BUSCADOR RECOLECTA

Contiene además supplementary material.-- This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited., We have developed a wine fermentation procedure that takes advantage of the metabolic features of a previously characterized Metschnikowia pulcherrima strain in order to reduce ethanol production. It involves the use of M. pulcherrima/Saccharomyces cerevisiae mixed cultures, controlled oxygenation conditions during the first 48 h of fermentation, and anaerobic conditions thereafter. The influence of different oxygenation regimes and initial inoculum composition on yeast physiology and final ethanol content was studied. The impact of oxygenation on yeast physiology goes beyond the first aerated step and influences yields and survival rates during the anaerobic stage. The activity of M. pulcherrima in mixed oxygenated cultures resulted in a clear reduction in ethanol yield, as compared to S. cerevisiae. Despite relatively low initial cell numbers, S. cerevisiae always predominated in mixed cultures by the end of the fermentation process. Strain replacement was faster under low oxygenation levels. M. pulcherrima confers an additional advantage in terms of dissolved oxygen, which drops to zero after a few hours of culture, even under highly aerated conditions, and this holds true for mixed cultures. Alcohol reduction values about 3.7 % (v/v) were obtained for mixed cultures under high aeration, but they were associated to unacceptable volatile acidity levels. In contrast, under optimized conditions, only 0.35 g/L acetic acid was produced, for an alcohol reduction of 2.2 % (v/v), and almost null dissolved oxygen during the process., Authors would like to thank Cristina Juez Ojeda
andMiguel Ángel Fernández Recio for excellent technical assistance, the
YeSVitE consortium (EU project, 7FP-IRSES-GA no. 612441) for helpful
discussions, and Laura López Ocaña (CECT) for help with yeast
species confirmation. This work was supported by grants MINECO
AGL2012-32064 and INIA RM2012-00007-00-00 from the Spanish
Government. M.Q. was the recipient of a CSIC training contract, JAEDoc,
co-funded by the European Social Fund of the EU., Peer reviewed

This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited., Gassing pattern (gas valve status) employed in all the experiments after the initial assay., Authors would like to thank Cristina Juez Ojeda andMiguel Ángel Fernández Recio for excellent technical assistance, the YeSVitE consortium (EU project, 7FP-IRSES-GA no. 612441) for helpful discussions, and Laura López Ocaña (CECT) for help with yeast species confirmation. This work was supported by grants MINECO AGL2012-32064 and INIA RM2012-00007-00-00 from the Spanish Government. M.Q. was the recipient of a CSIC training contract, JAEDoc, co-funded by the European Social Fund of the EU., Peer reviewed

The growing interest of the winemaking industry on the use of non-Saccharomyces starters has prompted several studies about the physiological features of this diverse group of microorganisms. The fact that the proposed use of these new starters will almost invariably involve either simultaneous or sequential inoculation with Saccharomyces cerevisiae has also driven the attention to the potential biological interactions between different starters during wine fermentation. Our current understanding is that alternative yeast starters will affect wine features by both direct and indirect mechanisms (through metabolic or other types of interactions with S. cerevisiae). There are still few studies addressing the question of yeast–yeast interactions in winemaking by a transcriptomic approach. In a previous report, we revealed early responses of S. cerevisiae and Torulaspora delbrueckii to the presence of each other under anaerobic conditions, mainly the overexpression of genes related with sugar consumption and cell proliferation. We have now studied the response, under aerobic conditions, of S. cerevisiae to other two non-Saccharomyces species, Hanseniaspora uvarum and Candida sake, keeping T. delbrueckii as a reference; and always focusing on the early stages of the interaction. Results point to some common features of the way S. cerevisiae modifies its transcriptome in front of other yeast species, namely activation of glucose and nitrogen metabolism, being the later specific for aerobic conditions., This work was supported by grant AGL2015-63629-R (MINECO/FEDER, UE), YeSVitE consortium (EU project, 7FP-IRSES-GA no. 612441), JC is the recipient of a MINECO Formación Postdoctoral contract from the Spanish Government., Peer reviewed

