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.

Development of a Descriptive Profile and References for the Assessment of Taste and Mouthfeel Descriptors of Protected Designation of Origin Wines.

Producers of PDO (Protected Designation of Origin) wines must submit to the EU authorities' technical specifications that include the specific sensory description of each product typology, to be subsequently checked by the competent authority in each country. Unfortunately, there is no consensual and standardized approach for the development of sensory control methods for PDO wines. The aim of this work was to develop a sensory profile for the taste and mouthfeel descriptors that allows the characterization of wines from 11 existing PDOs in Catalonia (Spain), and with the purpose of advancing the process of harmonization of the official sensory analysis of wines. This paper includes the selection process of tasters, the procedure used for the definition and grouping of descriptors, and the development of references for the selected attributes. The use of this analytical tool should allow PDO/PGI product certification and control authorities to verify compliance with their specifications (descriptive and quantitative) based on objectively evaluated results.

Microanalytical flow system for the simultaneous determination of acetic acid and free sulfur dioxide in wines.

Free sulfur dioxide and volatile acidity are parameters related to the quality of wines. Traditional methods for their determination are tedious, time consuming and require analysis in decentralized laboratories, therefore corrective actions cannot be applied on time. This may be more complex in aging wine cellars, where hundreds of individual barrels containing almost finished wines should be monitored. To achieve this aim, a portable microanalytical flow system for the simultaneous detection of free SO2 and acetic acid during the ageing of wines is proposed in this work. The miniaturized system is based on the use of a gas-diffusion membrane and a pH-ISFET, and can be easily installed in barrels. The system was optimized in the range of 5-60 mg L-1 and 0.15-1.40 g L-1 for SO2 and acetic acid, respectively. It was validated with different sets of wine samples by comparing the results with standard methods, demonstrating a good agreement between methods.

Using an electronic nose and volatilome analysis to differentiate sparkling wines obtained under different conditions of temperature, ageing time and yeast formats.

Effect of yeast inoculation format (F), temperature (T), and "on lees" ageing time (t) factors were evaluated on the composition of sparkling wines by a quantitative fingerprint obtained from volatile metabolites and the response of an electronic nose (E-nose). Wines elaborated according the traditional method at 10 and 14 °C, free cells and yeast biocapsules formats were monitored at 15 and 24 months of ageing time. Sixty-six volatiles identified and quantified in the eight sampling lots were subjected to a pattern recognition technique. A dual criterion based on univariate (ANOVA) and multivariate analysis (PLS-DA) through the variable importance projection (VIP) values, allowed to identify ten volatiles as potential markers for T factor, eleven for t and twelve for F factors. The discriminant models based on E-nose dataset enable a 100% correct classification of samples, in relation with t and F factors and the 83% for T factor.

Compact analytical flow system for the simultaneous determination of L-lactic and L-malic in red wines.

During the malolactic fermentation of red wines, L-malic acid is mainly converted to L-lactic acid. Both acids should be precisely measured during the entire process to guarantee the quality of the final wine, thus making real-time monitoring approaches of great importance in the winemaking industry. Traditional analytical methods based on laboratory procedures are currently applied and cannot be deployed on-site. In this work, we report on the design and development of a bi-parametric compact analytical flow system integrating two electrochemical biosensors that could be potentially applied in this scenario. The developed flow-system will allow for the first time the simultaneous measurement of both acids in real scenarios at the real-time and in remote way. Miniaturized thin-film platinum four-electrode chips are fabricated on silicon substrates by standard photolithographic techniques and further implemented in a polymeric fluidic structure. This includes a 15 µL flow cell together with the required fluidic channels for sample and reagent fluid management. The four-electrode chip includes counter and pseudo-reference electrodes together with two working electrodes. These are sequentially modified with electropolymerized polypyrrole membranes that entrap the specific receptors for selectively detecting both target analytes. The analytical performance of both biosensors is studied by chronoamperometry, showing a linear range from 5 × 10-6 to 1 × 10-4 M (LOD of 3.2 ± 0.3 × 10-6 M) and from 1 × 10-7 to 1 × 10-6 M (LOD of 6.7 ± 0.2 × 10-8 M) for the L-lactate and the L-malate, respectively. Both biosensors show long-term stability, retaining more than the 90% of their initial sensitivity after more than 30 days, this being a prerequisite for monitoring the whole process of the malolactic fermentation of the red wines (time between 20 and 40 days). The flow system performance is assessed with several wine samples collected during the malolactic fermentation process of three red wines, showing an excellent agreement with the results obtained with the standard method.

