Biodiversity and brewing attitude of non-Saccharomyces strains isolated from Uruguayan vineyards and other ecosystems.

In recent years, interest in non-Saccharomyces yeasts for the innovation and development of different and alternative beer styles has been increasing, especially for the microbrewing industry. This work studied the biodiversity of non-Saccharomyces yeasts based on isolates from grapes of different Uruguayan vineyards, craft breweries and raw materials, with the aim of selecting autochthonous non-Saccharomyces yeasts with a brewing attitude. Brewing tests were performed on synthetic wort developed for this purpose, and the evolution of alcoholic fermentation was monitored by measuring glucose, maltose, maltotriose consumption, ethanol and glycerol production and final sensory analysis. A total of two hundred seventy-one yeast strains belonging to different genera were evaluated according to these parameters. After evaluating alcoholic fermentation performance, a native yeast strain belonging to the species Starmerella meliponinorum was selected due to its high maltotriose consumption and glycerol production, making it a very promising brewing yeast, especially for production of low carbohydrate beers.

Effect of Saccharomyces and non-Saccharomyces native yeasts on beer aroma compounds.

Recently, the increase in microbreweries and the consequent production of craft beers have reached exponential growth. The interest in non-conventional yeasts for innovation and a unique selling feature in beer fermentation is increasing. This work studied the autochthonous Saccharomyces and non-Saccharomyces yeasts, isolated from various food sources, with the ability to modify and improve the fermentative and aromatic profiles during alcoholic fermentation. The ability to ferment maltose and produce desirable aroma compounds were considered as the key characters for the screening selection. A synthetic beer wort was developed for this purpose, to simulate beer wort composition. A total of forty-seven yeast strains belonging to different genera were analysed according to their fermentation profile, volatile compounds production and sensory analysis. Three native strains of Saccharomyces cerevisiae, Zygoascus meyerae and Pichia anomala were selected to evaluate their aromatic profile in single and mixed fermentations. The strains produced 4-vinylguaiacol, ß-phenylethyl alcohol, and isoamyl alcohol at levels significantly above the sensory threshold, making them interesting for wheat and blond craft beer styles. The native Hanseniaspora vineae was also included in a co-fermentation treatment, resulting in a promising yeast to produce fruity beers.

Overproduction of isoprenoids by Saccharomyces cerevisiae in a synthetic grape juice medium in the absence of plant genes.

The objective of this work is to demonstrate if the hexaprenyl pyrophosphate synthetase Coq1p might be involved in monoterpenes synthesis in Saccharomyces cerevisiae, although its currently known function in yeast is to catalyze the first step in ubiquinone biosynthesis. However, in a BY4743 laboratory strain, the presence of an empty plasmid in a chemically defined grape juice medium results in a statistically significant increase of linalool, (E)-nerolidol and (E,E)-farnesol. When COQ1 is overexpressed from a plasmid, the levels of the volatile isoprenoids are further increased. Furthermore, overexpression of COQ1 in the same genetic context but with a mutated farnesyl pyrophosphate synthetase (erg20 mutation K197E), results in statistically significant higher levels of linalool (above 750 µg/L), geraniol, α-terpineol, and the sesquiterpenes, farnesol and nerolidol (total concentration of volatile isoprenoids surpasses 1300 µg/L). We show that the levels of monoterpenes and sesquiterpenes that S. cerevisiae can produce, in the absence of plant genes, depend on the composition of the medium and the genetic context. To the best of our knowledge, this is the highest level of linalool produced by S. cerevisiae up to now. Further research will be needed for understanding how COQ1 and the medium composition might interact to increase flavor complexity of fermented beverages.

Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae.

Discovery, characterisation and use of novel yeast strains for winemaking is increasingly regarded as a way for improving quality and to provide variation, including subtle characteristic differences in fine wines. The objective of this work was to evaluate the use of a native apiculate strain, selected from grapes, Hanseniaspora vineae (H. vineae) 02/5A. Fermentations were done in triplicate, working with 225 L oak barrels, using a Chardonnay grape must. Three yeast fermentation strategies were compared: conventional inoculation with a commercial Saccharomyces cerevisiae strain, ALG 804, sequential inoculation with H. vineae and then strain ALG 804 and spontaneous fermentation. Yeast strain identification was performed during fermentation, in which the apiculate strain was found to be active, until 9% of alcohol in volume, for the co-fermentation and the spontaneous fermentation was completed by three native S. cerevisiae strains. Basic winemaking parameters and some key chemical analysis, such as concentration of glycerol, biogenic amines, organic acids, and aroma compounds were analysed. Sensory analysis was done using a trained panel and further evaluated with professional winemakers. Sequential inoculation with H. vineae followed by S. cerevisiae resulted in relatively dry wines, with increased aroma and flavour diversity compared with wines resulting from inoculation with S. cerevisiae alone. Wines produced from sequential inoculations were considered, by a winemaker's panel, to have an increased palate length and body. Characteristics of wines derived from sequential inoculation could be explained due to significant increases in glycerol and acetyl and ethyl ester flavour compounds and relative decreases in alcohols and fatty acids. Aroma sensory analysis of wine character and flavour, attributed to winemaking using H. vineae, indicated a significant increase in fruit intensity described as banana, pear, apple, citric fruits and guava. GC analysis of the relative accumulation of 23 compounds to significantly different concentrations for the three fermentation strategies is discussed in relation to aroma compound composition.

