Fermentative processes for the upcycling of xylose to xylitol by immobilized cells of Pichia fermentans WC1507.

Xylitol is a pentose-polyol widely applied in the food and pharmaceutical industry. It can be produced from lignocellulosic biomass, valorizing second-generation feedstocks. Biotechnological production of xylitol requires scalable solutions suitable for industrial scale processes. Immobilized-cells systems offer numerous advantages. Although fungal pellet carriers have gained attention, their application in xylitol production remains unexplored. In this study, the yeast strain P. fermentans WC 1507 was employed for xylitol production. The optimal conditions were observed with free-cell cultures at pH above 3.5, low oxygenation, and medium containing (NH4)2SO4 and yeast extract as nitrogen sources (xylitol titer 79.4 g/L, YP/S 66.3%, and volumetric productivity 1.3 g/L/h). Yeast cells were immobilized using inactive Aspergillus oryzae pellet mycelial carrier (MC) and alginate beads (AB) and were tested in flasks over three consecutive production runs. Additionally, the effect of a 0.2% w/v alginate layer, coating the outer surface of the carriers (cMC and cAB, respectively), was examined. While YP/S values observed with both immobilized and free cells were similar, the immobilized cells exhibited lower final xylitol titer and volumetric productivity, likely due to mass transfer limitations. AB and cAB outperformed MC and cMC. The uncoated AB carriers were tested in a laboratory-scale airlift bioreactor, which demonstrated a progressive increase in xylitol production in a repeated batch process: in the third run, a xylitol titer of 63.0 g/L, YP/S of 61.5%, and volumetric productivity of 0.52 g/L/h were achieved. This study confirmed P. fermentans WC 1507 as a promising strain for xylitol production in both free- and entrapped-cells systems. Considering the performance of the wild strain, a metabolic engineering intervention aiming at further improving the efficiency of xylitol production could be justified. MC and AB proved to be viable supports for cell immobilization, but additional process development is necessary to identify the optimal bioreactor configuration and fermentation conditions.

Differentiation through E-nose and GC-FID data modeling of rosé sparkling wines elaborated via traditional and Charmat methods.

BACKGROUND: The growing demand for rosé sparkling wine has led to an increase in its production. Traditional or Charmat wine-making influence the aromatic profiles in wine. An analysis such as gas chromatography makes an accurate assessment of wines based on volatile detection but is resource intensive. On the other hand, the electronic nose (E-nose) has emerged as a versatile tool, offering rapid, cost-effective discrimination of wines, and contributing insights into quality and production processes because of its aptitude to perform a global aromatic pattern evaluation. In the present study, rosé sparkling wines were produced using both methods and major volatile compounds and polyols were measured. Wines were tested by E-nose and predictive modelling was performed to distinguish them. RESULTS: Volatile profiles showed differences between Charmat and traditional methods, especially at 5 months of aging. A partial least square discriminant analysis (PLS-DA) was carried out on E-nose detections, obtaining a model that describes 94% of the variability, separating samples in different clusters and correctly identifying different classes. The differences derived from PLS-DA clustering agree with the results obtained by gas-chromatography. Moreover, a principal components regression model was built to verify the ability of the E-nose to non-destructively predict the amount of different volatiles analyzed. CONCLUSION: Production methods of Rosé sparkling wine affect the final wine aroma profiles as a result of the differences in terms of volatiles. The PLS-DA of the data obtained with E-nose reveals that distinguishing between Charmat and traditional methods is possible. Moreover, predictive models using gas chromatography-flame ionization detection analysis and E-nose highlight the possibility of fast and efficient prediction of volatiles from the E-nose. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Microbial diversity in sherry wine biofilms and surrounding mites.

