Dissipation of three fungicides and their effects on anthocyanins and color of monastrell red wines

The effect of fungicides on fermentation is of paramount importance to control the quality and safety of wines. In this work, the quality (enological parameters, color, phenolic content, antioxidant activity, and fungicide residues) of wines from Monastrell grapes fortified with iprovalicarb, mepanipyrim, and tetraconazole fungicides was evaluated. Along the winemaking process, initial residues of mepanipyrim and tetraconazole were removed in more than 90% while the dissipation of iprovalicarb was around 73%. Significant statistical differences were found in the presence of iprovalicarb and mepanipyrim residues, especially at the highest concentration assayed. For both fungicides, increases in the volatile acidity (between 4 and 8.6 times), the lactic acid content (between 8.6 and 20.5 times), the percentage of polymeric anthocyanins (between 1.3 and 1.7 times), and also a slight increase of the total phenolic index and the total anthocyanin content determined by spectrophotometry were observed. On the contrary, the total monomeric anthocyanins content decreased about 16.3% and 28.6% in the presence of iprovalicarb and mepanipyrim, respectively. These results could be related to a higher development of acetic acid or lactic bacteria in the presence of these fungicides. The color of the final wines was also different in comparison with the control, with a higher yellow component, color intensity, tonality, and hue angle because of pH changes in the medium. Tetraconazole fermentations had a more similar trend to the control wine, probably due to the lower concentration of this fungicide in the grape must at the initial time. No effects on the antioxidant activity was observed for any of the target fungicides. A multivariate statistical analysis was done to view the interrelationships between different variables (color and anthocyanins profile). The obtained model allowed the wines to be separated according to the fungicide treatment applied.Interreg | Ref. 0377_IBERPHENOL_6_EMinisterio de Economía y Competitividad | Ref. AGL2015-66491-C2-1-

Effect of two anti-fungal treatments (metrafenone and boscalid plus kresoxim-methyl) applied to vines on the color and phenol profile of different red wines

The effect of two anti-fungal treatments (metrafenone and boscalid + kresoxim-methyl) on the color and phenolic profile of Tempranillo and Graciano red wines has been studied. To evaluate possible modifications in color and phenolic composition of wines, control and wines elaborated with treated grapes under good agricultural practices were analyzed. Color was assessed by Glories and CIELab parameters. Color changes were observed for treated wines with boscalid + kresoxim-methyl, leading to the production of wines with less color vividness. Phenolic profile was characterized by HPLC analysis. Boscalid + kresoxim-methyl treatment promoted the greatest decrease on the phenolic content in wines.Ministerio de Ciencia e Innovación | Ref. AGL2011-30378-C03-0

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-

Effects of new generation fungicides on the proteome of fermentative yeasts and the phenolic metabolome with influence on the color of red wines

