Wine yeast selection in the Iberian Peninsula: Saccharomyces and non-Saccharomyces as drivers of innovation in Spanish and Portuguese wine industries.

Yeast selection for the wine industry in Spain started in 1950 for the understanding of the microbial ecology, and for the selection of optimal strains to improve the performance of alcoholic fermentation and the overall wine quality. This process has been strongly developed over the last 30 years, firstly on Saccharomyces cerevisiae, and, lately, with intense activity on non-Saccharomyces. Several thousand yeast strains have been isolated, identified and tested to select those with better performance and/or specific technological properties. The present review proposes a global survey of this massive ex-situ preservation of eukaryotic microorganisms, a reservoir of biotechnological solutions for the wine sector, overviewing relevant screenings that led to the selection of strains from 12 genera and 22 species of oenological significance. In the first part, the attention goes to the selection programmes related to relevant wine-producing areas (i.e. Douro, Extremadura, Galicia, La Mancha and Uclés, Ribera del Duero, Rioja, Sherry area, and Valencia). In the second part, the focus shifted on specific non-Saccharomyces genera/species selected from different Spanish and Portuguese regions, exploited to enhance particular attributes of the wines. A fil rouge of the dissertation is the design of tailored biotechnological solutions for wines typical of given geographic areas.

Impact of the application of monosilicic acid to grapevine (Vitis vinifera L.) on the chemical composition of young red Mencia wines.

Impact of applying monosilicic acid to grapevines during ripening on chemical composition of Mencía red wines in an area where fungal diseases during summer are common was examined. The foliar application of monosilicic acid to grapevines led to a less oxidized wine, with lower levels of acetic acid, acetaldehyde, ethyl acetate and diacethyl; this should be considered as positive from a sensory point of view. Wines made with silicon-treated grapes also contained lower levels of gluconic acid and glycerol, which are chemical markers of wines made with botrytized grapes, as well as higher contents of total phenols, anthocyanins and tannins. Furthermore, the contents of several mid-chain alcohols were higher (p < 0.05) in wines made with grapes from silicon-treated plants.

A high-entropy manganite in an ordered nanocomposite for long-term application in solid oxide cells.

The implementation of nano-engineered composite oxides opens up the way towards the development of a novel class of functional materials with enhanced electrochemical properties. Here we report on the realization of vertically aligned nanocomposites of lanthanum strontium manganite and doped ceria with straight applicability as functional layers in high-temperature energy conversion devices. By a detailed analysis using complementary state-of-the-art techniques, which include atom-probe tomography combined with oxygen isotopic exchange, we assess the local structural and electrochemical functionalities and we allow direct observation of local fast oxygen diffusion pathways. The resulting ordered mesostructure, which is characterized by a coherent, dense array of vertical interfaces, shows high electrochemically activity and suppressed dopant segregation. The latter is ascribed to spontaneous cationic intermixing enabling lattice stabilization, according to density functional theory calculations. This work highlights the relevance of local disorder and long-range arrangements for functional oxides nano-engineering and introduces an advanced method for the local analysis of mass transport phenomena.

Characterization of polymeric pigments and pyranoanthocyanins formed in microfermentations of non-Saccharomyces yeasts.

AIMS: To assess the influence of non-Saccharomyces yeasts on the pyranoanthocyanins and polymeric pigments formation after the addition of (+)-catechin and procyanidin B2 to fresh red grape must. METHODS AND RESULTS: The fermentation of red grape musts was done with non-Saccharomyces yeasts either alone or in sequential fermentations with the Saccharomyces cerevisiae species. The characterization of both pyranoanthocyanin and polymeric pigments has been carried out with liquid chromatography coupled to mass spectroscopy (HPLC-DAD-ESI/MS). Red wines were also characterized by infrared spectroscopy (FTIR), gas chromatography (GC-FID) and spectrophotometry (UV-Vis). It has been observed that fermentation with the species Schizosaccharomyces pombe led to higher concentrations of pigments of all types: anthocyanins, polymeric pigments and pyranoanthocyanins, particularly vitisin A. CONCLUSIONS: The use of non-Saccharomyces yeasts improve the formation of stable pigments in red wines thanks to the differences in the microbial metabolism from among the yeasts studied. SIGNIFICANCE AND IMPACT OF THE STUDY: Colour stability as one of the main organoleptic properties in red wines, may be improved by the controlled use of selected non-Saccharomyces yeasts during red must fermentation.

