Impact of Fungicide Residues on Polymerase Chain Reaction and on Yeast Metabolism

ABSTRACT The indiscriminate use of pesticides on grape crops is harmful for consumers´ healthin "in natura" consumption and in the ingestion of wine and grape juice. During winemaking, a rapid and efficient fermentation stage is critical to avoid proliferation of contaminating microorganisms and to guarantee the product´s quality. Polymerase chain reaction (PCR) has the advantage of detecting these contaminants in the early stages of fermentation. However,this enzymatic reaction may also be susceptible to specific problems, reducing its efficiency. Agricultural practices, such as fungicide treatments, may be a source of PCR inhibiting factors and may also interfere in the normal course of fermentation.The action of the pesticides captan and folpet on PCR and on yeast metabolism was evaluated, once these phthalimide compounds are widely employed in Brazilian vineyards. DNA amplification was only observed at 75 and 37.5 µg/mL of captan concentrations, whereas with folpet, amplification was observed only in the two lowest concentrations tested (42.2 and 21.1µg/mL).Besides the strong inhibition on Taq polymerase activity, phthalimides also inhibited yeast metabolism at all concentrations analyzed.Grape must containing captan and folpet residues could not be transformed into wine due to stuck fermentation caused by the inhibition of yeast metabolism. Non-compliance with the waiting period for phthalimide fungicides may result in financial liabilities to the viticulture sector.The use of yeasts with high fungicide sensitivity should be selected for must fermentation as a strategy for sustainable wine production and to assure that products comply with health and food safety standards.

Characterization of autochthonous wine yeasts isolated in vineyards of the State of Paraná, Brazil / Caracterização de leveduras vínicas autóctones isoladas em vinhedos do Estado do Paraná, Brasil

The transformation of grape must into wine is a complex microbiological process and is the product of the combined action of several genera and species of yeasts, dominated in the intermediate and final stages of fermentation by an alcohol-tolerant Saccharomyces sp. Current assay characterizes 42 autochthonous yeasts, isolated from the state of Paraná, southern Brazil, according to the following oenological properties: H2S production, fermentation rate, flocculation capacity, and killer phenotype (killer, sensitive and neutral characteristics). Current analysis is the first to evaluate killer phenotype in yeasts isolated from the State of Paraná, Brazil. With regard to their oenological traits, the yeasts evaluated were not suitable for winemaking and suggested that, depending on the harvest, the winemakers may face problems during the spontaneous wine production process.

A transformação do mosto de uva em vinho é um processo microbiológico complexo, resultado da ação combinada de diferentes gêneros e espécies de leveduras, no qual, entretanto, prevalece uma levedura, Saccharomyces sp. álcool-tolerante nos estágios intermediário e final da fermentação alcoólica. O objetivo deste trabalho foi caracterizar 42 leveduras autóctones isoladas da região sul do Brasil (Estado do Paraná) de acordo com as seguintes características enológicas: produção de H2S, taxa de fermentação, capacidade de floculação e fenótipo killer (características killer, sensível e neutra). Este estudo é o primeiro a avaliar fenótipo killer em leveduras isoladas do estado do Paraná. Com relação às características enológicas, as leveduras avaliadas não se mostraram promissoras para vinificação, sugerindo que, conforme a safra, o vinicultor pode enfrentar problemas durante o processo de fermentação espontânea das uvas para a elaboração de vinho.

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

The purpose of this work was to study a rapid yeast DNA extraction by boiling and freeze-thawing processes without using chemical reagents or any purification procedures, to obtain a high grade PCR-product. A specific DNA fragment of the 18S region of Dekkera bruxellensis and Saccharomyces cerevisiae was chosen. The described boiling and freeze-thawing protocols generated the PCR-grade product preparations and could be used to process many samples. The amplification of the fragments could be observed after 30 and 35 cycles. These processes of extraction without using any kind of chemical reagents, especial water, and purification procedures proved to be efficient, reproducible, simple, fast, and inexpensive.

