicio das fermentacións ou incluso producirse paradas fermentativas (Cabras e col., 1987). Por outra banda, dado que os lévedos desempeñan un papel crucial na formación dos principais compostos volátiles responsables da base aromática dos viños, e no caso dos lévedos do xénero Saccharomyces chegan a participar na liberación de aromas primarios a través de enzimas glucosidasas (Gamero e col., 2014), os residuos de funxicidas poden afectar ás rutas de biosíntese de compostos aromáticos e, alterar, consecuentemente, o perfil sensorial dos viños (González-Álvarez e col., 2012). Durante a fermentación, os lévedos empregan os nutrientes presentes no mosto para o seu desenvolvemento e crecemento. O mosto contén compostos aromáticos que se atopan ben en forma libre (olorosos per se) ou en forma ligada (non volátiles; precursores do aroma). Dentro dos precursores do aroma, pódense distinguir aqueles compostos específicos (glicosídicos, cisteínicos e glutatiónicos) que poden orixinar volátiles olorosos mediante unha ou dúas fragmentacións da molécula, quedando aínda recoñecible a estrutura do seu precursor (Salinas e col., 2013) e os compostos inespecíficos, como é o caso da maior fonte nitroxenada do mosto, os aminoácidos (precursores metabólicos dos principais compostos aromáticos do viño), ademais de ácidos graxos e carotenoides. O contido en nitróxeno, en calquera das súas formas químicas, orgánica (aminoácidos, péptidos e proteínas) e inorgánica (ión amonio) é moi variable, dependendo de distintos factores como a variedade de uva, o seu grao de maduración, características edafoclimáticas e diversos aspectos tecnolóxicos do proceso de vinificación (Mas e col., 2013). O nitróxeno desempeña un papel importante en moitas funcións e procesos biolóxicos, como o crecemento e división celular dos lévedos e a biosíntese de compostos aromáticos (alcohois superiores e ésteres de acetato) a través do metabolismo secundario dos lévedos, concretamente mediante a vía catabólica denominada ruta de Ehrlich (descarboxilación e posterior redución dos α-cetoácidos obtidos por transaminación dos aminoácidos) (Ayräpää, 1971). Todo o exposto até o de agora, evidencia a tripla interacción entre a composición do substrato fermentable (mosto), o lévedo empregado e a presenza de residuos de funxicidas sobre a biosíntese de compostos aromáticos. Sen embargo, neste momento, os mecanismos moleculares de sobreexpresión/represión xenética mediante os cales os funxicidas provocan ditos cambios, non se coñecen con exactitude. O principal obxectivo do presente proxecto é proporcionar un maior coñecemento sobre a influenza dos funxicidas habitualmente empregados no cultivo da vide sobre o transcurso do proceso fermentativo e o metabolismo secundario dos lévedos e, polo tanto, sobre o perfil aromático dos viños obtidos.The active substances of antifungal treatments applied during grape cultivation, can be transferred to the must and, despite being reduced during the winemaking process, may remain at small levels in the final wine. The presence of this residues in the must, although still present below their Maximum Residual Limits (LMR) as set by European legislation for vinification grapes (regulation 396/2005 and later amendments), could produce changes in the organoleptic quality of the wines. Thus, the metabolism of yeasts may be altered by exposure to stressful situations such as the presence of fungicides during the growth stage and as a result, it is possible to slow down the beginning of the fermentations or to produce stops in the fermentation process (Cabras et al., 1987). On the other hand, due to the yeasts have a key role in the biosynthesis of aromatic compounds responsible of aromatic base of wines and, in the case of Saccharomyces yeasts, they even participate in the release of primary aromas through glucosidase enzimes (Gamero et al., 2014), the fungicide residues can affect the biosynthetic routes of aromatic compounds and, alter, consequently, the aromatic profile of wines (González-Álvarez et al., 2012). During fermentation process, yeasts use the nutrients present in grape juice for their development and growth. The must contains aromatic compounds that are free form (odorous by themselves) and others in a linked form (non-volatile; aroma precursors). Belonging to precursors of the aroma, it can distinguish those specific compounds (glycosidic, cysteinic and glutationic) that can cause odorous volatiles by one or two fragmentations of the molecule, still recognizable the structure of its precursor (Salinas et al., 2013), and non-specific compounds, as in the case of the largest nitrogen source of the must: the amino acids, metabolic precursors of the main compounds of wine, as well as fatty acids and carotenoids. The nitrogen content, in any of its chemical forma (organic: amino acids, peptides and proteins) and inorganic (ammonium ion) is very variable, depending of maturation, edafoclimatic characteristics and various technological aspects of the winemaking process (Mas et al., 2013). The nitrogen plays a key role in many of the biological functions and processes, as well as the growth and cellular division of the yeasts and the biosynthesis of aromatic compounds (higher alcohols and acetate esters) through secondary metabolism, particularly through the catabolic route named Erhlich (decarboxylation and later reduction of α-ketoacids obtained though transamination of amino acids) (Ayräpää, 1971). Everything exposed up to now, evidences the triple interaction between the composition of the fermentable substrate (must), yeast employed and the presence of fungicide residues on the biosynthesis of aromatic compounds. However, at this time, the molecular mechanisms of genetic overexpression/repression by which fungicides cause such changes are not exactly known. The main objective of this study is to provide greater knowledge about the influence of fungicides commonly used in the cultivation of the vine on the fermentation course and in the secondary metabolism of yeasts and, therefore, on the aromatic profile of obtained wines.Xunta de Galicia | Ref. EM2013/004Ministerio de Economía y Competitividad | Ref. AGL2015-66491-C2-1-

