The life cycle of most non-conventional yeasts, such as Torulaspora delbrueckii (Td), is not as well-understood as that of Saccharomyces cerevisiae (Sc). Td is generally assumed to be haploid, which detracts from some biotechnological properties compared to diploid Sc strains. We analyzed the life cycle of several Td wine strains and found that they were mainly diploid during exponential growth in rich medium. However, most cells became haploid in stationary phase, as observed for Sc haploid heterothallic strains. When transferred and incubated in nutrient-deficient media, these haploid cells became polymorphic, enlarged, and transitioned to diploid or polyploid states. The increased ploidy, that mainly results from supernumerary mitosis without cytokinesis, was followed by sporulation. A similar response was observed in yeasts that remained alive during the second fermentation of base wine for sparkling wine making, or during growth in ethanol-supplemented medium. This response was not observed in the Sc yeast populations under any of the experimental conditions assayed, which suggests that it is a specific adaptation of Td to the stressful fermentation conditions. This response allows Td yeasts to remain alive and metabolically active longer during wine fermentation. Consequently, we designed procedures to increase the cell size and ploidy of haploid Td strains. Td inocula with increased ploidy showed enhanced fermentation efficiency compared to haploid inocula of the same strains.

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 Ucles, 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.

The yeasts Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) may show a killer phenotype that is encoded in dsRNA M viruses (V-M), which require the helper activity of another dsRNA virus (V-LA or V-LBC) for replication. Recently, two TdV-LBCbarr genomes, which share sequence identity with ScV-LBC counterparts, were characterized by high-throughput sequencing (HTS). They also share some similar characteristics with Sc-LA viruses. This may explain why TdV-LBCbarr has helper capability to maintain M viruses, whereas ScV-LBC does not. We here analyze two stretches with low sequence identity (LIS I and LIS II) that were found in TdV-LBCbarr Gag-Pol proteins when comparing with the homologous regions of ScV-LBC. These stretches may result from successive nucleotide insertions or deletions (indels) that allow compensatory frameshift events required to maintain specific functions of the RNA-polymerase, while modifying other functions such as the ability to bind V-M (+)RNA for packaging. The presence of an additional frameshifting site in LIS I may ensure the synthesis of a certain amount of RNA-polymerase until the new compensatory indel appears. Additional 5′- and 3′-extra sequences were found beyond V-LBC canonical genomes. Most extra sequences showed high identity to some stretches of the canonical genomes and can form stem-loop structures. Further, the 3′-extra sequence of two ScV-LBC genomes contains rRNA stretches. The origin and possible functions of these extra sequences are here discussed.

Non-conventional yeasts such as Torulaspora delbrueckii (Td) have been proposed for sparkling winemaking. Unfortunately, this yeast has poor efficiency in completing wine fermentation as compared to Saccharomyces cerevisiae (Sc). New mutants with increased resistance to SO2, ethanol, and high CO2 pressure were previously isolated from spore clones of Td. Although these mutants showed improved capability for base wine fermentation, there is still room for genetic improvement of Td yeasts until the fermentative capacity of Sc is achieved. As an alternative approach, yeast mixture for eventual hybridization of Td with Sc was assayed in this study. The new yeast mixture clones (Sc-mixed Td) showed an intermediate phenotype between both parent yeasts for some relevant biotechnological properties, such as resistance to SO2, ethanol, copper, high CO2 pressure, and high temperature, as well as flocculation potential. These properties varied depending on the specific Sc-mixed Td clone. Several mixture clones showed improved capability for base wine fermentation as compared to the Td parent strain, approaching the fermentation capability of the Sc parent strain. The organoleptic quality of sparkling wine was also improved by using some mixture clones and this improved quality coincided with an increased amount of acetate and ethyl esters. The genetic stability of some Sc-mixed Td clones was good enough for commercial yeast production and winery applications.