Standalone ethanol micro-reformer integrated on silicon technology for onboard production of hydrogen-rich gas.

A novel design of a silicon-based micro-reformer for onboard hydrogen generation from ethanol is presented in this work. The micro-reactor is fully fabricated with mainstream MEMS technology and consists of an active low-thermal-mass structure suspended by an insulating membrane. The suspended structure includes an embedded resistive metal heater and an array of ca. 20k vertically aligned through-silicon micro-channels per square centimetre. Each micro-channel is 500 µm in length and 50 µm in diameter allowing a unique micro-reformer configuration that presents a total surface per projected area of 16 cm(2) cm(-2) and per volume of 320 cm(2) cm(-3). The walls of the micro-channels become the active surface of the micro-reformer when coated with a homogenous thin film of Rh-Pd/CeO2 catalyst. The steam reforming of ethanol under controlled temperature conditions (using the embedded heater) and using the micro-reformer as a standalone device are evaluated. Fuel conversion rates above 94% and hydrogen selectivity values of ca. 70% were obtained when using operation conditions suitable for application in micro-solid oxide fuel cells (micro-SOFCs), i.e. 750 °C and fuel flows of 0.02 mlL min(-1) (enough to feed a one watt power source).

Use of Schizosaccharomyces strains for wine fermentation-Effect on the wine composition and food safety.

Schizosaccharomyces was initially considered as a spoilage yeast because of the production of undesirable metabolites such as acetic acid, hydrogen sulfide, or acetaldehyde, but it currently seems to be of great value in enology.o ced Nevertheless, Schizosaccharomyces can reduce all of the malic acid in must, leading to malolactic fermentation. Malolactic fermentation is a highly complicated process in enology and leads to a higher concentration of biogenic amines, so the use of Schizosaccharomyces pombe can be an excellent tool for assuring wine safety. Schizosaccharomyces also has much more potential than only reducing the malic acid content, such as increasing the level of pyruvic acid and thus the vinylphenolic pyranoanthocyanin content. Until now, few commercial strains have been available and little research on the selection of appropriate yeast strains with such potential has been conducted. In this study, selected and wild Sc. pombe strains were used along with a Saccharomyces cerevisiae strain to ferment red grape must. The results showed significant differences in several parameters including non-volatile and volatile compounds, anthocyanins, biogenic amines and sensory parameters.

Synthesis of mesoporous nanocomposites for their application in solid oxide electrolysers cells: microstructural and electrochemical characterization.

Fabrication routes for new SOEC mesoporous nanocomposite materials as electrodes are presented in this paper. NiO-CGO and SDC-SSC are described as possible new materials and their synthesis and the cell fabrication are discussed. The obtained materials are microstructurally characterised by SEM, TEM and XRD, where the mesoporous structures are observed and analysed. The obtained samples are electrochemically analysed by I-V polarisation curves and EIS analysis, showing a maximum current density of 330 mA cm(-2) at 1.7 V. A degradation of the cell performance is evidenced after a potentiostatic test at 1.4 V after more than 40 hours. Oxygen electrode delaminating is detected, which is most probably the main cause of ageing. Even taking into account the lack of durability of the fabricated cells, the mesoporous electrodes do not seem to be altered, opening the possibility for further studies devoted to this high stability material for SOEC electrodes.

Co-electrolysis of steam and CO2 in full-ceramic symmetrical SOECs: a strategy for avoiding the use of hydrogen as a safe gas.

