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The main objective of this work was to isolate and characterize viruses that infect both Saccharomyces and other Flor veil-forming genera and suggest the possible consequences they can have on the processes of formation of the Flor veil of typical D.O. Jerez-Xèrés-Sherry wines. Results obtained show that different Flor veil yeast strains isolated in Fino wines and in Amontillados presented a great resistance to ethanol and all of them presented infection by Totiviruses. These were identified after the extraction of double-stranded RNA and amplified by multiplex PCR with specific primers for the most common viruses involved in the killer factor. Subsequently, the same strains of both the S. cerevisiae species and Wickerhamomyces anomalus were subjected to an antiviral treatment with ribavirin. After microbiological analysis with Methylene Blue (MB) plates and by rt-PCR, it was determined that most of them did not show killer factor and the viruses involved in it had been correctly eliminated under the treatment with ribavirin. Subsequently, competition and implantation experiments of the treated and untreated yeasts were carried out against their controls. The wines inoculated with yeasts infected by Totiviruses showed to be dominant in all cases.

Abstract The wines produced in the Jerez-Xérès-Sherry Designation of Origin fall within the category of Fortified Wines, according to Regulation 1308/2013 of the European Parliament, which stipulates that they must be exclusively fortified with wine alcohol, with an acquired alcoholic strength between 95.0% and 96.0%. However, there are other alternative alcohols whose behavior is unknown but could be equally effective in the development of biological aging, with an impact on the final product quality and lower economic costs. This study examines the effect of using malt alcohols, grape pomace, agave, sugarcane, and cereal alcohols, as well as wine alcohols obtained through different distillation methods and at varying alcohol concentrations, for fortifying wines subjected to biological aging. The development of the "flor" yeast veil will be evaluated under each aging condition and isolated and characterized using molecular techniques. Additionally, the base wine fortified with the different alcohols will be monitored and analyzed both physicochemically and organoleptically.

The EC 2019/187 shows the rules for the usage of ethyl alcohols to fortify wines. These must be obtained from products derived from agriculture and the use of alcohols of mineral or chemical origin is not authorized. The ethyl alcohols authorized for the fortification in the D.O. Sherry have wine origin. But, there are other alcohols that could be equally productive and at a lower economic cost, such as beet or sugar cane alcohol. The effect that different types of ethyl alcohols may have on the yeasts of flor remains unknown. Our goal was to analyze the effects of other alcohols (beet, sugar cane, etc.) on the development of the veil of flor. In this way, the yeasts that constitute the veil of flor of Fino wines were isolated and identified by microbiological techniques and characterized by molecular tools and by biochemical and microbiological tests such as cellular hydrophobicity, flocculation, acetic acid production, etc. Then, base wines were fortified with different distilled alcohols, such as malt brandy, tequila, among others. Biological aging under the veil of flor was monitored to later determine the volatile fractions. Alcohol from sugar cane could be a promising alternative to fortify Fino wines.

During alcoholic fermentation, the yeasts responsible for the process produce various metabolites, including urea. The reaction of urea with ethanol spontaneously generates ethyl carbamate in wine, a carcinogenic metabolite whose concentration needs to be regulated. Yeasts produce urea during the arginine metabolic pathway. For this purpose, adaptive evolution has been used by using a toxic analogue of l-arginine, l-canavanine, for selective pressure, once increasing the genetic variability of the population by sporulation and subsequent conjugation. Using this strategy, five putative variants of the parental yeast have been obtained and seem to have reduced urea production, thus being able to achieve a lower concentration of ethyl carbamate.

Vitis vinifera L. ssp. Sylvestris(Gmelin) Hegi is recognized as the dioecious parental generation of today's cultivars. Climatic change and the arrival in Europe of pathogens and pests have led it to be included on the IUCN Red List of Threatened Species in 1997. At best of our knowledge, no studies on microbial populations of grape-berry surfaces have been done. The present work has been focused on the study of yeast occurrence and diversity on grape-berries collected from wild vines. Final outputs have allowed: ito obtain precise information about yeast communities; ii) to provide an objective framework for the classification of the broadest range of species according to their extinction risk; iii) to select attractive yeast strains for their biotechnological potential, offering new opportunities to winemakers. Sampling plan was performed in Azerbaijan, Georgia, Italy, Romania and Spain. In all, 3180 yeast colonies were isolated and identified as belonging to 50 species, including Saccharomyces cerevisiae, by 26S rDNA D1/D2 domains and ITS region sequencing. Isolates of S. cerevisiaewere also analysed by SSR-PCR obtaining 163 different genotypes. This study highlights the biodiversity potential of pristine environments that still represent a fascinating source to face common problems in winemaking.