[Analysis of a putative protection against free radicals by grape derivatives (Vitis vinifera L. Cv. Tannat) in Saccharomyces cerevisiae]. / Exploración del efecto protector frente a radicales libres de derivados de la uva (Vitis vinifera L. Cv. Tannat) en Saccharomyces cerevisiae.

The aim of this work was to analyse a possible genome protection provided by a grape derivative (Tannat wine) in yeast cell populations exposed to H2O2. Haploid and diploid strains of Saccharomyces cerevisiae were used as eukaryotic model. Cell samples were exposed to H2O2 in a nutrient medium. Chromosomal DNA was analysed after isolation and separation by pulsed field electrophoresis. Double strand breaks were determined by laser densitometry and application of Poisson distribution. Both haploid and diploid cells showed H2O2 dose dependent DNA fractionation, as well as an increase of lethal -and mutation- events. Upon combination of the Tannat wine and H2O2 a significant decrease of double strand breaks was observed, in association with an increase in surviving fractions. No mutagenic effect was observed after wine exposure. Part of the observations regarding protective wine effect were simulated by exposure to high concentrations of alpha-tocopherol. Present results indicate that a grape derivative could act as a genome protector increasing cell survival probabilities. Among others, the involved molecular targets could be components of transduction redox cascades as well as DNA repair enzymes.

Exploración del efecto protector frente a radicales libres de derivados de la uva (Vitis vinifera L. Cv. Tannat) en Saccharomyces cerevisiae / Analysis of a putative protection against free radicals by grape derivatives (Vitis vinifera L. Cv. Tannat) in Saccharomyces cerevisiae

Se exploró un posible efecto protector del genoma por parte de un derivado de la uva (vino Tannat). Se utilizaron poblaciones celulares haploides y diploides de Saccharomyces cerevisiae como modelo eucariota. Muestras celulares se expusieron a H2O2 en medio nutriente. El ADN se analizó por densitometría láser, luego de su aislamiento y separación por electroforesis con campos pulsados. Se aplicó la distribución de Poisson para la determinación de roturas dobles. El número de roturas dobles del ADN y la frecuencia mutagénica aumentaron en función de la dosis de H2O2, disminuyendo la probabilidad de sobrevida. La combinación de H2O2 con vino Tannat aumentó significa-tivamente la probabilidad de sobrevida y disminuyó el número de roturas dobles. No se observó efecto mutagénico por el vino Tannat. Estos efectos pudieron simularse utilizando altas concentraciones de α-tocoferol. Los resultados indican que un derivado de Vitis vinifera puede, en ciertas condiciones, disminuir las dobles roturas de ADN producidas por el H2O2 e incrementar las probabilidades de sobrevida celular. Los blancos involucrados podrían ser, entre otros, componentes intracelulares de las cascadas redox y/o enzimas de reparación del ADN.

The aim of this work was to analyse a possible genome protection provided by a grape derivative (Tannat wine) in yeast cell populations exposed to H2O2. Haploid and diploid strains of Saccharomyces cerevisiae were used as eukaryotic model. Cell samples were exposed to H2O2 in a nutrient medium. Chromosomal DNA was analysed after isolation and separation by pulsed field electrophoresis. Double strand breaks were determined by laser densitometry and application of Poisson distribution. Both haploid and diploid cells showed H2O2 dose dependent DNA fractionation, as well as an increase of lethal -and mutation- events. Upon combination of the Tannat wine and H2O2 a significant decrease of double strand breaks was observed, in association with an increase in surviving fractions. No mutagenic effect was observed after wine exposure. Part of the observations regarding protective wine effect were simulated by exposure to high concentrations of α-tocopherol. Present results indicate that a grape derivative could act as a genome protector increasing cell survival probabilities. Among others, the involved molecular targets could be components of transduction redox cascades as well as DNA repair enzymes.

Differentiation of industrial wine yeast strains using microsatellite markers.

AIMS: To differentiate nine industrial wine strains of Saccharomyces cerevisiae using microsatellite (simple sequence repeats, SSR) markers. METHODS AND RESULTS: Six of the strains were indigenous yeasts currently used as high-density starter monocultures by the Uruguayan wine industry. Unequivocal differentiation of these six native strains and three commercial S. cerevisiae wine strains was achieved by PCR amplification and polymorphism analysis of loci containing microsatellite markers. CONCLUSION: We recommend the use of this reproducible and simple molecular method to routinely discriminate wine yeast strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Microsatellites are superior to other methods for typing yeasts because the results can be exchanged as quantitative data. Knowledge of the frequencies of the alleles for different SSR markers will eventually lead to an accurate typing method to identify industrial wine yeast strains.

Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.