Anna Puig-Pujol

In recent years, the wine industry has been researching how to improve wine quality along the production value chain. In this scenario, we present here a new tool, MicroVi, a cost-effective chip-sized microscopy solution to detect and count yeast cells in wine samples. We demonstrate that this novel microscopy setup is able to measure the same type of samples as an optical microscopy system, but with smaller size equipment and with automated cell count configuration. The technology relies on the top of state-of-the-art computer vision pipelines to post-process the images and count the cells. A typical pipeline consists of normalization, feature extraction (i.e., SIFT), image composition (to increase both resolution and scanning area), holographic reconstruction and particle count (i.e., Hough transform). MicroVi achieved a 2.19 µm resolution by properly resolving the G7.6 features from the USAF Resolving Power Test Target 1951. Additionally, we aimed for a successful calibration of cell counts for Saccharomyces cerevisiae. We compared our direct results with our current optical setup, achieving a linear calibration for measurements ranging from 0.5 to 50 million cells per milliliter. Furthermore, other yeast cells were qualitatively resolved with our MicroVi microscope, such as, Brettanomyces bruxellensis, or bacteria, like, Lactobacillus plantarum, thus confirming the system’s reliability for consistent microbial assessment.

Despite the large number of existing varieties of Vitis vinifera L., only few occupy a large niche in today’s highly globalized wine market. The increasing consumer demand for diversified products, as well as the changing climatic conditions, make establishing a process of varietal diversification essential to achieve both challenges. It is for this reason that the study of minority varieties, which have a higher level of adaptation to each area of origin, is of particular interest. With the main objective of achieving an in-depth knowledge of minority varieties in Spain, the national research project ‘Valorization of Minority Grapevine Varieties for their Potential for Wine Diversification and Resilience to Climate Change’ (MINORVIN), has been proposed. Within this extensive project, the present study describes the aroma profiles of 60 single-variety wines, corresponding with 44 different varieties, with 12 of these varieties being studied at the same time in several Spanish regions. Volatile compounds were determined through three consecutive vintages using gas chromatography-mass spectrometry (GC-MS) and gas chromatography–flame ionization detector (GC-FID). Compounds were grouped into major compounds, including alcohols, C₆ compounds, esters, acetates, acids, carbonyl compounds, and other type of compounds, and minor compounds, including lactones, terpenes, and C₁₃-norisoprenoids, according to their concentration in the wines being analyzed. Among this last group of compounds, lactones were quantitatively the most abundant, followed by terpenes. This study reflects that minority variety wines show distinctive aromatic profiles, supporting the importance of valuing and promoting the autochthonous minority grapevine varieties for the Spanish winemaking industry.

Downy mildew is one of the most destructive diseases affecting grapevines (Vitis vinifera L.). Caused by the oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, it can appear anywhere where vines are cultivated. It is habitually controlled by the application of phytosanitary agents (copper-based or systemic) at different stages of the vine growth cycle. This, however, is costly, can lead to reduced yields, has a considerable environmental impact, and its overuse close to harvest can cause fermentation problems. All grapevines are susceptible to this disease, although the degree of susceptibility differs between varieties. Market demands and European legislation on viticulture and the use of phytosanitary agents (art. 14 of Directive 128/2009/EC) now make it important to know the sensitivity of all available varieties, including minority varieties. Such knowledge allows for a more appropriate use of phytosanitary agents, fosters the commercial use of these varieties and thus increases the offer of wines associated with different terroirs, and helps identify material for use in crop improvement programmes via crossing or genetic transformation, etc. Over 2020–2021, the susceptibility to P. viticola of 63 minority vine varieties from different regions of Spain was examined in the laboratory using the leaf disc technique. Some 87% of these varieties were highly susceptible and 11% moderately susceptible; just 2% showed low susceptibility. The least susceptible of all was the variety Morate (Madrid, IMIDRA). Those showing intermediate susceptibility included the varieties Sanguina (Castilla la Mancha, IVICAM), Planta Mula (Comunidad Valenciana, ITVE), Rayada Melonera (Madrid, IMIDRA), Zamarrica (Galicia, EVEGA), Cariñena Roja (Cataluña, INCAVI), Mandrègue (Aragón, DGA) and Bastardo Blanco (Extremadura, CICYTEX). The highly susceptible varieties could be differentiated into three subgroups depending on sporulation severity and density.

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.

