Eduardo Dellacassa

Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains

The contribution of yeast fermentation metabolites to the aromatic profile of wine is well documented; however, the biotechnological application of this knowledge, apart from strain selection, is still rather limited and often contradictory. Understanding and modeling the relationship between nutrient availability and the production of desirable aroma compounds by different strains must be one of the main objectives in the selection of industrial yeasts for the beverage and food industry. In order to overcome the variability in the composition of grape juices, we have used a chemically defined model medium for studying yeast physiological behavior and metabolite production in response to nitrogen supplementation so as to identify an appropriate yeast assimilable nitrogen level for strain differentiation. At low initial nitrogen concentrations, strain KU1 produced higher quantities of esters and fatty acids whereas M522 produced higher concentrations of isoacids, gamma-butyrolactone, higher alcohols and 3-methylthio-1-propanol. We propose that although strains KU1 and M522 have a similar nitrogen consumption profile, they represent useful models for the chemical characterization of wine strains in relation to wine quality. The differential production of aroma compounds by the two strains is discussed in relation to their capacity for nitrogen usage and their impact on winemaking. The results obtained here will help to develop targeted metabolic footprinting methods for the discrimination of industrial yeasts.

Essential oils of Mentha pulegium and Mentha rotundifolia from Uruguay

Essential oils obtained by hydrodistillation from leaves of Mentha pulegium L. and Mentha rotundifolia (L.) Huds. from Uruguay were analysed by GC-FID and GC-MS. Oxygen-containing monoterpenes were the main group of constituents in both oils. Pulegone, isomenthone and menthone were the major components in the oil of M. pulegium, whereas piperitenone oxide and (Z)-sabinene hydrate were the major ones in M. rotundifolia. Enantiomerically pure (-)-menthone, (+)-isomenthone, (+)-isomenthol, (-)-menthol and (+)-pulegone were detected by multidimensional gas chromatography in the case of M. pulegium oil.

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 winemakers 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.

Growth of non-Saccharomyces yeasts affects nutrient availability for Saccharomyces cerevisiae during wine fermentation.

Yeast produces numerous secondary metabolites during fermentation that impact final wine quality. Although it is widely recognized that growth of diverse non-Saccharomyces (NS) yeast can positively affect flavor complexity during Saccharomyces cerevisiae wine fermentation, the inability to control spontaneous or co-fermentation processes by NS yeast has restricted their use in winemaking. We selected two NS yeasts from our Uruguayan native collection to study NS-S. cerevisiae interactions during wine fermentation. The selected strains of Hanseniaspora vineae and Metschnikowia pulcherrima had different yeast assimilable nitrogen consumption profiles and had different effects on S. cerevisiae fermentation and growth kinetics. Studies in which we varied inoculum size and using either simultaneous or sequential inoculation of NS yeast and S. cerevisiae suggested that competition for nutrients had a significant effect on fermentation kinetics. Sluggish fermentations were more pronounced when S. cerevisiae was inoculated 24h after the initial stage of fermentation with a NS strain compared to co-inoculation. Monitoring strain populations using differential WL nutrient agar medium and fermentation kinetics of mixed cultures allowed for a better understanding of strain interactions and nutrient addition effects. Limitation of nutrient availability for S. cerevisiae was shown to result in stuck fermentations as well as to reduce sensory desirability of the resulting wine. Addition of diammonium phosphate (DAP) and a vitamin mix to a defined medium allowed for a comparison of nutrient competition between strains. Addition of DAP and the vitamin mix was most effective in preventing stuck fermentations.

The High Polyphenol Content of Grapevine Cultivar Tannat Berries Is Conferred Primarily by Genes That Are Not Shared with the Reference Genome

The Tannat grape berry is used to produce high-quality wines with an intense purple color and remarkable antioxidant properties. Through reference-guided assembly of the genome combined with de novo assembly of the transcriptome, we found that the variety-specific genes that might contribute substantially to the unique characteristics of the Tannat berry are not present in the reference genome. The grapevine (Vitis vinifera) cultivar Tannat is cultivated mainly in Uruguay for the production of high-quality red wines. Tannat berries have unusually high levels of polyphenolic compounds, producing wines with an intense purple color and remarkable antioxidant properties. We investigated the genetic basis of these important characteristics by sequencing the genome of the Uruguayan Tannat clone UY11 using Illumina technology, followed by a mixture of de novo assembly and iterative mapping onto the PN40024 reference genome. RNA sequencing data for genome reannotation were processed using a combination of reference-guided annotation and de novo transcript assembly, allowing 5901 previously unannotated or unassembled genes to be defined and resulting in the discovery of 1873 genes that were not shared with PN40024. Expression analysis showed that these cultivar-specific genes contributed substantially (up to 81.24%) to the overall expression of enzymes involved in the synthesis of phenolic and polyphenolic compounds that contribute to the unique characteristics of the Tannat berries. The characterization of the Tannat genome therefore indicated that the grapevine reference genome lacks many genes that appear to be relevant for the varietal phenotype.

The effect of bacterial strain and aging on the secondary volatile metabolites produced during malolactic fermentation of Tannat red wine.