The use of cermets as fuel electrodes for solid oxide electrolysis cells requires permanent circulation of reducing gas, e.g. H2 or CO, so called safe gas, in order to avoid oxidation of the metallic phase. Replacing metallic based electrodes by pure oxides is therefore proposed as an advantage for the industrial application of solid oxide electrolyzers. In this work, full-ceramic symmetrical solid oxide electrolysis cells have been investigated for steam/CO2 co-electrolysis. Electrolyte supported cells with La(0.75)Sr(0.25)Cr(0.5)Mn(0.5)O3-δ reversible electrodes have been fabricated and tested in co-electrolysis mode using different fuel compositions, from pure H2O to pure CO2, at temperatures between 850-900 °C. Electrochemical impedance spectroscopy and galvanostatic measurements have been carried out for the mechanistic understanding of the symmetrical cell performance. The content of H2 and CO in the product gas has been measured by in-line gas micro-chromatography. The effect of employing H2 as a safe gas has also been investigated. Maximum density currents of 750 mA cm(-2) and 620 mA cm(-2) have been applied at 1.7 V for pure H2O and for H2O : CO2 ratios of 1 : 1, respectively. Remarkable results were obtained for hydrogen-free fuel compositions, which confirmed the interest of using ceramic oxides as a fuel electrode candidate to reduce or completely avoid the use of safe gas in operation minimizing the contribution of the reverse water shift reaction (RWSR) in the process. H2 : CO ratios close to two were obtained for hydrogen-free tests fulfilling the basic requirements for synthetic fuel production. An important increase in the operation voltage was detected under continuous operation leading to a dramatic failure by delaminating of the oxygen electrode.

Towards a full integration of vertically aligned silicon nanowires in MEMS using silane as a precursor.

Silicon nanowires present outstanding properties for electronics, energy, and environmental monitoring applications. However, their integration into microelectromechanical systems (MEMS) is a major issue so far due to low compatibility with mainstream technology, which complicates patterning and controlled morphology. This work addresses the growth of 〈111〉 aligned silicon nanowire arrays fully integrated into standard MEMS processing by means of the chemical vapor deposition-vapor liquid solid method (CVD-VLS) using silane as a precursor. A reinterpretation of the galvanic displacement method is presented for selectively depositing gold nanoparticles of controlled size and shape. Moreover, a comprehensive analysis of the effects of synthesis temperature and pressure on the growth rate and alignment of nanowires is presented for the most common silicon precursor, i.e., silane. Compared with previously reported protocols, the redefined galvanic displacement together with a silane-based CVD-VLS growth methodology provides a more standard and low-temperature (<650 °C) synthesis scheme and a compatible route to reliably grow Si nanowires in MEMS for advanced applications.

Role of an intermittent claudication questionnaire for the diagnosis of PAD in ambulatory patients with type 2 diabetes.

AIM: In epidemiological studies, peripheral arterial disease is assessed by the measurement of the ankle/brachial index (ABI), thus enabling detection of asymptomatic disease. Our aim was to evaluate the diagnostic accuracy of a validated questionnaire on intermittent claudication for peripheral arterial disease in a clinical setting. METHODS: We administered the Edinburgh questionnaire on intermittent claudication and measured ABI using a portable Doppler in 456 outpatients with type 2 diabetes. Subjects with intermittent claudication and an ABI above 0.9 were examined with color Doppler ultrasound imaging. Peripheral arterial disease was considered to be present when the ABI was <0.9 or the color Doppler arterial waveform was monophasic. RESULTS: Thirty-five (7.6%) of the 456 patients had intermittent claudication. Of these, 22 (63%) had an ABI <0.9 and the remaining 13 (37%) had an ABI >0.9. Of these latter, 12 were reexamined and 3 (25%) were found to have monophasic waveforms in color Doppler, thus being diagnosed with peripheral arterial disease. The overall accuracy of intermittent claudication for peripheral arterial disease was 75% (95% CI, 71-79). CONCLUSION: Among patients with type 2 diabetes, a normal ABI does not rule out peripheral arterial disease; the use of an intermittent claudication questionnaire is able to identify correctly the disease in 3 out of 4 patients with diabetes. Our results suggest incorporating the intermittent claudication questionnaire into the general consultation instead of the general screening of the ABI.