Biological ageing is an essential process for obtaining some distinctive Sherry wines, such as Fino and Manzanilla. It occurs after the fermentation of the grape must due to the appearance of a biofilm on the surface of the wine called "veil of flor". Yeasts belonging to the Saccharomyces cerevisiae species mainly comprise such biofilm. Although other species have also been found, these have been traditionally considered spoilage. Indeed, it has even been hypothesised that they may not be able to form biofilm on their own under such conditions. In the present work, four different non-Saccharomyces yeasts isolated from barrels in the Jerez area under biological ageing have been characterised through their physiological abilities, including extracellular enzymatic and biofilm-forming capabilities. Results showed not only a surprising ethanol tolerance, above 15.5% in all cases, but also a significant degree of extracellular enzyme production, highlighting the urease and proteolytic activities found in Pichia manshurica, as well as lipolytic activity in Pichia kudriavzevii, Pichia membranifaciens and Wicherhamomyces anomalus. As a conclusion, these non-Saccharomyces could be very interesting in the oenological field, beyond improving the organoleptic characteristics as well as technological features in these wines. [ABSTRACT FROM AUTHOR]

There is growing interest in yeast selection for industrial fermentation applications since it is a factor that protects a wine's identity. Although it is strenuous evaluating the oenological characteristics of yeasts in selection processes, in many cases the most riveting yeasts produce some undesirable organoleptic characteristics in wine. The aim of the present work is to improve an industrial yeast strain by reducing its hydrogen sulfide (H2S) production. To accomplish this, two different improvement approaches were used on said yeast: hybridization by mass mating and adaptive laboratory evolution, both performed through spore generation and conjugation, thus increasing genetic variability. Three evolved variants with lower H2S production were obtained and used as starters to carry out fermentation at an industrial level. Wine quality was analyzed by its principal oenological parameters and volatile aroma compounds, which were both corroborated by sensory evaluations. Significant differences between the produced wines have been obtained and a substantial improvement in aromatic quality has been achieved. Both hybrids were the most different to the control due to terpenes and esters production, while the evolved strain was very similar to the parental strain. Not only have organoleptic defects been reduced at an industrial level, more floral and fruitier wines have been produced. [ABSTRACT FROM AUTHOR]

Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi is recognized as the dioecious parental generation of today's cultivars. Climatic change and the arrival of pathogens and pests in Europe led it to be included on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species in 1997. The present work focused on the study of culturable yeast occurrence and diversity of grape berries collected from wild vines. Sampling was performed in 29 locations of Azerbaijan, Georgia, Italy, Romania, and Spain. In total, 3431 yeast colonies were isolated and identified as belonging to 49 species, including Saccharomyces cerevisiae, by 26S rDNA D1/D2 domains and ITS region sequencing. Isolates of S. cerevisiae were also analyzed by SSR–PCR obtaining 185 different genotypes. Classical ecology indices were used to obtain the richness (S), the biodiversity (H'), and the dominance (D) of the species studied. This study highlights the biodiversity potential of natural environments that still represent a fascinating source of solutions to common problems in winemaking. [ABSTRACT FROM AUTHOR]

In this sense, food and fermented beverage safety is a systemic approach to hygiene and accountability that concerns every aspect of the global food and beverages industry. The vast majority of fermented beverages is in practice similar to winemaking, but beer, cider and other similar beverages typically contain much lower ethanol levels (around 5-7%) compared to wine (11-15%). After a microbiological analysis of the native yeast population and chemically studying the wine composition, their results showed that I L. thermotolerans i Lt93 was unable to overgrow wild yeast population in "Treixadura" white must. [Extracted from the article]

