Preliminary Study on Yeasts Associated with the Production of "Tostado"-a Traditional Sweet Wine from Galicia (NW Spain).

BACKGROUND: Tostado is a traditional sweet wine from the Designations of Origins (DOs) of Ribeiro and Valdeorras in Galicia (NW Spain). The harvested grapes are air-dried and pressed to increase the concentrations of sugars, acids, and flavour compounds. Therefore, knowledge of the yeasts involved in fermentation under these conditions is essential to guarantee the quality and uniqueness of the valuable, distinctive, and expensive Tostado wines. METHODS: Saccharomyces and non-Saccharomyces yeasts were identified using Wallerstein Laboratory (WL) Nutrient Agar and lysine plating, followed by polymerase chain reaction (PCR) amplification, enzymatic digestion, and sequencing. Saccharomyces cerevisiae isolates were further characterised at the strain level using mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP). Statistical analyses were also performed, including different diversity indices, Similarity Percentage (SIMPER) analysis, principal component analysis (PCA), neighbor-joining clustering, parsimony-phylogram, and network plot. In addition, the total acidity, volatile acidity, reducing sugars, and alcoholic strength by volume of the Tostado wines were analysed. RESULTS: A wide diversity of autochthonous yeasts was found, which were predominantly species of oenological relevance, such as Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, Debaryomyces hansenii, Torulaspora delbrueckii, Pichia spp., and Saccharomyces cerevisiae from the must and paste stages of Tostado wine. In addition, 19 different S. cerevisiae strains were identified. This high yeast diversity, which changed from the early stages of fermentation, could contribute to the distinctive characteristics observed in Tostado wine. CONCLUSIONS: Characteristic and differentiating chemical and microbiological profiles were found as early as the pre-fermentation stages, which adds value to these special wines that have rarely been studied.

Physicochemical Properties and Microbiome of Vineyard Soils from DOP Ribeiro (NW Spain) Are Influenced by Agricultural Management.

Agricultural management influences the soil ecosystem by affecting its physicochemical properties, residues of pesticides and microbiome. As vineyards grow crops with the highest incidence of pesticides, the aim of this study was to evaluate the impact of conventional and sustainable management systems of vineyards from DOP Ribeiro on the soil's condition. Samples from soils under three different management systems were collected, and the main soil physicochemical properties were evaluated. A selection of 50 pesticides were investigated by liquid chromatography with tandem mass spectrometry. The bacterial and fungal microbiomes were characterized through amplicon sequencing. The results show that organic agriculture positively influences soil pH and the concentration of some nutrients compared to conventional management. Our microbiome analysis demonstrated that transitioning from conventional to organic management significantly improves several BeCrop® indexes related to key microbial metabolism and soil bio-sustainability. Such a transition does not affect soil alpha diversity, but leads to a higher interconnected microbial network structure. Moreover, differential core genera and species for each management system are observed. In addition, the correlation of the microbiome with geographical distance is evidence of the existence of different microbial terroirs within DOP Ribeiro. Indeed, sustainable management leads to higher nutrient availability and enhances soil health in the short term, while lowering pesticide usage.

Characterization of Indigenous Non-Saccharomyces Yeast Strains with Potential Use in Winemaking.

BACKGROUND: The vineyard is a great reservoir of autochthonous yeast strains whose composition is defined by different regional (edaphology, orography or climatology) and anthropological factors (cultivation systems or cultural practices). Most of this yeast diversity corresponds to non-Saccharomyces strains, some of which have potential use in winemaking. METHODS: The oenological potential of 29 different native non-Saccharomyces strains belonging to 4 species (Lachancea thermotolerans, Torulaspora delbrueckii, Starmerella bacillaris and Metschnikowia spp.) was evaluated, using the autochthonous Saccharomyces cerevisiae XG3 strain as a control. Microfermentations with pure culture of each strain were performed in duplicate and the basic parameters and major volatiles of wines were analysed following official methodology. The best strain within each species was selected using a quantification matrix including the relevant oenological characteristics. RESULTS: The fermentative ability of non-Saccharomyces was lower than S. cerevisiae in all cases, but with differences among species. L. thermotolerans and T. delbrueckii showed higher fermentation rates than Starm. bacillaris, whereas Metschnikowia spp. presented a low fermentative power. At chemical level all non-Saccharomyces strains reduced the alcoholic content, the higher alcohols and the volatile acidity of wines and increased the content of glycerol, with differences among strains within a given species. T. delbrueckii and L. thermotolerans increased the total acidity of wines. The latter and Metschnikowia spp. strains produced lactic acid, which decreased the wine pH in the case of L. thermotolerans. According to their oenological traits the best rated strains of each species were Lt93, Td315, Mf278 and Sb474. In addition, the data obtained in pure fermentations were correlated to those chemical and aromatic compounds obtained with these non-Saccharomyces strains in sequential fermentations. CONCLUSIONS: Autochthonous strains of non-Saccharomyces yeast species contribute distinctive chemical characteristics to the wines. The correlations observed between wines fermented with the different non-Saccharomyces indigenous strains in pure and sequential fermentations suggest that their contribution to wine properties remains stable regardless of must composition or winemaking techniques.

Can Satellite Remote Sensing Assist in the Characterization of Yeasts Related to Biogeographical Origin?