Effect of Saccharomyces strains on the quality of red wines aged on lees.

Ageing on lees involves ageing the wine in contact with yeast cells after fermentation. If combined with the addition of oak chips, it can soften the wood flavour and increase the aromatic complexity of wine. The aim of the present work is to optimise both ageing techniques through selection of an adequate Saccharomyces cerevisiae strain. The study lasted 6 months and content of polysaccharides, anthocyanins, proanthocyanidins, volatile compounds, colour parameters and sensory analysis, were periodically evaluated. Among the strains tested, G37 showed the highest release of polysaccharides (24.4±5.5 mg l(-1)). Vanillin, syringaldehyde and furfuryl alcohol increased with ageing time in 7VA2 treatment. The wine aged with CTPL14 strain presented fewer monomeric and oligomeric proanthocyanidins (12.4±0.6 and 83.4±8.3 mg l(-1), respectively), and showed the lowest astringency and bitterness sensations. Results show an improvement in the sensory profile of the red wine aged with a combination of these two techniques.

Reduction of 4-ethylphenol production in red wines using HCDC+ yeasts and cinnamyl esterases.

Hydroxycinnamate decarboxylase (HCDC) activity has been evaluated in several commercial yeast strains. The combined effect of using cinnamyl esterases (CE) and HCDC+ Saccharomyces cerevisiae strains has been studied in the formation of vinylphenolic pyranoanthocyanins (VPAs) during fermentation, analysing the kind and concentration of pigments formed according to the yeast strain used. Wines fermented with yeasts HCDC+ were contaminated with Dekkera bruxellensis and afterwards analysed to evaluate the formation of ethylphenols (EPs). The musts treated with CE and later fermented with HCDC+ yeast strains showed lower contents of 4-ethylphenol than those fermented with HCDC- strains. This reduction in the EP content is due to the transformation of hydroxycinnamic acids in stable VPAs pigments. The associated use of CEs and HCDC+ Saccharomyces strains is a natural strategy to reduce the formation of EPs in wines contaminated by Dekkera/Brettanomyces.

Formation of pyranoanthocyanins by Schizosaccharomyces pombe during the fermentation of red must.

Schizosaccharomyces pombe is a non-Saccharomyces yeast strain that can ferment grape musts with high sugar contents - but it also has other metabolic and physiological properties that render it of great interest to wine biotechnologists. This work compares the production of pyranoanthocyanins by S. pombe, Saccharomyces cerevisiae and Saccharomyces uvarum during fermentation. Total pyranoanthocyanins ranged from 11.9 to 19.4 mg/l depending on the strain of S. pombe used. On average, S. pombe produced more pyruvic acid than did either Saccharomyces species; as a consequence it also formed more vitisin A-type pigments. S. pombe 938 produced the largest quantity of vitisin A (11.03±0.82 mg/l). The formation of large amounts of pyranoanthocyanins intensifies the post-fermentation colour of wines somewhat, a phenomenon that helps them maintain their colour over ageing as the natural grape anthocyanins become degraded. Some of the S. pombe strains showed hydroxycinnamate decarboxylase activity, which favours the formation of vinylphenolic pyranoanthocyanins. Fermentation with S. pombe therefore provides an interesting way of increasing the overall pyranoanthocyanin content of red wines, and of stabilising their colour during ageing.

Minimization of ethylphenol precursors in red wines via the formation of pyranoanthocyanins by selected yeasts.