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.

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.

Use of a flor yeast strain for the second fermentation of sparkling wines: Effect of endogenous CO2 over-pressure on the volatilome.

Saccharomyces cerevisiae flor yeast is used for the first time in sparkling wine-making. Twenty-six oenological variables and fifty-three volatile metabolites are quantified in the middle (P = 3 bar) and at the end (P = 6 bar) of the second fermentation, carried out in open and closed bottles. A heat-map of volatiles and the fingerprints obtained for ten chemical families and ten odorant series visualize the changes for each condition. Terpenes, fatty acids and volatile phenols increased their contents by pressure effect at the end of the study by 25.0, 7.8 and 2.2%, respectively. The remaining families decrease between 17.4% and 30.1% for furanic compounds and esters in the same stage. A Principal Component Analysis established that nine volatiles are mainly affected by pressure and five by fermentation stage. The use of ethanol-tolerant flor yeasts constitutes an innovative procedure for the enhancement of the sparkling wines diversification.

Influence of two yeast strains in free, bioimmobilized or immobilized with alginate forms on the aromatic profile of long aged sparkling wines.

Production of sparkling wines involve a second alcoholic fermentation and contact with yeast less over an extended period of time, which influences the aroma composition and sensory quality of the resulting wines. Sparkling wines obtained with two yeast strains inoculated as free cells, immobilized in alginate bed and bioimmobilized as biocapsules, were aged during 32 months. Among the volatile compounds, high Odor Activity Values were obtained with isoamyl acetate, ethyl propanoate, ethyl butanoate, ethyl 3-methylbutanoate, ethyl hexanoate, ethyl octanoate, hexanol, 2-methoxy-4-vinylphenol, decanal, octanoic acid, decanoic acid and TDN. Taken together these contribute more than 70% of the overall aromatic series value. Although some results rely more on the yeast strain than the inoculation format, specific aroma compounds were associated with the immobilization format, allowing the classification of sparkling wines by PCA. As a result the aroma quality of sparkling wines could be improved using immobilized yeasts.

Changes in sparkling wine aroma during the second fermentation under CO2 pressure in sealed bottle.

High quality sparkling wine made by the traditional method requires a second alcoholic fermentation of a base wine in sealed bottles, followed by an aging time in contact with yeast lees. The CO2 overpressure released during this second fermentation has an important effect on the yeast metabolism and therefore on the wine aroma composition. This study focuses on the changes in chemical composition and 43 aroma compounds released by yeast during this fermentation carried out under two pressure conditions. The data were subjected to statistical analysis allowing differentiating between the base wine and the wine samples taken in the middle and at the end of fermentation. The differentiation among wines obtained to the end of fermentation with or without CO2 pressure is only achieved by a principal component analysis of 15 selected minor compounds (mainly ethyl dodecanoate, ethyl tetradecanoate, hexyl acetate, ethyl butanoate and ethyl isobutanoate).

Analysis of free and total sulfur dioxide in wine by using a gas-diffusion analytical system with pH detection.

The use of sulfur dioxide as preservative in winemaking industry has a direct impact on wine quality. The standard methods to analyze this parameter require several processes and are time consuming. In this paper a simple and rapid analytical method for free and total sulfur dioxide detection is proposed. This method is based on the separation of the analyte from the sample with a permeable gas diffusion membrane and its indirect detection with a pH sensor. The system has been validated and optimized for free sulfur dioxide detection in the range of 1-60mgL-1 and for total sulfur dioxide in the range of 30-300mgL-1 with a limit of detection of 0.5mgL-1. Validation of the system has been carried out using a total of 70 samples of white and red wines and two standard methods, the Ripper and the Paul method. The obtained values have demonstrated a good agreement for both methods.

Robust l-malate bienzymatic biosensor to enable the on-site monitoring of malolactic fermentation of red wines.