Biogeography is a key concept associated with microbial terroir, which is responsible for the differentiation and uniqueness of wines. One of the factors influencing this microbial terroir is the vegetation, which in turn is influenced by climate, soil, and cultural practices. Remote sensing instruments can provide useful information about vegetation. This study analyses the relationship between NDVI, calculated using Sentinel-2 and Landsat-8 satellite images of different veraison dates, and microbial data obtained in 2015 from 14 commercial (organic and conventional) vineyards belonging to four Designations of Origin (DOs) from Galicia (northwest Spain). Microbial populations in grapes and musts were identified using PCR techniques and confirmed by sequencing. Statistical analyses were made using PCA, CCA, TB-PLS, and correlation analyses. This study confirms that the NDVI is positively correlated with the diversity of yeasts, both in grapes' surface and must samples. Moreover, the results of this study show: (i) Sentinel-2 images, as well as Landsat-8 images, can establish differences in NDVI related to yeast terroir in grapes and musts, as it is the most relevant DO factor, (ii) Sentinel-2 NDVI and yeast biogeography are moderately to strongly correlated, (iii) Sentinel-2 achieved a better delimitation of the DOs than Landsat-8 and can establish more accurate differences in NDVI-yeast terroir correlations, and (iv) a higher NDVI was associated with the yeast biogeographical patterns of the DOs with higher species richness (S) consisting of weakly fermenting yeasts (Hanseniaspora uvarum, Pichia spp., Starmerella bacillaris, and Zygosaccharomyces spp). However, NDVI values did not correlate well with biogeographic patterns of yeasts previously studied at frequency level (proportion or percentage of each species) in each particular DO. This study suggests that satellite imagery has the potential to be a valuable tool for wine quality management and a decision-making instrument for DO regulators and winegrowers.

Oenological potential of non-Saccharomyces yeasts to mitigate effects of climate change in winemaking: impact on aroma and sensory profiles of Treixadura wines.

The effects of climate change on wine include high-alcohol content, low acidity and aroma imbalance. The potential of several non-Saccharomyces wine yeasts to mitigate these effects was evaluated by sequential fermentation of Treixadura grape must. Fermentations with only Saccharomyces cerevisiae ScXG3 and a spontaneous process were used as control assays. All yeast strains were obtained from the yeast collection of Estación de Viticultura e Enoloxía de Galicia (EVEGA), Galicia, Spain. Fermentation kinetics as well as yeast dynamics and implantation ability varied depending on inoculated yeasts. In addition, the results showed significant differences in the chemical composition of wine. Starmerella bacillaris 474 reduced the alcohol content (1.1% vol) and increased the total acidity (1.2 g L-1) and glycerol of wines. Fermentation with Lachancea thermotolerans Lt93 and Torulaspora delbrueckii Td315 also decreased the alcohol content, although to a lesser extent (0.3% and 0.7% vol, respectively); however, their effect on wine acidity was less significant. The wines also differed in their concentration of volatile compounds and sensory characteristics. Thus, wines made with Metschnikowia fructicola Mf278 and S. cerevisiae ScXG3 had higher content of esters, acetates and some acids than other wines, and were most appreciated by tasters due to their fruity character and overall impression.

Private versus social incentives for pharmaceutical innovation.

We provide a theoretical framework to contribute to the current debate regarding the tendency of pharmaceutical companies to direct their R&D toward marketing products that are "follow-on" drugs of already existing drugs, rather than toward the development of breakthrough drugs. We construct a model with a population of patients who can be treated with drugs that are horizontally and vertically differentiated. In addition to a pioneering drug, a new drug can be marketed as the result of an innovative process. We analyze physician prescription choices and the optimal pricing decision of an innovative firm. We also characterize the incentives of the innovative firm to conduct R&D activities, disentangling the quest for breakthrough drugs from the firm effort to develop follow-on drugs. Our results offer theoretical support for the conventional wisdom that pharmaceutical firms devote too many resources to conducting R&D activities that lead to incremental innovations.

Effects of injection of botulinum toxin type B into the external anal sphincter on anal pressure of horses.

OBJECTIVE: To determine effects on anal pressure of horses after local injection of the external anal sphincter with Clostridium botulinum toxin type B. ANIMALS: 11 healthy adult horses. PROCEDURE: Peak and resting anal sphincter pressures were measured with a custom-made rectal probe that was connected to a pressure transducer. Pressures were measured before treatment and after injection with botulinum toxin type B (BTB) or saline (0.9% NaCl) solution. Dose titration with 500, 1,000, 1,500, and 2,500 U of BTB was completed. Physical changes, behavior, and anal pressure were recorded for each horse. RESULTS: Injection of 1,000 U of BTB caused a significant reduction in peak anal pressure from days 2 to 84, compared with pressure in control horses. Maximal effect of the toxin was observed within the first 15 days after injection, followed by a slow return to baseline during the 168-day period. Injection of 2,500 U of BTB in the anal sphincter in 1 horse resulted in lethargy, generalized weakness, and dysphagia for 14 days. Adverse clinical effects were not observed in horses after injections with 500, 1,000, or 1,500 U of BTB. CONCLUSIONS AND CLINICAL RELEVANCE: The effect of focal intramuscular injection of BTB in horses is similar to that reported for other species. However, horses appear to be more sensitive to BTB, compared with other species, and clinical signs of botulism may develop at doses exceeding 1,500 U. Injections of BTB in the external anal sphincter of mares may be useful to reduce incisional dehiscence after repair of perineal lacerations.