In Andalusia (Spain), there are different wine regions that have a great recognized tradition. In these regions, the cultivation of the vine is ancient and there are still vineyards planted with local varieties of Vitis vinifera L. that have not yet been identified. The aim of this research study was to identify 49 accessions of grapevine collected in the districts of four provinces in Andalusia (Spain). All samples were genotyped with 20 microsatellite markers in order to ascertain the identity and analyze the genetic diversity of the collected material. In total, 30 different genotypes were obtained, 22 of them which were identified with named, known varieties by comparison to the Spanish or European microsatellite databases, and eight which are referred to as new genotypes. All loci were polymorphic, and a total of 159 alleles were detected, ranging from 4 to 12 alleles per locus, with an average allele number of 7.95. The overall observed heterozygosity was 0.763 and was slightly higher than expected (0.715), while the gene diversity per locus varied between 0.167 (VVIN73) and 0.967 (VVMD5). A dendrogram representing the genetic similarities among cultivars was depicted using the UPGMA method to investigate their relationships. The eight new genotypes identified in this research work could represent ancient local varieties in danger of extinction. These new cultivars may be used to determine original wines.

Non-anthocyanin compounds (NAN) such as flavonol, flavanol, and phenolic acids should be considered in the characterization of minority red grapevine varieties because these compounds are involved in copigmentation reactions and are potent antioxidants. Sixteen NAN were extracted, identified, and quantified by High Performance Liquid Chromatography (HPLC) from grapes of 28 red genotypes of Vitis vinifera L. grown in Galicia (Northwest of Spain) in 2018 and 2019 vintages. The percentage of total NAN with respect to the total polyphenol content (TPC) values was calculated for each sample and established into three categories: high percentage NAN varieties (NANV), those varieties showing low percentages of NAN (ANV), and finally those varieties showing medium percentages of NAN (NANAV). ‘Xafardán’ and ‘Zamarrica’, classified as NANAV, had high values of TPC and showed good percentages of flavonol and flavanol compounds. Principal component analyses (PCA) were performed with flavonol, flavanol, and phenolic acid profiles. The flavonol and flavanol profiles allowed a good discrimination of samples by variety and year, respectively. The flavonol profile should therefore be considered as a potential varietal marker. The results could help in the selection of varieties to be disseminated and in the identification of the most appropriate agronomic and oenological techniques that should be performed on them.

The main phenological stages (budburst, flowering, veraison, and ripeness) of 53 Spanish minority varieties were studied to determine their potential to help winegrowers adapt to climate change conditions. In total, 43 varieties were studied in the same location in Spain (Alcalá de Henares, in the Madrid region) and 10 varieties in 5 other regions (Galicia, Navarre, Catalonia, Extremadura, and Andalusia). Other traits of agronomic and oenological interest, such as yield and acidity, were also monitored. The results allow for the grouping of the varieties into several clusters according to the time of ripeness (very early—only for red varieties—and early, intermediate, and late, for both red and white varieties) and yield (high, medium, and low). The total acidity in the grape juice ranged from 3 to 11 g of tartaric acid/L. The average temperatures were higher (up to 3–4 °C during summer) compared to historical averages during the 1957–2021 time period. Advanced phenology phases and reduced acidity are regarded as negative effects of climate change for winegrowing practices. Since some minority varieties showed late or intermediate ripening, high acidity, and high (1 Kg/shoot) or medium (0.5 Kg/shoot) yield, our findings suggest that they may be cultivated in the coming years by winegrowers as an approach to mitigate climate change effects.

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.

The traditional method for sparkling wine making consists of a second fermentation of a base wine followed by ageing in the same bottle that reaches the consumers. Nitrogen metabolism is the second most important process after carbon and takes place during wine fermentation by yeast. Amino acids are the most numerous nitrogen compounds released by this process. They contribute to the organoleptic properties of the wines and, therefore, to their sensory quality. The main objective of this study is to compare the differential proteomic response of amino acid metabolism, specifically their proteins and their interactions in the G1 strain (unconventional yeast) during sparkling wine production versus the conventional P29 strain. One of the new trends in winemaking is the improvement of the organoleptic diversity of wine. We propose the use of unconventional yeast that shows desirable characteristics for the industry. For this purpose, these two yeasts were grown at sealed bottle conditions for the second fermentation (Champenoise method). No differences were obtained in the middle of fermentation between the yeast strains. The number of proteins identified, and the relationships established, were similar, highlighting lysine metabolism. At the end of the second fermentation, the difference between each strain was remarkable. Hardly any proteins were identified in unconventional versus conventional yeast. However, in both strains, the metabolism of sulfur amino acids, methionine, and cysteine obtained a greater number of proteins involved in these processes. The release of these amino acids to the medium would allow the yeast to balance the internal redox potential by reoxidation of NADPH. This study is focused on the search for a more complete knowledge of yeast metabolism, specifically the metabolism of amino acids, which are key compounds during the second fermentation.

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.