José V. Gil

Studies on acetate ester production by non-Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora (Hanseniaspora guilliermondii and H. uvarum) and Pichia (Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non-Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.

Acetate ester formation in wine by mixed cultures in laboratory fermentations.

Two non-Saccharomyces wine yeast strains, Hanseniaspora guilliermondii 11104 and Pichia anomala 10590, selected as good producers of acetate esters when grown on synthetic microbiological medium, have been tested in wine fermentations as mixed cultures together with Saccharomyces cerevisiae. Wines produced using mixed cultures showed levels of acetaldehyde, acetic acid, glycerol and total higher alcohols within the ranges described for wine, whereas an increase in acetate ester concentrations was found. Ethyl acetate was the main ester produced, and isoamyl acetate and 2-phenylethyl acetate made up the next largest group of ester compounds in the wines analysed. H. guilliermondii 11104 was found to be a strong producer of 2-phenylethyl acetate in both pure and mixed cultures whereas S. cerevisiae was the best producer of ethyl esters. Mixed cultures did not influence ethyl ester levels at all.

Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits.

Thirty-eight yeast strains belonging to the genera Candida, Hanseniaspora, Pichia, Torulaspora and Zygosaccharomyces were screened for ester formation on synthetic microbiological medium. The genera Hanseniaspora and Pichia stood out as the best acetate ester producers. Based on the ester profile Hanseniaspora guilliermondii 11027 and 11102, Hanseniaspora osmophila 1471 and Pichia membranifaciens 10113 and 10550 were selected for further characterization of enological traits. When growing on must H. osmophila 1471, which displayed a glucophilic nature and was able to consume more than 90% of initial must sugars, produced levels of acetic acid, medium chain fatty acids and ethyl acetate, within the ranges described for wine. On the other hand, it was found to be a strong producer of 2-phenylethyl acetate. Our data suggest that H. osmophila 1471 is a good candidate for mixed starters, although the possible interactions with S. cerevisiae deserve further research.

Construction of a Genetically Modified Wine Yeast Strain Expressing the Aspergillus aculeatus rhaA Gene, Encoding an -L-Rhamnosidase of Enological Interest

ABSTRACT The Aspergillus aculeatus rhaA gene encoding anα -l-rhamnosidase has been expressed in both laboratory and industrial wine yeast strains. Wines produced in microvinifications, conducted using a combination of the genetically modified industrial strain expressing rhaA and another strain expressing a β-glucosidase, show increased content mainly of the aromatic compound linalool.

The use of transgenic yeasts expressing a gene encoding a glycosyl-hydrolase as a tool to increase resveratrol content in wine.

Resveratrol, a phenolic compound produced in grapes, exhibits properties that may contribute to the reduction of the incidence of coronary heart disease and other human health related processes. Recombinant yeast strains expressing the Aspergillus niger abfB gene encoding an alpha-L-arabinofuranosidase or the Candida molischiana bgIN gene encoding a beta-glucosidase have been used in vinifications as tools to increase the resveratrol content of white wine. Glycosylated resveratrol isomers (trans- and cis-piceid) and free resveratrol isomers (trans- and cis-resveratrol) were detected and quantified in white wines. Wines fermented with the strain expressing BgiN showed an increased amount of total resveratrol derivatives, particularly the non-glycosylated forms.

Quantitative Comparison of Free and Bound Volatiles of Two Commercial Tomato Cultivars (Solanum lycopersicum L.) during Ripening

The changes in the profile of both free and glycosidically bound volatiles were studied in Moneymaker and Raf tomato cultivars during fruit ripening. The concentrations of 20 of 24 and of 27 of 30 compounds detected in the free volatile fraction (FVF) and glycosidically bound fraction (GBF), respectively, differed significantly between cultivars during ripening. Most free and bound volatiles increased during ripening in both cultivars. The contribution of each free volatile compound to the overall aroma was estimated by calculating its log U value, which indicated that only 11 compounds seem to exert a strong influence. Nine volatiles were detected exclusively in the GBF, among them geraniol, beta-citronellol, alpha-terpineol, and trans- and cis-linalool oxides. Nine other compounds were found to be more abundant in the GBF than in the FVF, their absolute levels varying between cultivars and stages of ripening. According to the log U values reached, of these nine compounds, linalool, 3-methyl-1-butanol, trans-2-hexenal, eugenol, and 2-phenylethanol may have an impact on tomato aroma upon release from their glycosidic conjugates. Sugars resulting from the enzymatic hydrolysis of the GBF were detected by high-performance liquid chromatography. Rhamnose was the most abundant followed by arabinose, glucose, and xylose.

Measurement of alcohol acetyltransferase and ester hydrolase activities in yeast extracts

Abstract A method has been developed for measurement in vitro of the simultaneous activities alcohol acetyltransferase (AATase) and ester hydrolase (EHase) in yeast extracts taking into account the possibility of interaction between the two opposite activities or the rapid inactivation of the AATase activity. A mathematical model, including as parameters a first order kinetic constant corresponding to the EHase activity and the inactivation constant of AATase, is proposed for the evaluation of AATase activity. To determine ester concentrations, the Headspace-SPME-GC technique has been used. The method has been successfully applied to three yeast strains belonging to the species Pichia anomala, Pichia heedii and Saccharomyces cerevisiae and the corresponding parameters for AATase and EHase have been calculated.

Evaluation of the Ability of Polyphenol Extracts of Cocoa and Red Grape to Promote the Antioxidant Response in Yeast Using a Rapid Multiwell Assay

Saccharomyces cerevisiae has been used as a model organism to study the capacity of cocoa and red grape extracts to trigger an antioxidant response. A methodology adapted to microtiter plates has been developed to monitor yeast growth after culture preincubation with food ingredients and exposure to oxidative stress by hydrogen peroxide and menadione. This methodology proved effective in measuring the ability of cocoa and red grape extracts to promote an antioxidant response in yeast, and also the prospect of conducting dose-response studies. Additionally, the method has proven useful to perform studies with mutant strains lacking genes that may be related to the mechanism of action underlying the antioxidant properties. Thus, in a single assay, it is possible to elucidate the sensitivity of strains to oxidative stress, the ability of an ingredient to promote an antioxidant response, and the possible implication of certain genes. Results of assays using strain hst3Δ showed that the antioxidant protection provided by exposure to cocoa and red grape extracts was not present in the strain lacking gene HST3 when H2 O2 and menadione were used as oxidizing agents. This effect was previously reported for cocoa extract only, with H2 O2 as stressor. Moreover, the results showed that the mutant strain hst3Δ is more resistant to menadione and H2 O2 in the absence of preincubation with cocoa and red grape extract, hinting at the possible implication of sirtuin Hst3 in the antioxidant cellular response.