This study investigates the effects of simultaneous inoculation of a selected Saccharomyces cerevisiae yeast strain with two different commercial strains of wine bacteria Oenococcus oeni at the beginning of the alcoholic fermentation on the kinetics of malolactic fermentation (MLF), wine chemical composition, and organoleptic characteristics in comparison with spontaneous MLF in Tempranillo grape must from Castilla-La Mancha (Spain). Evolution of MLF was assessed by the periodic analysis of L-malic acid through the enzymatic method, and most common physiochemical parameters and sensory traits were evaluated using a standardized sensory analysis. The samples were analyzed by GC/MS in SCAN mode using a Trace GC gas chromatograph and a DSQII quadrupole mass analyzer. Co-inoculation reduced the overall fermentation time by up to 2 weeks leading to a lower increase in volatile acidity. The fermentation-derived wine volatiles profile was distinct between the co-inoculated wines and spontaneous MLF and was influenced by the selected wine bacteria used in co-inoculation. Co-inoculation allows MLF to develop under reductive conditions and results in wines with very few lactic and buttery flavors, which is related to the impact of specific compounds like 2,3-butanedione. This compound has been also confirmed as being dependent on the wine bacteria used. [ABSTRACT FROM AUTHOR]

Certain acetic and lactic acid bacteria are major causes of quality defects in musts and wines, giving rise to defects such as a "vinegary," "sharp, like nail polish-remover" taste or preventing alcoholic and/or malolactic fermentation. Sulfur dioxide is the major tool currently used in the control of these bacteria in wine. The aim of this work was to isolate bacteriophages from musts and wine of different grape varieties that were able to eliminate lactic and acetic acid bacteria spoilages at the laboratory scale. Musts obtained from grape-berries of Vitis vinifera cv. Chardonnay and Moscatel and a red wine made with V. vinifera cv. Tintilla de Rota were used to isolate bacteriophages. Bacteriophages were obtained from each of the musts and the wine and belonged to the order Caudovirals and the family Tectivirals. They were isolated by classical virology methods and identified by electron microscopy. The host bacteria used in the study were lactic acid bacteria of the species Lactobacillus hilgardii , Lactobacillus plantarum , and Oenococcus oeni and the acetic bacteria Acetobacter aceti. A comparative study was performed by adding phage titrations and SO2 to musts and wines, which had been previously inoculated with bacteria, to study the effectiveness of bacteriophages against bacteria. The comparative study showed that some bacteriophages were as effective as sulfur dioxide at low concentrations. [ABSTRACT FROM AUTHOR]

The principal role of wine yeast is to transform efficiently the grape-berries' sugars to ethanol, carbon dioxide, and other metabolites, without the production of off-flavors. Wine yeast strains are able to fermentmusts, while other commercial or laboratory strains fail to do so. The genetic differences that characterize wine yeast strains in contrast to the biological ageing of the veil-forming yeasts in Sherry wines are poorly understood. Saccharomyces cerevisiae strains frequently exhibit rather specific phenotypic features needed for adaptation to a special environment, like fortified wines with ethanol up to 15% (v/v), known as Sherry wines. Factors that affect the correct development of the veil of flor during ageing are also reviewed, along with the related aspects of wine composition, biofilm formation processes, and yeast autolysis. This review highlights the importance of yeast ecology and yeast metabolic reactions in determining Sherry wine quality and the wealth of untapped indigenous microorganisms co-existing with the veil-forming yeast strains. It covers the complexity of the veil forming wine yeasts' genetic features, and the genetic techniques often used in strain selection and monitoring during fermentation or biological ageing. Finally, the outlook for new insights to protect and to maintain the microbiota of the Sherry wines will be discussed. [ABSTRACT FROM AUTHOR]

The increasing level of hazardous residues in the environment and food chains has led the European Union to restrict the use of chemical fungicides. Thus, exploiting new natural antagonistic microorganisms against fungal diseases could serve the agricultural production to reduce pre- and post-harvest losses, to boost safer practices for workers and to protect the consumers' health. The main aim of this work was to evaluate the antagonistic potential of epiphytic yeasts against Botrytis cinerea, Aspergillus carbonarius, and Penicillium expansum pathogen species. In particular, yeast isolation was carried out from grape berries of Vitis vinifera ssp sylvestris populations, of the Eurasian area, and V. vinifera ssp vinifera cultivars from three different farming systems (organic, biodynamic, and conventional). Strains able to inhibit or slow the growth of pathogens were selected by in vitro and in vivo experiments. The most effective antagonist yeast strains were subsequently assayed for their capability to colonize the grape berries. Finally, possible modes of action, such as nutrients and space competition, iron depletion, cell wall degrading enzymes, diffusible and volatile antimicrobial compounds, and biofilm formation, were investigated as well. Two hundred and thirty-one yeast strains belonging to 26 different species were isolated; 20 of them, ascribed to eight species, showed antagonistic action against all molds. Yeasts isolated from V. vinifera ssp sylvestris were more effective (up to 50%) against B. cinerea rather than those isolated from V. vinifera ssp vinifera. Six strains, all isolated from wild vines, belonging to four species (Meyerozyma guilliermondii, Hanseniaspora uvarum, Hanseniaspora clermontiae, and Pichia kluyveri) revealed one or more phenotypical characteristics associated to the analyzed modes of antagonistic action. [ABSTRACT FROM AUTHOR]