Sherry wines are film wines produced in the Jerez-Xérès-Sherry and Montilla-Moriles regions in southern Spain which require an aging process under flor biofilms, known as "biological aging". The presence of mites in Sherry wine wineries has been reported and associated with improved wine volatile properties. This work analyzes the microbial diversity in flor biofilms and mites in Sherry wine wineries using Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) and ITS/gene amplification. Two mite species, Carpoglyphus lactis and Tyrophagus putrescentiae, were spotted in the sampled winery and 32 microorganism species were identified in their exoskeleton or surrounding biofilms. To our knowledge, 26 of these species were never described before in sherry wine environments. We hypothesized that mites feed on the flor biofilms as well as another type of biofilm located in barrel cracks, known by winemakers as "natas" (cream in English). These non-studied biofilms showed the highest microbiome diversity among all samples (followed by C. lactis spotted nearby) thus, representing a niche of microorganisms with potential biotechnological interest. Besides mites, Drosophila flies were spotted in the sampling areas. The role of flies and mites as vectors that transport microorganisms among different niches (i.e., flor biofilms and natas) is discussed.

Flor yeast immobilization in microbial biocapsules for Sherry wine production: microvinification approach.

Sherry wine is a pale-yellowish dry wine produced in Southern-Spain which features are mainly due to biological aging when the metabolism of biofilm-forming yeasts (flor yeasts) consumes ethanol (and other non-fermentable carbon sources) from a previous alcoholic fermentation, and produces volatile compounds such as acetaldehyde. To start aging and maintain the wine stability, a high alcohol content is required, which is achieved by the previous fermentation or by adding ethanol (fortification). Here, an alternative method is proposed which aims to produce a more economic, distinctive Sherry wine without fortification. For this, a flor yeast has been pre-acclimatized to glycerol consumption against ethanol, and later confined in a fungal-based immobilization system known as "microbial biocapsules", to facilitate its inoculum. Once aged, the wines produced using biocapsules and free yeasts (the conventional method) exhibited chemical differences in terms of acidity and volatile concentrations. These differences were evaluated positively by a sensory panel. Pre-acclimatization of flor yeasts to glycerol consumption was not successful but when cells were immobilized in fungal pellets, ethanol consumption was lower. We believe that immobilization of flor yeasts in microbial biocapsules is an economic technique that can be used to produce high quality differentiated Sherry wines.

Endogenous CO2 Overpressure Effect on Higher Alcohols Metabolism during Sparkling Wine Production.

Higher alcohols produced by yeast during the fermentation of sparkling wine must have the greatest impact on the smell and taste of wine. At present, the metabolic response to methanol and higher alcohols formation of Saccharomyces cerevisiae under endogenous CO2 overpressure has not been fully elucidated. In this work, a proteomics and metabolomics approach using a OFFGEL fractionator and the LTQ Orbitrap for the protein identification, followed by a metabolomic study for the detection and quantification of both higher alcohols (GC-FID and SBSE-TD-GC-MS) and amino acids (HPLC), was carried out to investigate the proteomic and metabolomic changes of S. cerevisiae in relation to higher alcohols formation under a CO2 overpressure condition in a closed bottle. The control condition was without CO2 overpressure in an open bottle. Methanol and six higher alcohols were detected in both conditions, and we have been able to relate to a total of 22 proteins: 15 proteins in the CO2 overpressure condition and 22 proteins in the control condition. As for the precursors of higher alcohols, 18 amino acids were identified in both conditions. The metabolic and proteomic profiles obtained in both conditions were different, so CO2 overpressure could be affecting the metabolism of higher alcohols. Furthermore, it was not possible to establish direct correlations in the condition under CO2 overpressure; however, in the condition without pressure it was possible to establish relationships. The data presented here can be considered as a platform that serves as a basis for the S. cerevisiae metabolome-proteome with the aim of understanding the behavior of yeast under conditions of second fermentation in the production of sparkling wines.

Yeast cell vacuum infusion into fungal pellets as a novel cell encapsulation methodology.

