Analytical and Chemometric Characterization of Fino and Amontillado Sherries during Aging in Criaderas y Solera System

Fino and Amontillado are Sherry wines, produced in Marco de Jerez area (southern Spain), and aged in Criaderas y Solera system. Fino Sherry wine follows a biological aging process, under a veil of flor yeasts, while Amontillado Sherry wine shares the same biological aging firstly, followed by oxidative aging, which gives them special features. Organic acids, esters, higher alcohols, phenolic compounds and total dry extract of Sherries evolve during aging due to evaporation processes, physical-chemical reactions, wood contributions and microbiological activity. During aging, Sherry wines improve their organoleptic profile, as could be proved in the tasting sessions. Hierarchical Cluster Analysis and Factor Analysis with factor extraction using Principal Components of Sherry wines studied were carried out and natural groupings of the wines according to the type of aging and their age were observed. A strong correlation between the parameters analyzed and the aging of each wine has been seen in the Multiple Linear Regression studies, establishing two different models, one for each type of Sherry wine, that, with only four of all the variables studied estimated the wine age with more than 99% of confidence. This constitutes a useful tool to control the age of these Sherry wines in the winery.

Flor Yeasts Rewire the Central Carbon Metabolism During Wine Alcoholic Fermentation

The identification of natural allelic variations controlling quantitative traits could contribute to decipher metabolic adaptation mechanisms within different populations of the same species. Such variations could result from human-mediated selection pressures and participate to the domestication. In this study, the genetic causes of the phenotypic variability of the central carbon metabolism of Saccharomyces cerevisiae were investigated in the context of the enological fermentation. The genetic determinism of this trait was found out by a quantitative trait loci (QTL) mapping approach using the offspring of two strains belonging to the wine genetic group of the species. A total of 14 QTL were identified from which 8 were validated down to the gene level by genetic engineering. The allelic frequencies of the validated genes within 403 enological strains showed that most of the validated QTL had allelic variations involving flor yeast specific alleles. Those alleles were brought in the offspring by one parental strain that contains introgressions from the flor yeast genetic group. The causative genes identified are functionally linked to quantitative proteomic variations that would explain divergent metabolic features of wine and flor yeasts involving the tricarboxylic acid cycle (TCA), the glyoxylate shunt and the homeostasis of proton and redox cofactors. Overall, this work led to the identification of genetic factors that are hallmarks of adaptive divergence between flor yeast and wine yeast in the wine biotope. These results also reveal that introgressions originated from intraspecific hybridization events promoted phenotypic variability of carbon metabolism observed in wine strains.

Metabolic Changes by Wine Flor-Yeasts with Gluconic Acid as the Sole Carbon Source

Gluconic acid consumption under controlled conditions by a Saccharomyces cerevisiae flor yeast was studied in artificial media. Gluconic acid was the sole carbon source and the compounds derived from this metabolism were tracked by endo-metabolomic analysis using a Gas Chromatography-Mass Spectrometry (GC-MSD) coupled methodology. After 6 days, about 30% of gluconic acid (1.5 g/L) had been consumed and 34 endo-metabolites were identified. Metabolomic pathway analysis showed the TCA cycle, glyoxylate-dicarboxylate, glycine-serine-threonine, and glycerolipid metabolic pathway were significantly affected. These results contribute to the knowledge of intracellular metabolomic fluctuations in flor yeasts during gluconic acid uptake, opening possibilities for future experiments to improve their applications to control gluconic acid contents during the production of fermented beverages.

Flor yeasts rewire the central carbon metabolism during wine alcoholic fermentation

The identification of natural allelic variations controlling quantitative traits could contribute to decipher metabolic adaptation mechanisms within different populations of the same species. Such variations could result from man-mediated selection pressures and participate to the domestication. In this study, the genetic causes of the phenotypic variability of the central carbon metabolism Saccharomyces cerevisiae were investigated in the context of the enological fermentation. Carbon dioxide and glycerol production as well as malic acid consumption modulate the fermentation yield revealing a high level of genetic complexity. Their genetic determinism was found out by a multi environment QTL mapping approach allowing the identification of 14 quantitative trait loci from which 8 of them were validated down to the gene level by genetic engineering. Most of the validated genes had allelic variations involving flor yeast specific alleles. Those alleles were brought in the offspring by one parental strain that is closely related to the flor yeast genetic group while the second parental strain is part of the wine group. The causative genes identified are functionally linked to quantitative proteomic variations that would explain divergent metabolic features of wine and flor yeasts involving the tricarboxylic acid cycle (TCA), the glyoxylate shunt and the homeostasis of proton and redox cofactors. Overall, this work led to the identification of genetic factors that are hallmarks of adaptive divergence between flor yeast and wine yeast in the wine biotope. These alleles can also be used in the context of yeast selection to improve oenological traits linked to fermentation yield.