In recent years, the spread of antibiotic-resistant bacteria and efforts to preserve food microbiota have induced renewed interest in phage therapy. Phage cocktails, instead of a single phage, are commonly used as antibacterial agents since the hosts are unlikely to become resistant to several phages simultaneously. While the spectrum of activity might increase with cocktail complexity, excessive phages could produce side effects, such as the horizontal transfer of genes that augment the fitness of host strains, dysbiosis or high manufacturing costs. Therefore, cocktail formulation represents a compromise between achieving substantial reduction in the bacterial loads and restricting its complexity. Despite the abovementioned points, the observed bacterial load reduction does not increase significantly with the size of phage cocktails, indicating the requirement for a systematic approach to their design. In this work, the information provided by host range matrices was analyzed after building phage-bacteria infection networks (PBINs). To this end, we conducted a meta-analysis of 35 host range matrices, including recently published studies and new datasets comprising Escherichia coli strains isolated during ripening of artisanal raw milk cheese and virulent coliphages from ewes’ feces. The nestedness temperature, which reflects the host range hierarchy of the phages, was determined from bipartite host range matrices using heuristic (Nestedness Temperature Calculator) and genetic (BinMatNest) algorithms. The latter optimizes matrix packing, leading to lower temperatures, i.e., it simplifies the identification of the phages with the broadest host range. The structure of infection networks suggests that generalist phages (and not specialist phages) tend to succeed in infecting less susceptible bacteria. A new metric (Φ), which considers some properties of the host range matrices (fill, temperature, and number of bacteria), is proposed as an estimator of phage cocktail size. To identify the best candidates, agglomerative hierarchical clustering using Ward’s method was implemented. Finally, a cocktail was formulated for the biocontrol of cheese-isolated E. coli, reducing bacterial counts by five orders of magnitude.

The killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded in the genome of medium-size dsRNA viruses (V-M). Killer strains also contain a helper large size (4.6 kb) dsRNA virus (V-LA) which is required for maintenance and replication of V-M. Another large-size (4.6 kb) dsRNA virus (V-LBC), without known helper activity to date, may join V-LA and V-M in the same yeast. T. delbrueckii Kbarr1 killer strain contains the killer virus Mbarr1 in addition to two L viruses, TdV-LAbarr1 and TdV-LBCbarr1. In contrast, the T. delbrueckii Kbarr2 killer strain contains two M killer viruses (Mbarr1 and M1) and a LBC virus (TdV-LBCbarr2), which has helper capability to maintain both M viruses. The genomes of TdV-LBCbarr1 and TdV-LBCbarr2 were characterized by high-throughput sequencing (HTS). Both RNA genomes share sequence identity and similar organization with their ScV-LBC counterparts. They contain all conserved motifs required for translation, packaging, and replication of viral RNA. Their Gag-Pol amino-acid sequences also contain the features required for cap-snatching and RNA polymerase activity. However, some of these motifs and features are similar to those of LA viruses, which may explain that at least TdV-LBCbarr2 has a helper ability to maintain M killer viruses. Newly sequenced ScV-LBC genomes contained the same motifs and features previously found in LBC viruses, with the same genome location and secondary structure. Sequence comparison showed that LBC viruses belong to two clusters related to each species of yeast. No evidence for associated co-evolution of specific LBC with specific M virus was found. The presence of the same M1 virus in S. cerevisiae and T. delbrueckii raises the possibility of cross-species transmission of M viruses.

The use of Torulaspora delbrueckii has been repeatedly proposed to improve a wine's organoleptic quality. This yeast has lower efficiency in completing wine fermentation than Saccharomyces cerevisiae since it has less fermentation capability and greater sensitivity to SO2, ethanol, and CO2 pressure. Therefore, the completion of fermentation is not guaranteed when must or wine is single-inoculated with T. delbrueckii. To solve this problem, new strains of T. delbrueckii with enhanced resistance to winemaking conditions were obtained. A genetic study of four wine T. delbrueckii strains was carried out. Spore clones free of possible recessive growth-retarding alleles were obtained from these yeasts. These spore clones were used to successively isolate mutants resistant to SO2, then those resistant to ethanol, and finally those resistant to high CO2 pressure. Most of these mutants showed better capability for base wine fermentation than the parental strain, and some of them approached the fermentation capability of S. cerevisiae. The genetic stability of the new mutants was good enough to be used in industrial-level production in commercial wineries. Moreover, their ability to ferment sparkling wine could be further improved by the continuous addition of oxygen in the culture adaptation stage prior to base wine inoculation.