The use of non-Saccharomyces strains in aerated conditions has proven effective for alcohol content reduction in wine during lab-scale fermentation. The process has been scaled up to 20 L batches, in order to produce lower alcohol wines amenable to sensory analysis. Sequential instead of simultaneous inoculation was chosen to prevent oxygen exposure of Saccharomyces cerevisiae during fermentation, since previous results indicated that this would result in increased acetic acid production. In addition, an adaptation step was included to facilitate non-Saccharomyces implantation in natural must. Wines elaborated with Torulaspora delbrueckii or Metschnikowia pulcherrima in aerated conditions contained less alcohol than control wine (S. cerevisiae, non-aerated). Sensory and aroma analysis revealed that the quality of mixed fermentations was affected by the high levels of some yeast amino acid related byproducts, which suggests that further progress requires a careful selection of non-Saccharomyces strains and the use of specific N-nutrients., This work was supported by grants MINECO/FEDER RTC-2014-2186-2 (SOUNDWINE) developed in collaboration with Agrovin S.A., and AGL2015-63629-R, and EU project YeSVitE. (7FP-IRSES-GA n° 612441). JAC and PSN are the recipients of a MINECO Formación Postdoctoral contract from the Spanish Government., Peer reviewed

Trabajo presentado en el 33rd International Specialised Symposium on Yeast (ISSY33), celebrado en Cork (Irlanda), del 26 al 29 de junio de 2017, Sulphur dioxide is used at several stages in winemaking, mainly as antioxidants and as antimicrobial, even before
the onset of fermentation. Sulphites are also used as preservatives in many canned and processed foods. Its
usefulness during wine fermentation depends on the relatively high tolerance to sulphites of wine strains of
Saccharomyces cerevisiae. Sulphite tolerance in wine yeast strains has been related to mutations that boost
transcription of SSU1, the gene coding for a plasma membrane sulphite pump required for efficient sulphite
efflux. We used competition of bar-coded Yeast Knockout (YKO) collections to better understand microbial
sulphite resistance, and sulphite cellular targets. Our results point to some of the cell functions that are more
specifically required to survive sulphite-induced stress. For example, we found increased sulphite sensitivity for
most KO strains involving genes required for autophagy, so pointing to this process as a key one in the
detoxification of sulphite, probably through recycling of damaged proteins. In addition, some deletion strains,
gained some competitiveness in the presence of sulphite (usually strains that were already impaired under
standard growth conditions). These late functions are probably targets of sulphite (i.e. specifically sensitive to
it)., MINECO(AGL2015-63629-R, RTC-2014-2186-2) EU(7FP-IRSES-GA612441); Junta de
Andalucía(P10-AGR6544), Peer reviewed

Trabajo presentado en el 33rd International Specialised Symposium on Yeast (ISSY33), celebrado en Cork (Irlanda), del 26 al 29 de junio de 2017 -- Trabajo presentado al 7th Congress of European Microbiologists (FEMS), celebrado en Valencia (España) del 9 al 13 de julio de 2017, Advances in microbial wine biotechnology have led to the recent commercialization of several non-Saccharomyces starter cultures. These are intended to be used in either simultaneous or sequential inoculation with Saccharomyces cerevisiae. We analysed the transcriptional response to co-cultivation of S. cerevisiae and Torulaspora delbrueckii. The study is focused in the initial stages of wine fermentation with the goal of better understand the microbial interactions that can be stablished during wine fermentation with mixed-starters. Fermentations were carried out in bioreactors using synthetic grape must to mimic industrial conditions. Experiments were carried out in triplicate for fermentation kinetics, CO2 performance and RNAseq analysis.
Both species showed a clear response to the presence of each other, even though the portion of the genome showing altered transcriptional levels was relatively small. Changes in the transcription pattern suggested a stimulation of metabolic activity and growth, as a consequence of the presence of competitors in the same medium. The response of S. cerevisiae seems to take place earlier, as compared to T. delbrueckii. Enhanced glycolytic activity of the mixed culture was confirmed by the CO2 production profile during these early stages of fermentation. Interestingly, HSP12 expression appeared induced by co-cultivation for both of S. cerevisiae and Torulaspora delbrueckii in the two time points studied. This might be related with a recently described role of Hsp12 in intercellular communication in yeast. Expression of S. cerevisiae PAU genes was also stimulated in mixed cultures., MINECO (AGL2015-63629-R,RTC-2014-2186-2); EU (7FP-IRSES-GA612441); (JdC- JA.Curiel)., Peer reviewed

