Biodisponibilidad in vitro de residuos de fungicidas en vinosinfluencia de su presencia en la capacidad antioxidante y biodisponibilidad de compuestos fenólicos

Resumo

ABSTRACT This paper studies the influence of fungicide residues (metrafenone, boscalid+kresoxim-methyl mix) applied in the field under good agricultural practice conditions (GAP and CAP) and those added in the winery prior to vinification (cyazofamid, fenhexamid and mepanipyrim) on antioxidant activity, phenolic compounds and their bioavailability in wines made from Tempranillo and Graciano grapes (D.O. Ca. Rioja), during the wine harvests of 2012, 2013 and 2014. First the analytical methodology was validated for the determination of fungicides in grape and wine using the QuEChERS multiresidue extraction method with acetonitrile and HPLC-MS/MS (triple quadrupole) analysis up to a maximum quantification of 0.01 ppm, a response linearity (R2 >0,998) and an accuracy (70-110%) and precision with SD< 20%. In the wines made from grapes treated in the field under GAP, the LMR established for grapes were not surpassed. In the Tempranillo varieties under CPA treatments, kresoxim-metyl and metrafenone slightly exceeded the el LMR (1.48 and 0.84 versus 1 and 0.5 ppm, respectively). In the wines fortified in the winery with fenhexamid, mepanipyrim and cyazofamid, the LMR were not exceeded. The presence of fungicide residues in the Tempranillo wines had no significant effect on their antioxidant activity. In the Graciano wines, boscalid+kresoxim-methyl and metrafenone presented significant differences in their antioxidant capacity. Residues of boscalid+kresoxim-methyl, fenhexamid and cyazofamid reduce the total polyphenols (Folin-Ciocalteu index) in Tempranillo. In contrast, metrafenone slightly increases it in Graciano. The most notable phenolic fraction in Tempranillo and Graciano is the antocianos (70.45 and 77.50 % respectively), followed by the hidroxicinnamic derivatives (18.31 and 13.64 %) and flavonols (14.62 and 9.95 %). The stilbenes in Graciano wines show slightly higher percentages than in Tempranillo (6.34 versus 4.98%). The in vitro bioavailability of the fungicides in Graciano wine shows a positive matrix effect with respect to Tempranillo. Dialysis percentages range from 6.1% for kresoxim-metil in Tempranillo to 36.6% for boscalid in Graciano. Mepanipyrim in Tempranillo and boscalid in Graciano present the greatest bioavailability. It is confirmed that the clarification has a slight positive effect on bioavailability. In wines made from grapes treated under CPA, the dialyzation percentages of boscalid and kresoxim-methyl stood between 21 and 31%, while metrafenone did not present dialyzation. No dialyzation occurred for cyazofamid added to the must. It has been shown that fungicide residues affect the bioavailability of both the phenolic fraction and the antioxidant activity, but no clear differences were found in the dialyzation of the phenolic fraction for either wine. The stilbenes were the phenolic compounds that showed the highest percentage of dialyzation (50%). The total polyphenol content after the in vitro digestion of wine fell on average by 10% with respect to the initial content in Tempranillo treated in the field and by more than 18% in the case of cyazofamid added prior to vinification. The results were similar for Graciano, except in the treatments with the boscalid and kresoxim-methyl mix, where the bioavailability of the polyphenols increased by up to 25%. The presence of the fungicides studied affects the bioavailability of the antioxidant activity of the Tempranillo wines, but not that of the Graciano ones. Finally, it has been shown that, in general, for all the pesticides studied the in vitro bioavailability does not exceed 37% dialyzation with respect to the initial concentration present in the wine, which indicates a clear safety margin in toxicological terms.