Immobilized yeast cells are used industrially in winemaking processes such as sparkling wine and Sherry wine production. Here, a novel approach has been explored for the infusion and immobilization of yeast cells into filamentous fungal pellets, which serve as a porous natural material. This was accomplished through vacuum application to force the yeast cells towards the core of the fungal pellets followed by culture in YPD medium to promote their growth from the interior. This method represents an improved variation of a previous approach for the assembly of "yeast biocapsules," which entailed the co-culture of both fungal and yeast cells in the same medium. A comparison was made between both techniques in terms of biocapsule productivity, cell retention capacity, and cell biological activity through an alcoholic fermentation of a grape must. The results indicated a substantial increase in biocapsule productivity (37.40-fold), higher cell retention within the biocapsules (threefold), and reduction in cell leakage during fermentation (twofold). Although the majority of the chemical and sensory variables measured in the produced wine did not exhibit notable differences from those produced utilizing suspended yeast cells (conventional method), some differences (such as herbaceous and toasted smells, acidity, bitterness, and persistence) were perceived and wines positively evaluated by the sensory panel. As the immobilized cells remain functional and the encapsulation technique can be expanded to other microorganisms, it creates potential for additional industrial uses like biofuel, health applications, microbe encapsulation and delivery, bioremediation, and pharmacy. KEY POINTS: • New approach improves biocapsule productivity and cell retention. • Immobilized yeast remains functional in fermentation. • Wine made with immobilized yeast had positive sensory differences.

Effect of Bentonite Addition to Pedro Ximénez White Grape Musts before Their Fermentation with Selected Yeasts on the Major Volatile Compounds and Polyols of Wines and Tentative Relationships with the Sensorial Evaluation.

In this work, we study the effect of bentonite addition to the grape must before alcoholic fermentation on the chemical composition and sensorial profile of the obtained wines. Fermentations were carried out with two Saccharomyces cerevisiae commercial active dry yeasts treated or not with bentonite and were compared with a control wine obtained by spontaneous fermentation (using the grape must microbiota). Several significant effects on the chemical and sensorial attributes were established by statistical treatments. The selection by multiple variable analysis of seven volatile molecules (ethyl acetate; methanol; 1-propanol; isobutanol; 2-methyl-1-butanol; 3-metyl-1-butanol and 2-phenylethanol) provided several footprints that provide an easy visualization of bentonite effects on wine volatile compounds. A Principal Component Analysis carried out with all the compounds quantified by Gas-Chromatography revealed that the first two Principal Components explain 60.15 and 25.91%, respectively, of the total variance and established five groups that match with the five wines analyzed. Lastly, predictive models at p ≤ 0.05 level for the attributes sight, smell and taste were obtained by Partial Least Squared regression analysis of selected chemical variables.

Use of yeast biocapsules as a fungal-based immobilized cell technology for Indian Pale Ale-type beer brewing.

Immobilized cell technologies (ICT) have been used in wort fermentation, beer maturation, or production of alcohol-free or low-alcohol beer. The purpose of ICT is to restrict intact cells to a specific location while allowing biological function. It improves cell stability, operational flexibility, and control in brewing, as well as ease in executing continuous operations. We investigated the use of yeast biocapsules for Indian Pale Ale (IPA) type beer wort fermentation, a novel ICT in brewing. Yeast biocapsules are a spherical yeast immobilization system in which yeast cells are encapsulated and connected to the hyphae of an inactivated hollow filamentous fungus pellet. Fermentations with yeast encapsulated in alginate beads, as the standard immobilization practice, and in free (non-immobilized) forms were carried out in parallel. We found that yeast biocapsules are a better option for cell reutilization than alginate beads, but worse for beer must clarity. Beer brewed with yeast biocapsules differed in concentration for five volatile compounds (acetaldehyde, diacetyl, ethyl acetate, 1,1-diethoxyethane, and isoamyl alcohol) and three sensory characters (persistency of the foam, malt, and yeast character). KEY POINTS: • Yeast biocapsules were investigated for beer wort fermentation • Biocapsules improve cell reutilization but are limited for beer clarification • Beer brewed with biocapsules is chemically different than conventional beer • Most sensory features did not differ between biocapsule and control beer.

Assessing Edible Filamentous Fungal Carriers as Cell Supports for Growth of Yeast and Cultivated Meat.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Impact of CO2 overpressure on yeast mitochondrial associated proteome during the "prise de mousse" of sparkling wine production.