Correction: Noguerol-Pato, R., et al. Effect on the Aroma Profile of Graciano and Tempranillo Red Wines of the Application of Two Antifungal Treatments onto Vines. Molecules 2014, 19, 12173-12193

The authors wish to make the following correction to paper [1], doi:10.3390/molecules190812173, website: http://www.mdpi.com/1420-3049/19/8/12173.[...

Impact of mepanipyrim and tetraconazole in Mencía wines on the biosynthesis of volatile compounds during the winemaking process

The impact of fungicides mepanipyrim (Mep) and tetraconazole (Tetra) and their corresponding commercial formulations (Mep-Form and Tetra-Form) on the aroma composition of wines was assessed. Fungicide residues can affect the biotransformation of aroma precursors from grapes and/or the yeast metabolism. The results confirmed that both maximum residue levels (MRL and 2xMRL) of Mep promoted benzyl alcohol and 4-vinylguaiacol contents; while MRL and 2xMRL of Mep-Form promoted benzene derivatives (benzyl alcohol, benzaldehyde, and trans-isoeugenol), 2-phenylethanol and γ-nonalactone. The addition of Tetra (2xMRL) and Tetra-Form (MRL and 2xMRL) release higher contents of cis-3-hexen-1-ol and ethyl vanillate and affected yeast metabolism related to phenylacetaldehyde, 2-phenylethanol, methionol, capric acid, ethyl 2-methylbutyrate, ethyl isovalerate, ethyl monosuccionate, diethyl succinate and γ-butyrolactone production. Fungicide residues did not display higher variations in global odour activity values with respect to control wines, although some variations on the “floral”, “fruity”, “spicy” and “lactic” nuances could be sensed.Fondo Europeo de Desarrollo RegionalXunta de Galicia | Ref. EM2013/00

The effect of two antifungal commercial formulations on the metabolism of a commercial Saccharomyces cerevisiae strain and their repercussion on fermentation evolution and phenylalanine catabolism

The effect of two commercial formulations (incorporating mepanipyrim and tetraconazole as active substances) on the metabolism of Saccharomyces cerevisiae Lalvin T73™, growing on a synthetic grape must, and their influence on the alcoholic fermentation course and the biosynthesis of volatiles derived from phenylalanine catabolism was studied. No relevant effects were observed for mepanipyrim except for glycerol production. On the contrary, in the presence of tetraconazole many genes and some proteins related to cell cycle progression and mitosis were repressed. This fact could explain the lower biomass concentration and the lower sugar consumption registered for tetraconazole at the end of the study. However, the biomass-to-ethanol yield was higher in connection with the overexpression of the ADH1 gene. The presence of tetraconazole residues seems to accelerate the Ehrlich pathway. These results agree with the overexpression of several genes (BAT1, PDC1, PDC5, ADH1, SFA1, ATF2, PFK1, PFK2 and ARO3) and a higher abundance of two proteins (Gap1p and Atf2p) involved in this metabolic pathway.This work received financial support from European Union FEDER funds and the Spanish Ministry of Economy and Competitiveness (AGL 2015-66491-C2-1-R). T. Sieiro-Sampedro and N. Briz-Cid would like to thank their predoctoral fellowships by the Xunta de Galicia and the Spanish Researchers Resources Program, respectively.Peer reviewe

Tetraconazole alters the methionine and ergosterol biosynthesis pathways in Saccharomyces yeasts promoting changes on volatile derived compounds

The influence of antifungal tetraconazole residues (either as an active substance or as a commercial formulation product) on the fermentative activity of Saccharomyces cerevisiae yeast was evaluated in pasteurized Garnacha red must by using laboratory-scale fermentation assays. The presence of this fungicide promoted a slight decrease in glucose consumption. Volatile fermentative-derived compounds were evaluated in deep. Statistically significant changes were found in methionol (with a mean decrease of around 24%), fatty acids (with increments ranged from 23% to 66%), and ethyl esters (with increases ranged from 23% to 145%) contents when grape musts were enriched with the commercial formulation at both contamination levels assayed. Based on protein mass fingerprinting analysis, it was possible to relate these variations on volatiles content with changes in the activity of several enzymes (Met3p, Met14p, Adh2p, Hmg1p, Erg5p, Erg6p, Erg11p, and Erg20p) involved in the secondary metabolism of yeasts.Ministerio de Economía y Competitividad | Ref. AGL2015-66491-C2-1-RFondo Europeo de Desarrollo RegionalXunta de Galici