Different strains of Saccharomyces with different hydroxycinnamate decarboxylase (HCDC) activities, estimated by a bioconversion assay, were used for the fermentation of musts enriched with p-coumaric acid and grape anthocyanins, with the aim of favouring the formation of vinylphenolic pyranoanthocyanins, colour stabilization and (especially) the minimization of 4-ethylphenol. The development of anthocyanin-3-O-glucosides (precursors of vinylphenolic adducts), the decarboxylation of p-coumaric acid, and the formation of 4-vinylphenol, 4-ethylphenol and vinylphenolic pyranoanthocyanins were monitored by HPLC-DAD-ESI/MS. After fermentation, the wines were inoculated with large numbers (10(4) CFU/ml) of Dekkera bruxellensis to establish their potential for ethylphenol production. The HCDC activity of the strains significantly increased the formation of vinylphenolic pyranoanthocyanins and reduced the final concentration of 4-ethylphenol and 4-ethylguaiacol generated by the vinylreductase activity (VPhR) of D. bruxellensis. Early decarboxylation of hydroxycinnamates to vinylphenols, by means of Saccharomyces strains with strong HCDC activity, and their subsequent binding with anthocyanins to form stable pyranoanthocyanins, is a possible way to reduce the likelihood of ethylphenol production by Brettanomyces during in-barrel aging.

Factors affecting the hydroxycinnamate decarboxylase/vinylphenol reductase activity of dekkera/brettanomyces: application for dekkera/brettanomyces control in red wine making.

The growth of Dekkera/Brettanomyces yeasts during the ageing of red wines-which can seriously reduce the quality of the final product-is difficult to control. The present study examines the hydroxycinnamate decarboxylase/vinylphenol reductase activity of different strains of Dekkera bruxellensis and Dekkera anomala under a range of growth-limiting conditions with the aim of finding solutions to this problem. The yeasts were cultured in in-house growth media containing different quantities of growth inhibitors such as ethanol, SO(2), ascorbic acid, benzoic acid and nicostatin, different sugar contents, and at different pHs and temperatures. The reduction of p-coumaric acid and the formation of 4-ethylphenol were periodically monitored by HPLC-PDA. The results of this study allow the optimization of differential media for detecting/culturing these yeasts, and suggest possible ways of controlling these organisms in wineries.

A method for estimating Dekkera/Brettanomyces populations in wines.

AIMS: The formation of ethylphenols in wines, a consequence of Dekkera/Brettanomyces metabolism, can affect their quality. The main aims of this work were to further our knowledge of Dekkera/Brettanomyces with respect to ethylphenol production, and to develop a methodology for detecting this spoilage yeast and for estimating its population size in wines using differential-selective media and high performance liquid chromatography (HPLC). METHODS AND RESULTS: This work examines the reduction of p-coumaric acid and the formation of 4-vinylphenol and 4-ethylphenol (recorded by HPLC-DAD) in a prepared medium because of the activities of different yeast species and populations. A regression model was constructed for estimating the population of Dekkera/Brettanomyces at the beginning of fermentation via the conversion of hydroxycinnamic acids into ethylphenols. CONCLUSIONS: The proposed methodology allows the populations of Dekkera/Brettanomyces at the beginning of fermentation to be estimated in problem wines. Moreover, it avoids false positives because of yeasts resistant to the effects of the selective elements of the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This may help prevent the appearance of organoleptic anomalies in wines at the winery level.

Cell viability and prostacyclin release in cultured human umbilical vein endothelial cells.