The "prise de mousse" stage during sparkling wine elaboration by the traditional method (Champenoise) involves a second fermentation in a sealed bottle followed by a prolonged aging period, known to contribute significantly to the unique organoleptic properties of these wines. During this stage, CO2 overpressure, nutrient starvation and high ethanol concentrations are stress factors that affect yeast cells viability and metabolism. Since mitochondria are responsible for energy generation and are required for cell aging and response to numerous stresses, we hypothesized that these organelles may play an essential role during the prise de mousse. The objective of this study is to characterize the mitochondrial response of a Saccharomyces cerevisiae strain traditionally used in sparkling wine production along the "prise de mousse" and study the effect of CO2 overpressure through a proteomic analysis. We observed that pressure negatively affects the content of mitochondrion-related proteome, especially to those proteins involved in tricarboxylic acid cycle. However, proteins required for the branched-amino acid synthesis, implied in wine aromas, and respiratory chain, also previously reported by transcriptomic analyses, were found over-represented in the sealed bottles. Multivariate analysis of proteins required for tricarboxylic cycle, respiratory chain and amino acid metabolism revealed differences in concentrations, allowing the wine samples to group depending on the time and CO2 overpressure parameters. Ethanol content along the second fermentation could be the main reason for this changing behavior observed at proteomic level. Further research including genetic studies, determination of ROS, characterization of mitochondrial activity and targeted metabolomics analyses is required. The list of mitochondrial proteins provided in this work will lead to a better understanding of the yeast behavior under these conditions of special interest in the wine industry.

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.

Revealing the Yeast Diversity of the Flor Biofilm Microbiota in Sherry Wines Through Internal Transcribed Spacer-Metabarcoding and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Flor yeast velum is a biofilm formed by certain yeast strains that distinguishes biologically aged wines such as Sherry wine from southern Spain from others. Although Saccharomyces cerevisiae is the most common species, 5.8 S-internal transcribed spacer (ITS) restriction fragment length polymorphism analyses have revealed the existence of non-Saccharomyces species. In order to uncover the flor microbiota diversity at a species level, we used ITS (internal transcribed spacer 1)-metabarcoding and matrix-assisted laser desorption/Ionization time of flight mass spectrometry techniques. Further, to enhance identification effectiveness, we performed an additional incubation stage in 1:1 wine:yeast extract peptone dextrose (YPD) before identification. Six species were identified: S. cerevisiae, Pichia manshurica, Pichia membranifaciens, Wickerhamomyces anomalus, Candida guillermondii, and Trichosporon asahii, two of which were discovered for the first time (C. guillermondii and Trichosporon ashaii) in Sherry wines. We analyzed wines where non-Saccharomyces yeasts were present or absent to see any potential link between the microbiota and the chemical profile. Only 2 significant volatile chemicals (out of 13 quantified), ethanol and ethyl lactate, and 2 enological parameters (out of 6 quantified), such as pH and titratable acidity, were found to differ in long-aged wines. Although results show a low impact where the non-Saccharomyces yeasts are present, these yeasts isolated from harsh environments (high ethanol and low nutrient availability) could have a potential industrial interest in fields such as food microbiology and biofuel production.

A Differential Proteomic Approach to Characterize the Cell Wall Adaptive Response to CO2 Overpressure during Sparkling Wine-Making Process.

In this study, a first proteomic approach was carried out to characterize the adaptive response of cell wall-related proteins to endogenous CO2 overpressure, which is typical of second fermentation conditions, in two wine Saccharomyces cerevisiae strains (P29, a conventional second fermentation strain, and G1, a flor yeast strain implicated in sherry wine making). The results showed a high number of cell wall proteins in flor yeast G1 under pressure, highlighting content at the first month of aging. The cell wall proteomic response to pressure in flor yeast G1 was characterized by an increase in both the number and content of cell wall proteins involved in glucan remodeling and mannoproteins. On the other hand, cell wall proteins responsible for glucan assembly, cell adhesion, and lipid metabolism stood out in P29. Over-represented proteins under pressure were involved in cell wall integrity (Ecm33p and Pst1p), protein folding (Ssa1p and Ssa2p), and glucan remodeling (Exg2p and Scw4p). Flocculation-related proteins were not identified under pressure conditions. The use of flor yeasts for sparkling wine elaboration and improvement is proposed. Further research based on the genetic engineering of wine yeast using those genes from protein biomarkers under pressure alongside the second fermentation in bottle is required to achieve improvements.