Trabajo presentado en el 33rd International Specialised Symposium on Yeast (ISSY33), celebrado en Cork (Irlanda), del 26 al 29 de junio de 2017, Our group is interested in reducing ethanol yield during wine fermentation, to counter the growing sugar content in grapes due to climate change, and avoid the negative impact of increasing ethanol content in the wine market. While respiratory metabolism appears as an attractive alternative to pure fermentation, to redirect carbon flux towards sensory neutral products (CO2), both the Crabtree effect of Saccharomyces cerevisiae and the tendency to increased acetic acid production during aeration (as we have previously shown), constitute a hurdle to industrial application of respiration compatible fermentation conditions. However, mechanisms involved in acetic acid production by S. cerevisiae in the presence of oxygen are yet poorly understood. We addressed this topic by deletion of several genes, HXK2, REG1, PDE2, PDC1 (homozygous) and PYK1 (heterozygous) in the wine yeast strain FX0 (Laffort) background. Some of them had been previously related with the Crabtree effect. Our results show that oxygenation helps reducing ethanol content of wines. However, there is little impact of the deletions on this reduction. Interestingly, some of these deletions do results in a reduction in acetate yields during oxygenated fermentation of natural grape must., MINECO(AGL2015-63629-R, RTC-2014-2186-2) EU(7FP-IRSES-GA612441); Junta de Andalucía(P10-AGR6544), Peer reviewed

Trabajo presentado al 7th Congress of European Microbiologists (FEMS), celebrado en Valencia (España) del 9 al 13 de julio de 2017, Sulphur dioxide (often dosed as bisulphite salts) has had widespread use in winemaking for centuries. It is used at several stages of the process, and for several purposes, mainly as antioxidants and as antimicrobials, even before the onset of fermentation. Sulphites are also used as preservatives in many canned and processed foods. Its usefulness during wine fermentation depends on the relatively high tolerance to sulphites of industrial wine strains of the yeast Saccharomyces cerevisiae. Sulphite tolerance of these yeast strains is related to mutations that promote high level transcription of SSU1, the gene coding for a plasma membrane sulphite pump required for efficient sulphite efflux.

This work was designed, in order to better understand sulphite cellular targets in yeast, as well as microbial sulphite resistance.

By competition of bar-coded Yeast Knockout (YKO) collections we have identified some of the cell functions that are more specifically required in order to survive sulphite insult.

Our results show increased sulphite sensitivity for most KO strains involving genes required for autophagy, so pointing to this process as a key one in the detoxification of sulphite, probably through recycling of damaged proteins. Conversely, some deletion yeast strains, that were impaired under no-stressed conditions, recovered some competitiveness in the presence of sulphite. These functions are probably specifically sensitive to sulphite, so that already defective strains do not suffer from an additional impairment in the presence of sulphite., MINECO(AGL2015-63629-R, RTC-2014-2186-2) EU(7FP-IRSES-GA612441); Junta de Andalucía(P10-AGR6544), Peer reviewed

The dataset supporting the results of this article is available in the NCBI repository under BioProject PRJNA860570, Microbial diversity in vineyards and in grapes has generated significant scientific interest. From a biotechnological perspective, vineyard and grape biodiversity has been shown to impact soil, vine, and grape health and to determine the fermentation microbiome and the final character of wine. Thus, an understanding of the drivers that are responsible for the differences in vineyard and grape microbiota is required. The impact of soil and climate, as well as of viticultural practices in geographically delimited areas, have been reported. However, the limited scale makes the identification of generally applicable drivers of microbial biodiversity and of specific microbial fingerprints challenging. The comparison and meta-analysis of different datasets is furthermore complicated by differences in sampling and in methodology. Here we present data from a wide-ranging coordinated approach, using standardized sampling and data generation and analysis, involving four countries with different climates and viticultural traditions. The data confirm the existence of a grape core microbial consortium, but also provide evidence for country-specific microbiota and suggest the existence of a cultivar-specific microbial fingerprint for Cabernet Sauvignon grape. This study puts in evidence new insight of the grape microbial community in two continents and the importance of both location and cultivar for the definition of the grape microbiome., The YeSViTE project (FP7-IRSES-2013-GA612441) supported the secondments of J.T. and
F.V. to the Stellenbosch University (South Africa) and R.F. to the Agrarian University of Georgia
(Georgia), and the grape sampling in Tuscany carried out by D.F. This work was also supported by
Winetech grant SU IWBT 16-02.