Production of functional killer protein in batch cultures upon a shift from aerobic to anaerobic conditions

The aim of this work was to study the production of functional protein in yeast culture. The cells of Saccharomyces cerevisiae Embrapa 1B (K+R+) killed a strain of Saccharomyces cerevisiae Embrapa 26B (K-R-)in grape must and YEPD media. The lethal effect of toxin-containing supernatant and the effect of aeration upon functional killer production and the correlation between the products of anaerobic metabolism and the functional toxin formation were evaluated. The results showed that at low sugar concentration, the toxin of the killer strain of Sacch. cerevisiae was only produced under anaerobic conditions . The system of killer protein production showed to be regulated by Pasteur and Crabtree effects. As soon as the ethanol was formed, the functional killer toxin was produced. The synthesis of the active killer toxin seemed to be somewhat associated with the switch to fermentation process and with concomitant alcohol dehydrogenase (ADH) activity.

Influence of Brettanomyces custersianus upon the activity of Saccharomyces cerevisiae strains during the tumultuous phase of vinification

The aim of this work was to evaluate the influence of Brettanomyces custersianus on the metabolic activity of Saccharomyces cerevisiae during the tumultuous stage of wine production. The Cabernet Sauvignon grape must with the skin was inoculated with individual cultures of Sacch. cerevisiae and with mixed cultures of Sacch. cerevisiae and Br. custersianus. During the 6-day tumultuous phase of fermentation, the highest ethanol production and the highest sugar consumption were obtained with the strains without B. custersianus. Fermentations carried out with the addition of Brettanomyces metabolites, acetic acid and 4-ethylphenol, showed that only the former inhibited the growth of both Sacch. cerevisiae strains used. In some cases, Br. custersianus could affect the rate higher alcohols production and their final concentrations during the tumultuous phase of vinification.

Production of yellow-green fluorescent pigment by Pseudomonas fluorescens

Diversos microrganismos secretam substâncias com alta afinidade por ferro. Estas moléculas, sideróforos, transportam ferro para o interior das células. Como a produção destas moléculas depende da composição do meio, foi avaliada a influência do extrato de levedura (FYE), proveniente de resíduo de cervejaria, com e sem adição de sais minerais, sobre o crescimento de Pseudomonas fluorescens e sobre a formação de pigmento fluorescente verde-amarelado (YGFP). Observou-se que (i) FYE com sacarose (G7) e o extrato de levedura comercial (CYE) possuem um pico bem definido próximo a 260 nm; (ii) FYE, mas não CYE, promove alta formação de YGFP. Os meios de King's, usados para detectar a formação de sideróforo, se comportaram como indutores expressivamente mais fracos de YGFP que o meio FYE. Embora FYE e CYE possam ser usados para o crescimento de P. fluorescens, apenas FYE pode ser usado como meio para a detecção de linhagens formadoras de YGFP.

Produçäo de metabólitos no armazenamento de uvas ®Isabel¼ (Vitis labrusca L.) e sua relaçäo com temperatura e vácuo / Metabolites production during the storage of ®Isabela¼ grapes (Vitis labrusca L.) and its relationship to the temperature and vacuum

Este trabalho teve por objetivo avaliar as transformaçöes fisiológicas que ocorrem na uva ®Isabel¼ (Vitis labrusca L.), em diferentes condiçöes de armazenamento. As uvas foram conservadas em temperatura ambiente (25§C) e sob refrigeraçäo (4§C) e mantidas em dessecadores com aplicaçäo de vácuo (-60kPa), no início do processo de armazenamento, e sem aplicaçäo de vácuo. Os metabólitos produzidos foram analisados por cromatografia gás-líquida. Foi utilizado o delineamento experimental inteiramente casualizado, com duas repetiçöes. A refrigeraçäo reduziu a produçäo de etanol e de metanol. A formaçäo de acetato de etila näo foi afetada pelo vácuo ou pela temperatura isoladamente, mas depende da interaçäo desses dois fatores. A produçäo de etanol dependeu da interaçäo temperatura-vácuo. A aplicaçäo de vácuo aumentou a produçäo de etanol em temperatura ambiente. A refrigeraçäo diminuiu a quantidade de bagas danificadas e a aplicaçäo do vácuo teve um efeito oposto. Os resultados obtidos com a cultivar Isabel sugerem que o procedimento de aplicar vácuo no início do processo de armazenamento de uva pode induzir dano físico às bagas até mesmo sob refrigeraçäo e que, pela correlaçäo observada, a deterioraçäo da uva Isabel armazenada pode ser monitorada, analisando a concentraçäo de etanol, metanol e etanal.