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.

Biological Processes Highlighted in Saccharomyces cerevisiae during the Sparkling Wines Elaboration.

Sparkling wines elaboration has been studied by several research groups, but this is the first report on analysis of biological processes according to the Gene Ontology terms (GO terms) and related to proteins expressed by yeast cells during the second fermentation of sparkling wines. This work provides a comprehensive study of the most relevant biological processes in Saccharomyces cerevisiae P29, a sparkling wine strain, during the second fermentation under two conditions (without and with endogenous CO2 overpressure) in the middle and the end of second fermentation. Consequently, a proteomic analysis with the OFFGEL fractionator and protein identification with LTQ Orbitrap XL coupled to HPLC were performed. The classification of biological processes was carried out using the tools provided by the Saccharomyces Genome Database. Results indicate that a greater number of biological processes were identified under condition without CO2 overpressure and in the middle of the fermentation versus the end of the second fermentation. The biological processes highlighted under condition without CO2 overpressure in the middle of the fermentation were involved in the carbohydrate and lipid metabolic processes and catabolic and biosynthetic processes. However, under CO2 overpressure, specific protein expression in response to stress, transport, translation, and chromosome organization and specific processes were not found. At the end of fermentation, there were higher specific processes under condition without CO2 overpressure; most were related to cell division, growth, biosynthetic process, and gene transcription resulting in increased cell viability in this condition. Under CO2 overpressure condition, the most representative processes were related to translation as tRNA metabolic process, chromosome organization, mRNA processing, ribosome biogenesis, and ribonucleoprotein complex assembly, probably in response to the stress caused by the hard fermentation conditions. Therefore, a broader knowledge of the adaptation of the yeast, and its behavior under typical conditions to produce sparkling wine, might improve and favor the wine industry and the selection of yeast for obtaining a high-quality wine.

Metaproteomics of microbiota involved in submerged culture production of alcohol wine vinegar: A first approach.

Acetic acid bacteria form a complex microbiota that plays a fundamental role in the industrial production of vinegar through the incomplete oxidation reaction from ethanol to acetic acid. The organoleptic properties and the quality of vinegar are influenced by many factors, especially by the raw material used as acetification substrate, the microbial diversity and the technical methods employed in its production. The metaproteomics has been considered, among the new methods employed for the investigation of microbial communities, since it may provide information about the microbial biodiversity and behaviour by means of a protein content analysis. In this work, alcohol wine vinegar was produced through a submerged culture of acetic acid bacteria using a pilot acetator, operated in a semi-continuous mode, where the main system variables were monitored and the cycle profile throughout the acetification was obtained. Through a first approach, at qualitative level, of a metaproteomic analysis performed at relevant moments of the acetification cycle (end of fast and discontinuous loading phases and just prior to unloading phase), it is aimed to investigate the microbiota existent in alcohol wine vinegar as well as its changes during the cycle; to our knowledge, this is the first metaproteomics report carried out in this way on this system. A total of 1723 proteins from 30 different genera were identified; 1615 out of 1723 proteins (93.73%) belonged to the four most frequent (%) genera: Acetobacter, Gluconacetobacter, Gluconobacter and Komagataeibacter. Around 80% of identified proteins belonged to the species Komagataeibacter europaeus. In addition, GO Term enrichment analysis highlighted the important role of catalytic activity, organic cyclic compound binding, metabolic and biosynthesis processes throughout acetic acid fermentation. These findings provide the first step to obtain an AAB profile at omics level related to the environmental changes produced during the typical semi-continuous cycles used in this process and it would contribute to the optimization of operating conditions and improving the industrial production of vinegar.