The killer strains of Torulaspora delbrueckii can be used to improve the dominance of this yeast during must fermentation. The present work analyzes its usefulness for traditional sparkling wine making. T. delbrueckii killer strain dominated base wine fermentation better than non-killer strains and produced dried wines. The foam ability of T. delbrueckii base wines was very low compared to that of Saccharomyces cerevisiae. Significant positive correlations of foam parameters were found with some amounts of C4–C16 ethyl esters and proteins, and negative correlations with some antifoam alcohols. The organoleptic quality of T. delbrueckii base wines was considered unusual for cava making. While S. cerevisiae (single or mixed with T. delbrueckii) completed the second fermentation to produce dry sparkling wines with high CO2 pressure, single T. delbrueckii did not complete this fermentation, leaving sweet wines with low CO2 pressure. Death due to CO2 pressure was much higher in T. delbrueckii than in S. cerevisiae, making any killer effect of S. cerevisiae on T. delbrueckii irrelevant. However, the organoleptic quality of cava inoculated with mixtures of the two yeast species was better than that of wine inoculated exclusively with S. cerevisiae, and no deterioration in the quality of the foam was observed.

Wine killer yeasts such as killer strains of Torulaspora delbrueckii and Saccharomyces cerevisiae contain helper large-size (4.6 kb) dsRNA viruses (V-LA) required for the stable maintenance and replication of killer medium-size dsRNA viruses (V-M) which bear the genes that encode for the killer toxin. The genome of the new V-LA dsRNA from the T. delbrueckii Kbarr1 killer yeast (TdV-LAbarr1) was characterized by high-throughput sequencing (HTS). The canonical genome of TdV-LAbarr1 shares a high sequence identity and similar genome organization with its Saccharomyces counterparts. It contains all the known conserved motifs predicted to be necessary for virus translation, packaging, and replication. Similarly, the Gag-Pol amino-acid sequence of this virus contains all the features required for cap-snatching and RNA polymerase activity, as well as the expected regional variables previously found in other LA viruses. Sequence comparison showed that two main clusters (99.2–100% and 96.3–98.8% identity) include most LA viruses from Saccharomyces, with TdV-LAbarr1 being the most distant from all these viruses (61.5–62.5% identity). Viral co-evolution and cross transmission between different yeast species are discussed based on this sequence comparison. Additional 5′ and 3′ sequences were found in the TdV-LAbarr1 genome as well as in some newly sequenced V-LA genomes from S. cerevisiae. A stretch involving the 5′ extra sequence of TdV-LAbarr1 is identical to a homologous stretch close to the 5′ end of the canonical sequence of the same virus (self-identity). Our modeling suggests that these stretches can form single-strand stem loops, whose unpaired nucleotides could anneal to create an intramolecular kissing complex. Similar stem loops are also found in the 3′ extra sequence of the same virus as well as in the extra sequences of some LA viruses from S. cerevisiae. A possible origin of these extra sequences as well as their function in obviating ssRNA degradation and allowing RNA transcription and replication are discussed.

In recent years, the problem arising from the weed control in perennial crops is increasing, both in young woody crops during the first years of cultivation and in established plantations. The control of weeds through herbicides, the most widespread practice, presents many inconveniences, among which we can mention the appearance of weeds resistant or tolerant to them, crop phytotoxicities, especially in young plantations, or the significant reduction in the active materials allowed. Another aspect to take into account is the search for techniques that allow saving crop water consumption by reducing soil evaporation Therefore, in this work we evaluate the effect of three mixtures based on by-products derived from the agricultural sector, mixed with a binder and recycled paper paste and applied in liquid form on the ground with subsequent solidification (hydromulch), on different soil parameters (soil water content, temperatura and flow of CO2) in a young olive plantation in containers of 700 liters capacity. Additionally, two control treatments were included (manual weeding and a no-weeding treatments). In summary, and as preliminary results, hydromulches increased the soil water content, reduced slightly the soil temperature in the summer season and increased the flow of CO2, indicative of a higher soil microbial activity, closely related with the air temperature and the soil moisture. These preliminary results position hydromulches as an interesting alternative to herbicides and the conventional plastic mulches. Keywords: hydromulches, soil CO2 flow, soil temperatura, soil water content. Acknowledgements: Project RTA2015-00047-C05-03 - INIA (Spanish Ministry of Economy and Competitiveness).