Construction of efficient substitutes of human blood vessels is strongly dependent on the use of viable and fully functional cultured endothelial cells (ECs). However, very few reports have been published to date focused on the evaluation of cell viability of cultured ECs. In this work, we have determined cell viability, von Willebrand factor, and prostacyclin (PGI(2)) activity in primary cell cultures of human umbilical vein ECs, to identify the specific cell passage that is more appropriate for the development of artificial organs by tissue engineering. Cell viability was determined by quantification of the intracellular concentration of several ions by highly sensitive electron probe X-ray microanalysis, whereas von Willebrand was assayed by immunohistochemistry and PGI(2) release was quantified by radioimmunoassay. The results of our analyses demonstrate that the K/Na ratio was different for each cell passage (4.72 for the first passage, 4.55 for the second passage, and 7.82 for the third passage), suggesting that the highest cell viability corresponds to the third passage. In contrast, PGI(2) production was higher at the first two cell passages, with a significant decrease at the third passage (6.46 +/- 0.10, 5.98 +/- 0.08, and 1.62 +/- 0.05 ng/mL of supernatant for the first, second, and third passages, respectively), whereas von Willebrand expression was similar among the three cell passages analyzed in this work (64.12%, 66.66%, 65.93% of positive cells, respectively). These data suggest that cells corresponding to the second cell passage show the best ratio of viability to functionality and should therefore be used for tissue engineering protocols.

Effects of pH, temperature and SO2 on the formation of pyranoanthocyanins during red wine fermentation with two species of Saccharomyces.

The formation of vitisins A and B, p-coumaroyl and acetyl derivatives during the fermentation of red wine with two species of Saccharomyces was examined. One species, Saccharomyces cerevisiae strain 7VA was selected for its high production of acetaldehyde and pyruvic acid (7VA). The other (control) species, Saccharomyces uvarum strain S6U is used commercially for wine production. The final vitisins A and B concentrations produced with S. cerevisiae were, respectively, twice and three times that produced with S. uvarum. Models for the formation and accumulation of these vitisins are proposed. This is the first report that the formation of a vinylphenolic derivative of anthocyanin, malvidin-3-O-glucoside-4-vinylguaiacol, can be favored by fermentation with certain yeasts, possibly those with cinnamoyl decarboxylase activity. The effect of SO2, pH and temperature on the formation of pyranoanthocyanins during fermentation with S. cerevisiae and S. uvarum was also analyzed using High Pressure Liquid Chromatography (HPLC)/Photodiode Array Detection. The identification of these compounds was confirmed using HPLC/Electrospray Ionization-Mass Spectrometry.

Pyruvic acid and acetaldehyde production by different strains of Saccharomyces cerevisiae: relationship with Vitisin A and B formation in red wines.

The production of pyruvate and acetaldehyde by 10 strains of Saccharomyces cerevisiae was monitored during the fermentation of Vitis vinifera L. variety Tempranillo grape must to determine how these compounds might influence the formation of the pyroanthocyanins vitisin A and B (malvidin-3-O-glucoside-pyruvate acid and malvidin-3-O-glucoside-4 vinyl, respectively). Pyruvate and acetaldehyde production patterns were determined for each strain. Pyruvate production reached a maximum on day four of fermentation, while acetaldehyde production was at its peak in the final stages. The correlation between pyruvate production and vitisin A formation was especially strong (R (2) = 0.80) on day 4, when the greatest quantity of pyruvate was found in the medium. The correlation between acetaldehyde production and the formation of vitisin B was strongest (R (2) = 0.81) at the end of fermentation when the acetaldehyde content of the medium was at its highest. Identification and quantification experiments were performed by HPLC-DAD. The identification of the vitisins was confirmed by LC/ESI-MS.

Adsorption of anthocyanins by yeast cell walls during the fermentation of red wines.

This paper reports the anthocyanin adsorption profiles of the cell walls of different Saccharomyces strains isolated from grapes collected in the Spanish appellation controlée regions of La Rioja, Navarra, and Ribera del Duero. These strains are habitually used in red wine-making. The acyl derivatives of anthocyanins (acetyl and p-coumaryl compounds) were more strongly adsorbed than nonacyl derivatives. Peonidin-3G was also strongly adsorbed, as were its acyl derivatives. The greater presence of acetyl derivatives in the cell wall adsorbate leads to an increase in yellow color and a reduction in blue color with respect to the corresponding wine.