Mapping the intracellular metabolome of yeast biocapsules - Spherical structures of yeast attached to fungal pellets.

Co-culture conditions are beneficial for study due to the advances which arise from symbiotic interactions and which cannot be replicated under pure culture conditions. Here, the focus is on the connection between two fungi - a yeast, Saccharomyces cerevisiae, and a filamentous fungus, Penicillium chrysogenum - in a yeast immobilization system termed' yeast biocapsules', where the yeast and filamentous fungus are strongly attached to one another, forming spherical structures. This co-culture condition hinders filamentous fungal biomass growth, while immobilization of yeast cells continues to increase. The effect of the co-culture condition on endometabolites or intracellular metabolites were tracked during the beginning and end of the yeast biocapsule formation period, and metabolites analyzed by Gas Chromatography-Mass Spectrometry Detector (GC-MSD). Distinct metabolite profiles were found between single culture conditions, involving each organism separately, and with the co-culture condition, where there were differences in 54 endometabolites. Specifically, co-culture condition compounds such as fructose, glycolic acid and glyceric acid were present in higher concentrations at the end of biocapsule formation. These results shed light on the mechanisms and biochemical impact of the interaction between the yeast and filamentous fungus and serve as a basis to apply and further develop yeast biocapsules as a new biotechnological tool with benefits for industry.

Autophagic Proteome in Two Saccharomyces cerevisiae Strains During Second Fermentation for Sparkling Wine Elaboration.

A correlation between autophagy and autolysis has been proposed in order to acceleratethe acquisition of wine organoleptic properties during sparkling wine elaboration. In this context, aproteomic analysis was carried out in two industrial Saccharomyces cerevisiae strains (P29,conventional sparkling wine strain and G1, implicated in sherry wine elaboration) with the aim ofstudying the autophagy-related proteome and comparing the effect of CO2 overpressure duringsparkling wine elaboration. In general, a detrimental effect of pressure and second fermentationdevelopment on autophagy-related proteome was observed in both strains, although it was morepronounced in flor yeast strain G1. Proteins mainly involved in autophagy regulation andautophagosome formation in flor yeast G1, and those required for vesicle nucleation and expansionin P29 strain, highlighted in sealed bottle. Proteins Sec2 and Sec18 were detected 3-fold underpressure conditions in P29 and G1 strains, respectively. Moreover, 'fingerprinting' obtained frommultivariate data analysis established differences in autophagy-related proteome between strainsand conditions. Further research is needed to achieve more solid conclusions and design strategiesto promote autophagy for an accelerated autolysis, thus reducing cost and time production, as wellas acquisition of good organoleptic properties.

Differential Analysis of Proteins Involved in Ester Metabolism in two Saccharomyces cerevisiae Strains during the Second Fermentation in Sparkling Wine Elaboration.

The aromatic metabolites derived from yeast metabolism determine the characteristics of aroma and taste in wines, so they are considered of great industrial interest. Volatile esters represent the most important group and therefore, their presence is extremely important for the flavor profile of the wine. In this work, we use and compare two Saccharomyces cerevisiae yeast strains: P29, typical of sparkling wines resulting of second fermentation in a closed bottle; G1, a flor yeast responsible for the biological aging of Sherry wines. We aimed to analyze and compare the effect of endogenous CO2 overpressure on esters metabolism with the proteins related in these yeast strains, to understand the yeast fermentation process in sparkling wines. For this purpose, protein identification was carried out using the OFFGEL fractionator and the LTQ Orbitrap, following the detection and quantification of esters with gas chromatograph coupled to flame ionization detector (GC-FID) and stir-bar sorptive extraction, followed by thermal desorption and gas chromatography-mass spectrometry (SBSE-TD-GC-MS). Six acetate esters, fourteen ethyl esters, and five proteins involved in esters metabolism were identified. Moreover, significant correlations were established between esters and proteins. Both strains showed similar behavior. According to these results, the use of this flor yeast may be proposed for the sparkling wine production and enhance the diversity and the typicity of sparkling wine yeasts.