Felipe Palomero

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.

Combine Use of Selected Schizosaccharomyces pombe and Lachancea thermotolerans Yeast Strains as an Alternative to the Traditional Malolactic Fermentation in Red Wine Production

Most red wines commercialized in the market use the malolactic fermentation process in order to ensure stability from a microbiological point of view. In this second fermentation, malic acid is converted into l-lactic acid under controlled setups. However this process is not free from possible collateral effects that on some occasions produce off-flavors, wine quality loss and human health problems. In warm viticulture regions such as the south of Spain, the risk of suffering a deviation during the malolactic fermentation process increases due to the high must pH. This contributes to produce wines with high volatile acidity and biogenic amine values. This manuscript develops a new red winemaking methodology that consists of combining the use of two non-Saccharomyces yeast strains as an alternative to the traditional malolactic fermentation. In this method, malic acid is totally consumed by Schizosaccharomyces pombe, thus achieving the microbiological stabilization objective, while Lachancea thermotolerans produces lactic acid in order not to reduce and even increase the acidity of wines produced from low acidity musts. This technique reduces the risks inherent to the malolactic fermentation process when performed in warm regions. The result is more fruity wines that contain less acetic acid and biogenic amines than the traditional controls that have undergone the classical malolactic fermentation.

Selected Schizosaccharomyces pombe Strains Have Characteristics That Are Beneficial for Winemaking.

At present, wine is generally produced using Saccharomyces yeast followed by Oenococus bacteria to complete malolactic fermentation. This method has some unsolved problems, such as the management of highly acidic musts and the production of potentially toxic products including biogenic amines and ethyl carbamate. Here we explore the potential of the fission yeast Schizosaccharomyces pombe to solve these problems. We characterise an extensive worldwide collection of S. pombe strains according to classic biochemical parameters of oenological interest. We identify three genetically different S. pombe strains that appear suitable for winemaking. These strains compare favourably to standard Saccharomyces cerevisiae winemaking strains, in that they perform effective malic acid deacidification and significantly reduce levels of biogenic amines and ethyl carbamate precursors without the need for any secondary bacterial malolactic fermentation. These findings indicate that the use of certain S. pombe strains could be advantageous for winemaking in regions where malic acid is problematic, and these strains also show superior performance with respect to food safety.

Physiological features of Schizosaccharomyces pombe of interest in making of white wines

This work studies the physiology of Schizosaccharomyces pombe strain 938 in the production of white wine with high malic acid levels as the sole fermentative yeast, as well as in mixed and sequential fermentations with Saccharomyces cerevisiaeCru Blanc. The induction of controlled maloalcoholic fermentation through the use of Schizosaccharomyces spp. is now being viewed with much interest. The acetic, malic and pyruvic acid concentrations, relative density and pH of the musts were measured over the entire fermentation period. In all fermentations in which Schizo. pombe 938 was involved, nearly all the malic acid was consumed and moderate acetic concentrations produced. The urea content and alcohol level of these wines were notably lower than in those made with Sacch. cerevisiae Cru Blanc alone. The pyruvic acid concentration was significantly higher in Schizo. pombe fermentations. The sensorial properties of the different final wines varied widely.

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.

Oenological versatility of Schizosaccharomyces spp.

The biodiversity of non-Saccharomyces yeasts is currently a topic of great interest. The possibility of their use in winemaking has led to much research into the metabolic and structural properties of some of these yeasts, such as those belonging to Torulaspora, Pichia, Hanseniaspora and Hansenula. The present work reviews our knowledge of the genus Schizosaccharomyces, the use of which in winemaking has recently been discussed at the International Organisation of Vine and Wine. However, despite offering the advantage of malic dehydrogenase activity, plus a wall structure that ensures the autolytic release of mannoproteins and polysaccharides during ageing over lees, only one commercial strain of Schizosaccharomyces pombe is currently available.

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.

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.

Identifying yeasts belonging to the Brettanomyces/Dekkera genera through the use of selective-differential media

The purpose of this work was to compare and optimise different selective and differential media to aid in isolating spoilage yeasts belonging to the Brettanomyces/Dekkera genera. Growth media containing selective and differential factors were employed. These were inoculated with strains of yeast representing Spanish oenological microbiota. Lastly, some of these isolation media were successfully applied in 24 types of wine with a high ethylphenol content, all of which were from the Haro Oenological Station (La Rioja, Spain). p-coumaric acid was determined using High performance liquid chromatography-photodiode-array detection-electrospray ionization mass spectrometry (HPLC-DADESI/MS); 4-ethylphenol by using Solid phase micro extraction-gas chromatography-mass spectrometry (SPME-GC-MS); and the rest of the analysis was carried out using official OIV methodology. Actidione is the most effective selective factor for isolating Brettanomyces/Dekkera yeast genera. Other secondary selective factors (selective carbon sources, sorbic acid and ethanol as a microbicide agent) may be used successfully to eliminate potential false positivities; however, they slow growth and delay the time to obtain results.

Wood impregnation of yeast lees for winemaking.

This study develops a new method to produce more complex wines by means of an indirect diffusion of wood aromas from yeast cell-walls. An exogenous lyophilized biomass was macerated with an ethanol wood extract solution and subsequently dried. Different times were used for the adsorption of polyphenols and volatile compounds to the yeast cell-walls. The analysis of polyphenols and volatile compounds (by HPLC/DAD and GC-MS, respectively) demonstrate that the adsorption/diffusion of these compounds from the wood to the yeast takes place. Red wines were also aged with Saccharomyces cerevisiae lees that had been impregnated with wood aromas and subsequently dried. Four different types of wood were used: chestnut, cherry, acacia and oak. Large differences were observed between the woods studied with regards to their volatile and polyphenolic profiles. Sensory evaluations confirmed large differences even with short-term contact between the wines and the lees, showing that the method could be of interest for red wine making. In addition, the results demonstrate the potential of using woods other than oak in cooperage.