Comparative Study of the Proteins Involved in the Fermentation-Derived Compounds in Two Strains of Saccharomyces cerevisiae during Sparkling Wine Second Fermentation

Sparkling wine is a distinctive wine. Saccharomyces cerevisiae flor yeasts is innovative and ideal for the sparkling wine industry due to the yeasts’ resistance to high ethanol concentrations, surface adhesion properties that ease wine clarification, and the ability to provide a characteristic volatilome and odorant profile. The objective of this work is to study the proteins in a flor yeast and a conventional yeast that are responsible for the production of the volatile compounds released during sparkling wine elaboration. The proteins were identified using the OFFGEL fractionator and LTQ Orbitrap. We identified 50 and 43 proteins in the flor yeast and the conventional yeast, respectively. Proteomic profiles did not show remarkable differences between strains except for Adh1p, Fba1p, Tdh1p, Tdh2p, Tdh3p, and Pgk1p, which showed higher concentrations in the flor yeast versus the conventional yeast. The higher concentration of these proteins could explain the fuller body in less alcoholic wines obtained when using flor yeasts. The data presented here can be thought of as a proteomic map for either flor or conventional yeasts which can be useful to understand how these strains metabolize the sugars and release pleasant volatiles under sparkling wine elaboration conditions.

First Proteomic Approach to Identify Cell Death Biomarkers in Wine Yeasts during Sparkling Wine Production

Apoptosis and later autolysis are biological processes which take place in Saccharomyces cerevisiae during industrial fermentation processes, which involve costly and time-consuming aging periods. Therefore, the identification of potential cell death biomarkers can contribute to the creation of a long-term strategy in order to improve and accelerate the winemaking process. Here, we performed a proteomic analysis based on the detection of possible apoptosis and autolysis protein biomarkers in two industrial yeast strains commonly used in post-fermentative processes (sparkling wine secondary fermentation and biological aging) under typical sparkling wine elaboration conditions. Pressure had a negatively effect on viability for flor yeast, whereas the sparkling wine strain seems to be more adapted to these conditions. Flor yeast strain experienced an increase in content of apoptosis-related proteins, glucanases and vacuolar proteases at the first month of aging. Significant correlations between viability and apoptosis proteins were established in both yeast strains. Multivariate analysis based on the proteome of each process allowed to distinguish among samples and strains. The proteomic profile obtained in this study could provide useful information on the selection of wine strains and yeast behavior during sparkling wine elaboration. Additionally, the use of flor yeasts for sparkling wine improvement and elaboration is proposed.

The veil of flor's structure, composition and interactions in biological ageing wines

Biological ageing occurs after fermentation of the grape must and it is due to the appearance of a biofilm on the surface of the wine called “veil of flor”. Yeast involved in veil formation are mainly Saccharomyces cerevisiae and they have traditionally been divided into four races according to their ability to metabolize different sugars. The growth of flor yeasts depends on different factors, such as the aerobic assimilation of the wine ethanol, since the medium is deficient in both sugars and nitrogen. Actually, flor yeast metabolism is different from wine S. cerevisiaeyeast, but it hasn't been analysed yet. Thus, the aim of this work is to study the diversity of flor yeast strains and to analyse the composition and the structure of the veil of flor in Jerez-Xérés-Sherry D.O. The results of this work revealed 14 different genotypes of S. cerevisiaestrains using multiplex-microsatellite PCR and these strains showed 8 different biochemical profiles using a similar procedure than traditionally. In addition, mannose and glucose were found in veil of flor complex using UHPLC-MS.

Detection of protein markers for autophagy, autolysis and apoptosis processes in aSaccharomyces cerevisiaewine flor yeast strain when forming biofilm

Yeast autophagy, autolysis and apoptosis are triggered by nutrient starvation conditions that usually take place in winemaking. Biological aging of Sherry wines constitutes an enological environment suitable for the induction of these biological processes due to the scarcity of nutrients and formation of yeast social communities, i.e. biofilm; however, few studies have been carried out in this regard. Here, we perform a proteomic analysis to detect any autolysis/autophagy/apoptosis protein markers and/or proteins potentially related to these processes under flor forming and fermentative conditions. The scarce presence of autophagy proteins in flor biofilm forming conditions, the existence of autophagy inhibitors (e.g. Pph21p), and high quantity of crucial proteins for autolysis and apoptosis, Pep4p and Mca1p, respectively; indicate that autophagy may be silenced while autolysis and apoptosis are activated when the yeasts are forming flor. This is the first time that autophagy, autolysis and apoptosis have been studied as a whole in flor yeast to our knowledge.ImportanceFlor yeasts areSaccharomyces cerevisiaestrains traditionally used in winemaking and have the ability to survive under starvation conditions and form biofilm. These capabilities make flor yeast interesting organisms to study the biological processes of autophagy, autolysis and apoptosis. With this work, we aim to seek for evidences—protein markers— of these processes in a flor yeast when subjected to biofilm forming and fermentative conditions. Our results suggest that while autophagy may be silenced under biofilm conditions, autolysis and apoptosis are activated. The data provided improve the knowledge of yeast behavior under different enological conditions and can further improve quality of wines in a near future.