Adaptation to changes in osmolarity is fundamental for the survival of living cells, and has implications in food and industrial biotechnology. It has been extensively studied in the yeast Saccharomyces cerevisiae, where the Hog1 stress activated protein kinase was discovered about 20 years ago. Hog1 is the core of the intracellular signaling pathway that governs the adaptive response to osmotic stress in this species. The main endpoint of this program is synthesis and intracellular retention of glycerol, as a compatible osmolyte. Despite many details of the signaling pathways and yeast responses to osmotic challenges have already been described, genome-wide approaches are contributing to refine our knowledge of yeast adaptation to hypertonic media. In this work, we used a quantitative fitness analysis approach in order to deepen our understanding of the interplay between yeast cells and the osmotic environment. Genetic requirements for proper growth under osmotic stress showed both common and specific features when hypertonic conditions were induced by either glucose or sorbitol. Tolerance to high-glucose content requires mitochondrial function, while defective protein targeting to peroxisome, GID-complex function (involved in negative regulation of gluconeogenesis), or chromatin dynamics, result in poor survival to sorbitolinduced osmotic stress. On the other side, the competitive disadvantage of yeast strains defective in the endomembrane system is relieved by hypertonic conditions. This finding points to the Golgi-endosome system as one of the main cell components negatively affected by hyperosmolarity. Most of the biological processes highlighted in this analysis had not been previously related to osmotic stress but are probably relevant in an ecological and evolutionary context. [ABSTRACT FROM AUTHOR]

In Georgia, one of the most ancient vine-growing environment, the homemade production of wine is still very popular in every rural family and spontaneous fermentation of must, without addition of chemical preservatives, is the norm. The present work investigated the yeast biodiversity in five Georgian areas (Guria, Imereti, Kakheti, Kartli, Ratcha-Lechkhumi) sampling grapes and wines from 22 different native cultivars, in 26 vineyards and 19 family cellars. One hundred and eighty-two isolates were ascribed to 15 different species by PCR-ITS and RFLP, and partial sequencing of D1/D2 domain 26S rDNA gene. Metschnikowia pulcherrima (F' =0.56, I' = 0.32), Hanseniaspora guilliermondii (F' = 0.49, I' = 0.27), and Cryptococcus flavescens (F' = 0.31, I' = 0.11) were the dominant yeasts found on grapes, whereas Saccharomyces cerevisiae showed the highest prevalence into wine samples. Seventy four isolates with fermentative potential were screened for oenological traits such as ethanol production, resistance to SO2, and acetic acid, glycerol and H2S production. Three yeast strains (Kluyveromyces marxianus UMY207, S. cerevisiae UMY255, Torulaspora delbrueckii UMY196) were selected and separately inoculated in vinifications experiments at a Georgian cellar. Musts were prepared from healthy grapes of local varieties, Goruli Mtsvane (white berry cultivar) and Saperavi (black berry cultivar). Physical (Brix) and microbial analyses (plate counts) were performed to monitor the fermentative process. The isolation of indigenous S. cerevisiae yeasts beyond the inoculated strains indicated that a co-presence occurred during the vinification tests. Results from quantitative GC-FID analysis of volatile compounds revealed that the highest amount of fermentation flavors, such as 4-ethoxy-4-oxobutanoic acid (monoethyl succinate), 2-methylpropan-1-ol, ethyl 2-hydroxypropanoate, and 2-phenylethanol, were significantly more produced in fermentation conducted in Saperavi variety inoculated with K. marxianus, whereas other aromatic compounds like 3-methylbutyl acetate, ethyl hexanoate and dihydrofuran-2(3H)-one (γ- butyrolactone) showed a higher content in Goruli Mtsvane variety samples fermented by S. cerevisiae. The selected yeast strains have proved to be promising for enhancing the flavor potential in low aromatic Georgian cultivars. This work intends to be a knowledge contribution for a precision oenology toward the strategic concept of "one grape variety-one yeast". [ABSTRACT FROM AUTHOR]