During malolactic fermentation (MLF), lactic acid bacteria influence aroma and flavor of wines by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. In an effort to isolate these bacteria properties as advantages for winemaking, this study aimed to assess the relative contribution of two aspects: the effects of lactic acid bacteria activity on the volatiles compounds in Tannat wines and the consequences of aging in bottle on aroma compounds produced during MLF. To our knowledge, this is the first report related to the effect of wine aging in bottle on the aroma chemical compounds produced by MLF. Solid phase extraction complemented with chromatographic techniques was used to study the wine aroma compounds. A sensory evaluation of the wines was also performed through descriptive methods. We demonstrated modifications in the concentration of acetates, ethyl esters, and other secondary metabolites during MLF. Major sensorial differences between wines that had undergone MLF were also noted. In addition, some modifications detected in the composition of Tannat wines as a consequence of the aging in bottle contributed to the change in differences between wines with and without MLF and furthermore between strains. These changes probably influence its fruity character.

Effect of Saccharomyces cerevisiae inoculum size on wine fermentation aroma compounds and its relation with assimilable nitrogen content.

Different commercial Saccharomyces cerevisiae strains have been applied at the winemaking level, trying to establish a dominant population of selected strains from the start of fermentation and ensuring the complete consumption of sugars. Although a large population of active yeast cells can be introduced in the inoculated wines, resulting in a complete fermentation, this does not necessarily mean an improvement of the sensory characteristics of the wines. The impact of the size of the inocula in wine quality parameters has been very little studied, and in no case the nutrient balance of the grape must utilized was taken into account. In this work we present results obtained for wine aroma compounds at three inoculum levels (10(4), 10(5) and 10(6)cells/mL), and two different yeast assimilable nitrogen (YAN) in a white grape must, using two S. cerevisiae strains commonly used for winemaking. A significant effect in the final concentrations of higher alcohols, esters, fatty acids, free monoterpenes and lactones was attributed to the size of inoculum in both strains but not in an easily predictable way. However, a consistent increase of desired aroma compounds (esters, lactones and free monoterpenes), and a decrease of less desired compounds for white wine (higher alcohols and medium chain fatty acids), was shown at inoculum sizes of 10(5)cells/mL for both strains in real winemaking conditions. In a discriminant analysis six aroma compounds discriminate the three inoculum sizes for all wine samples: 1,8-terpine, hodiol I (trans-3,7-dimethyl-1,5-octadiene-3,7-diol), isobutyl alcohol, iso C4 acid, ethyl C6 ester and C8 acid.

Efeito fungicida dos óleos essenciais de Schinus molle L. e Schinus terebinthifolius Raddi, Anacardiaceae, do Rio Grande do Sul

Uma das alternativas para reduzir o uso de agrotoxicos pode ser a utilizacao de oleos essenciais para o controle de doencas e pragas que afetam a producao agricola. O objetivo do presente estudo foi verificar o efeito fungicida dos oleos essenciais de Schinus molle L. e Schinus terebinthifolius Raddi, especies nativas amplamente distribuidas na regiao. Os oleos foram extraidos por hidrodestilacao e analisados por cromatografia gasosa. Os fungos fitopatogenos Alternaria spp., Botrytis spp., Colletotrichum spp. e Fusarium spp., foram isolados a partir de gerberas e rosas com sintomas de doenca e os ensaios de atividade antifungica foram realizados atraves da tecnica de difusao em disco e os dados analisados pelo teste de Tukey e Duncan a 5%. Em geral o oleo essencial de S. molle foi efetivo para os quatro fungos testados, nas quatro diluicoes utilizadas e para todos os tempos avaliados e o oleo essencial de S. terebinthifolius apresenta efeito fungicida mais pronunciado contra Botrytis spp., a partir de testes in vitro.

Rosmarinus officinalis L. (Labiatae) Essential Oils from the South of Brazil and Uruguay

Abstract Rosemary oils from cultivars of Rosmarinus officinalis L. growing in different areas of Uruguay and South of Brazil (Rio Grande do Sul State) were analyzed by GC and GC/MS. The oils from Uruguay were found to be rich in αpinene (37.8–46.2%) and 1,8-cineole (13.4–13.8%). The oil from R. officinalis which was cultivated in Brazil contained αpinene (32.2%) and 1,8-cineole (14.7%), while the oil from wild plants found in Brazil contained αpinene (12.4%), myrcene (22.7%) and 1,8-cineole (15.3%).

Identification and quantification of 56 targeted phenols in wines, spirits, and vinegars by online solid-phase extraction - ultrahigh-performance liquid chromatography - quadrupole-orbitrap mass spectrometry

Phenolic compounds seriously affect the sensory and nutritional qualities of food products, both through the positive contribution of wood transfer in barrel-aged products and as off-flavours. A new targeted analytical approach combining on-line solid-phase extraction (SPE) clean-up to reduce matrix interference and rapid chromatographic detection performed with ultrahigh-performance liquid chromatography coupled with quadrupole/high-resolution mass spectrometry (Q-Orbitrap), was developed for the quantification of 56 simple phenols. Considering the advantages of using on-line SPE and a resolving power of 140,000, the proposed method was applied to define phenolic content in red (N=8) and white (8) wines, spirits (8), common (8) and balsamic (8) vinegars. The final method was linear from the limits of quantification (0.0001-0.001μgmL(-1)) up to 10μgmL(-1) with R(2) of at least 0.99. Recovery, used to define method accuracy, ranged from 80 to 120% for 89% of compounds. The method was suitable for analytical requirements in the tested matrices being able to analyse 46 phenols in red wines, 41 phenols in white wines and in spirits, 42 phenols in common vinegars and 44 phenols in balsamic vinegars.