Las uvas destinadas a vinificación pueden contener residuos de fungicidas en el momento de la vendimia como resultado de los tratamientos fitosanitarios que se llevan a cabo habitualmente en el viñedo para contrarrestar ciertas enfermedades fúngicas como el mildeu, el oídio y la botritis. Si bien estos residuos deben estar por debajo de los límites máximos de residuos (LMRs) establecidos en la legislación comunitaria (Reglamento 396/2005), su presencia puede repercutir en la calidad de los vinos obtenidos. Varios estudios científicos han demostrado que la presencia de residuos de fungicidas puede originar cambios en la evolución del proceso fermentativo, así como en los metabolitos generados. Los fungicidas pueden modificar la estructura de las membranas celulares de las levaduras y afectar a su función específica, causando un declive progresivo en la viabilidad de la población y, como consecuencia, la ralentización de la fermentación que, en casos extremos, podría paralizarse (Calhelha et al., 2006). Además, algunos estudios han demostrado que la presencia de residuos de fungicidas en las uvas puede llevar a cambios en el perfil polifenólico y en el color de los vinos obtenidos (Fernández et al., 2001; Oliva et al., 2009). Aunque actualmente se desconocen los mecanismos por los cuales se producen estos cambios, es probable que estén relacionados con cambios en el patrón de expresión genética de la levadura que posteriormente se traducen en cambios en su perfil proteico. Un aumento en el contenido de proteínas de respuesta al estrés, en particular enzimas antioxidantes, así como cambios en la abundancia de otras proteínas relacionadas con el metabolismo de los carbohidratos, la síntesis de proteínas y la degradación de proteínas dañadas ya fue observado por otros autores en presencia de fungicidas (Santos et al., 2009; Gil et al., 2014). Uno de los principales objetivos del presente proyecto es proporcionar un mayor conocimiento sobre la influencia de los fungicidas utilizados en el cultivo de la vid en el producto final, el vino y en el curso del proceso de fermentación. En concreto, se analizará el posible efecto de estas sustancias en las levaduras (microorganismos con un papel esencial durante la fermentación alcohólica del vino) y en la composición fenólica y el color de los vinos obtenidos.As uvas destinadas á vinificación poden conter residuos de funxicidas no momento da vendima como resultado dos tratamentos fitosanitarios que se efectúan habitualmente no viñedo para contrarrestar determinadas enfermidades fúnxicas como o mildeu, o oídio e a botrite. Aínda que estes residuos deben atoparse por debaixo dos límites máximos de residuos (LMRs) establecidos na lexislación comunitaria (Regulamento 396/2005) a súa presenza pode repercutir na calidade dos viños obtidos. Diversos estudos científicos mostraron que a presenza de residuos de funxicidas pode orixinar cambios na evolución do proceso fermentativo así coma nos metabolitos xerados. Os funxicidas poden modificar a estrutura das membranas celulares dos lévedos e afectar á súa función específica, ocasionando unha caída progresiva na viabilidade da poboación e, como consecuencia, a ralentización da fermentación que, en casos extremos, podería paralizarse (Calhelha et al., 2006). Ademais, algúns estudos puxeron de manifesto que a presenza de residuos de funxicidas nas uvas pode dar lugar a modificacións no perfil polifenólico e na cor dos viños obtidos (Fernández et al., 2001; Oliva et al., 2009). Aínda que actualmente se descoñecen os mecanismos polos que estas modificacións teñen lugar, é probable que estean relacionados con cambios no patrón de expresión xenética dos lévedos que posteriormente se traducen en cambios no seu perfil proteico. Un incremento do contido de proteínas de resposta ao estrés, en concreto, de enzimas antioxidantes, así como modificacións na abundancia de outras proteínas relacionadas co metabolismo de carbohidratos, a síntese proteica e a degradación de proteínas danadas xa foi observado por outros autores en presencia de funxicidas (Santos et al., 2009; Gil et al., 2014). Un dos principais obxectivos do presente proxecto é proporcionar un maior coñecemento sobre a influencia que teñen os funxicidas empregados no cultivo da vide sobre o produto final, o viño, e sobre o transcurso do proceso fermentativo. Concretamente, analizarase o posible efecto destas substancias sobre os lévedos (microorganismos cun papel imprescindible durante a fermentación alcohólica do viño) e sobre a composición fenólica e a cor dos viños obtidos.Winemaking grapes may contain residues of fungicides at the time of harvest as a result of phytosanitary treatments that are usually carried out in the vineyard to combat fungal diseases such as mildew, powdery mildew and botrytis. Although these residues must be under the maximum residue levels (MRLs) established by Community legislation (Regulation 396/2005), their presence may have an impact on the quality of the obtained wines. Several scientific studies have shown that the presence of fungicide residues can cause changes in the evolution of the fermentation process, as well as in the metabolites generated. Fungicides can modify the structure of yeast cell membranes and affect their specific function, causing a progressive decline in the viability of the population and, as a consequence, the slowing down of fermentation which, in extreme cases, could stop (Calhelha et al., 2006). In addition, some studies have shown that the presence of fungicide residues in grapes can lead to changes in the polyphenolic profile and the color of the obtained wines (Fernández et al., 2001; Oliva et al., 2009). Although the mechanisms by which these changes occur are currently unknown, it is likely that they are related to changes in the gene expression pattern of the yeast that later result in changes in their protein profile. An increase in the content of proteins of stress response, in particular antioxidant enzymes, as well as changes in the abundance of other proteins related to carbohydrate metabolism, protein synthesis and degradation of damaged proteins has already been observed by other authors in the presence of fungicides (Santos et al., 2009; Gil et al., 2014). One of the main objectives of the present project is to provide a greater knowledge about the influence of the fungicides used in vineyards in the final product, the wine, and in the course of the fermentation process. In particular, the possible effect of these substances on yeasts (microorganisms with an essential role during the wine's alcoholic fermentation) and on the phenolic composition and the color of the obtained wines will be analyzed.Ministerio de Ciencia e Innovación | Ref. AGL2011-30378-C03-01Ministerio de Economía y Competitividad | Ref. AGL2015-66491-C2-1-RComisión Europea. POCTEP| Ref. 0377_IBERPHENOL_6_

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

Effect of Two Anti-Fungal Treatments (Metrafenone and Boscalid Plus Kresoxim-methyl) Applied to Vines on the Color and Phenol Profile of Different Red Wines

The effect of two anti-fungal treatments (metrafenone and boscalid + kresoxim-methyl) on the color and phenolic profile of Tempranillo and Graciano red wines has been studied. To evaluate possible modifications in color and phenolic composition of wines, control and wines elaborated with treated grapes under good agricultural practices were analyzed. Color was assessed by Glories and CIELab parameters. Color changes were observed for treated wines with boscalid + kresoxim-methyl, leading to the production of wines with less color vividness. Phenolic profile was characterized by HPLC analysis. Boscalid + kresoxim-methyl treatment promoted the greatest decrease on the phenolic content in wines

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