Acetic acid, a byproduct formed during yeast alcoholic fermentation, is the main component of volatile acidity (VA). When present in high concentrations in wine, acetic acid imparts an undesirable 'vinegary' character that results in a significant reduction in quality and sales. Previously, it has been shown that sake yeast strains resistant to the antifungal cerulenin produce significantly lower levels of VA. In this study, we used a classical mutagenesis method to isolate a series of cerulenin-resistant strains, derived from a commercial diploid wine yeast. Four of the selected strains showed a consistent low-VA production phenotype after small-scale fermentation of different white and red grape musts. Specific mutations in YAP1, a gene encoding a transcription factor required for oxidative stress tolerance, were found in three of the four low-VA strains. When integrated into the genome of a haploid wine strain, the mutated YAP1 alleles partially reproduced the low-VA production phenotype of the dip-loid cerulenin-resistant strains, suggesting that YAP1 might play a role in (reg-ulating) acetic acid production during fermentation. This study offers prospects for the development of low-VA wine yeast starter strains that could assist winemakers in their effort to consistently produce wine to definable qual-ity specifications. [ABSTRACT FROM AUTHOR]

In view of the growing concern about the impact of synthetic fungicides on human health and the environment, several government bodies have decided to ban them. As a result, a great number of studies have been carried out in recent decades with the aim of finding a biological alternative to inhibit the growth of fungal pathogens. In order to avoid the large losses of fruit and vegetables that these pathogens cause every year, the biological alternative's efficacy should be the same as that of a chemical pesticide. In this review, the main studies discussed concern Saccharomyces and non-Saccharomyces yeasts as potential antagonists against phytopathogenic fungi of the genera Penicillium and Aspergillus and the species Botrytis cinerea on table grapes, wine grapes, and raisins. [ABSTRACT FROM AUTHOR]

The extensive use of synthetic fertilizers and pesticides has negative consequences in terms of soil microbial biodiversity and environmental contamination. Faced with this growing concern, a proposed alternative agricultural method is the use of microorganisms as biofertilizers. Many works have been focused on bacteria, but the limited literature on yeasts and their potential ability to safely promote plant growth is gaining particular attention in recent years. Thus, the objective of this review is to highlight the application of yeasts as biological agents in different sectors of sustainable agricultural practices through direct or indirect mechanisms of action. Direct mechanisms include the ability of yeasts to provide soluble nutrients to plants, produce organic acids and phytohormones (indole-3-acetic acid). Indirect mechanisms involve the ability for yeasts to act as biocontrol agents through their high antifungal activity and lower insecticidal and herbicidal activity, and as soil bioremediating agents. They also act as protective agents against extreme environmental factors by activating defense mechanisms. It is evident that all the aspects that yeasts offer could be useful in the creation of quality biofertilizers and biopesticides. Hence, extensive research on yeasts could be promising and potentially provide an environmentally friendly solution to the increased crop production that will be required with a growing population. [ABSTRACT FROM AUTHOR]

Fino wine is one of the most important Sherry wines and it is obtained through a complex and dynamic biological aging system. In this study, wine and veil of flor samples from fifty-two barrels with different aging levels and distributed in three different wineries from the Jerez-Xèrés-Sherry winemaking area have been analyzed during two years. Some of the wine compounds most deeply involved in flor yeast metabolism were analyzed to take into account the blending effect of this system. On the other hand, veil of flor was analyzed by molecular methods, finding five different species: S. cerevisiae , W. anomalus , P. membranaefaciens, P. kudriavzevii and P. manshurica, being the first time that the three last species have been reported in this biological aging system. Since S. cerevisiae was the vast majority of the isolates, its intraspecies variability was also analyzed by the simultaneous amplification of three microsatellite loci, obtaining nine different S. cerevisiae genotypes, also differentiated according to their physiological properties. Biodiversity analysis showed there were significant differences between the three wineries in the three aging scales, although the overall diversity was relatively low. Moreover, variations in the relative frequency of the different S. cerevisiae genotypes were found to be seasonal-dependent. • Biological aging in Jerez area is more diverse in yeasts than previously thought. • Is the first time that some non- Saccharomyces are described in this system. • Population biodiversity analysis revealed low differentiation among wineries. • S. cerevisiae populations dynamically changed due to climatic conditions. [ABSTRACT FROM AUTHOR]