Monitoring the malolactic fermentation process is strictly required to guarantee the sensorial quality and freshness of red wines. This could be achieved by in-field and real-time continuous measurements of l-malate concentration in the fermentation tanks. The potential of a miniaturized amperometric bienzymatic biosensor as an analytical tool to be applied in such scenario is described in this paper. The biosensor comprises a thin-film gold electrode as transducer, malate dehydrogenase (MDH) and diaphorase (DP) enzymes together with nicotinamide adenine dinucleotide (NAD+) cofactor as the selective receptor and an adequate redox mediator to record the corresponding amperometric signal. Three different biosensor architectures are studied, whose main differences lie in the immobilization of the different chemical components onto the electrode surface. In all cases a fast-electrosynthethized polypyrrole (PPy) membrane is generated for this purpose. The experimental conditions are optimized and the best architecture shows a sensitivity of 1365 ± 110 mA M-1 cm-2 and a detection limit of 6.3 × 10-8 M in a concentration range of 1 × 10-7 M - 1 × 10-6 M. The biosensor presents an excellent working stability as it retains above 90% of its sensitivity after 37 days, thus enabling the monitoring of the malolactic fermentation of three red wines. The obtained results show excellent agreement with the standard colorimetric method.

Electronic tongues to assess wine sensory descriptors.

This work reports the application of an electronic tongue as a tool towards the analysis of wine in tasks such as its discrimination based on the maturing in barrels or the prediction of the global scores assigned by a sensory panel. To this aim, red wine samples were first analysed with the voltammetric sensor array, without performing any sample pretreatment. Afterwards, obtained responses were preprocessed employing fast Fourier transform (FFT) for the compression and reduction of signal complexity, and obtained coefficients were then used as inputs to build the qualitative and quantitative models employing either linear discriminant analysis (LDA) or partial least squares regression (PLS), respectively. Satisfactory results were obtained overall, with a classification rate of 100% in the discrimination of the type of barrel used during wine maturing, a normalized NRMSE of 0.077 in the estimation of ageing time (months) or 0.11 in the prediction of the scores (0-10) from a trained sensory panel (all for the external test subset).

Portable Electronic Tongue Based on Microsensors for the Analysis of Cava Wines.

Cava is a quality sparkling wine produced in Spain. As a product with a designation of origin, Cava wine has to meet certain quality requirements throughout its production process; therefore, the analysis of several parameters is of great interest. In this work, a portable electronic tongue for the analysis of Cava wine is described. The system is comprised of compact and low-power-consumption electronic equipment and an array of microsensors formed by six ion-selective field effect transistors sensitive to pH, Na⁺, K⁺, Ca2+, Cl-, and CO32-, one conductivity sensor, one redox potential sensor, and two amperometric gold microelectrodes. This system, combined with chemometric tools, has been applied to the analysis of 78 Cava wine samples. Results demonstrate that the electronic tongue is able to classify the samples according to the aging time, with a percentage of correct prediction between 80% and 96%, by using linear discriminant analysis, as well as to quantify the total acidity, pH, volumetric alcoholic degree, potassium, conductivity, glycerol, and methanol parameters, with mean relative errors between 2.3% and 6.0%, by using partial least squares regressions.

Monitoring of malolactic fermentation in wine using an electrochemical bienzymatic biosensor for L-lactate with long term stability.

L-lactic acid is monitored during malolactic fermentation process of wine and its evolution is strongly related with the quality of the final product. The analysis of L-lactic acid is carried out off-line in a laboratory. Therefore, there is a clear demand for analytical tools that enabled real-time monitoring of this process in field and biosensors have positioned as a feasible alternative in this regard. The development of an amperometric biosensor for L-lactate determination showing long-term stability is reported in this work. The biosensor architecture includes a thin-film gold electrochemical transducer selectively modified with an enzymatic membrane, based on a three-dimensional matrix of polypyrrole (PPy) entrapping lactate oxidase (LOX) and horseradish peroxidase (HRP) enzymes. The experimental conditions of the biosensor fabrication regarding the pyrrole polymerization and the enzymes entrapment are optimized. The biosensor response to L-lactate is linear in a concentration range of 1 × 10(-6)-1 × 10(-4) M, with a detection limit of 5.2 × 10(-7) M and a sensitivity of - (13500 ± 600) µA M(-1) cm(-2). The biosensor shows an excellent working stability, retaining more than 90% of its original sensitivity after 40 days. This is the determining factor that allowed for the application of this biosensor to monitor the malolactic fermentation of three red wines, showing a good agreement with the standard colorimetric method.