Yeast mannoproteins have been thoroughly studied in recent years due to their contribution to different properties of wines. Our working group has the aim to establish the possible relationship between the structure of the mannoprotein (size and charge) and its effect on wines. For this, we have different non-transgenic mutants that synthesize mannoproteins with altered but known structures (mnn mutants). We have constructed double mutants with each of the available single mutants, thus obtaining a very diverse collection of mannoproteins that differ in size and charge. Since the mutants are unable to complete wine fermentations satisfactorily, an alternative is to grow mutants in the laboratory, purify their mannoproteins and add them later to wines. Another option would be to use co-inocula of the mentioned mutants, together with “killer” wine yeast strains capable of killing the mutants. In this work, experiments were carried out to determine the behavior of different “killer” wine strains with each one of the defective mutants in mannoprotein synthesis. The yeast Torulaspora delbrueckii Killer (Kbarr1) has been proved to be especially suitable for making this type of co-inoculum.

Torulaspora delbrueckii is probably the most suitable non-Saccharomyces yeast for winemaking. This is because it has a good fermentation performance compared to other non-Saccharomyces yeasts that might be considered for winemaking, such as Hanseniaspora uvarum, H. vineae, Candida zemplinina, C. pulcherrima, C. stellata, Schizosaccharomyces pombe, Hansenula anomala, Metschnikowia pulcherrima, Lachancea thermotolerans, Pichia fermentants, P. kluyveri, and Kazachstania aerobia. Additionally, it has been claimed that T. delbrueckii can be used to optimize some wine parameters with respect to usual S. cerevisiae wines. However, this option entails additional complications and economic costs in controlling must fermentation with respect to the use of conventional selected strains of S. cerevisiae, which is undoubtedly the most reliable yeast species for this purpose. Some of the complications of using T. delbrueckii in commercial wineries come from its physiological properties under the stressing conditions that are usual in the winemaking process. In this work, we review these specific properties, with a focus on their relevance for some applied aspects of winemaking. Many of the claimed advantages for Torulaspora are based on results from different research studies that are often contradictory, or non-reproducible. The easiest explanation is to attribute these discrepancies to possible differences in behavior of the different strains of T. delbrueckii used in different researches. However, it is quite possible that the different results found in the literature are due to the variable or unpredictable behavior of T. delbrueckii depending on these environmental conditions. Given this current situation, in order to obtain the required improvement of wine quality, we would propose for the time being to use single inoculation with T. delbrueckii under appropriate conditions to achieve its full dominance, and then to mix the resulting wines with other S. cerevisiae wines.

The cultivable microorganisms isolated from grapes and soils of two contiguous vineyards were analysed. Two types of management system were tested in each vineyard: conventional tillage (CT), and no-tillage with natural green cover vegetation (NV), both under semi-arid rainfed conditions. The main objective was to gather evidence as to whether the yeasts present in ripe grapes before harvest and those found in spontaneous wine fermentations came from the vineyard soil which could then be regarded as a natural reservoir for these yeasts. Bacteria isolated from the grapes all corresponded to three genera (Pseudomonas, Stenotrophomonas, and Bacillus) that were very abundant in soil samples taken just before grape harvest, indicative of probable contamination of the grapes with soil microorganisms. The amounts of fermentative yeasts in vineyard soil increased significantly during the dates close to harvest. Some yeasts were isolated from soils and spontaneous fermentations (Saccharomyces and Lanchacea), while others were only isolated from fermentations (Hanseniaspora, Metschnikowia, and Pichia) or from soils (Torulaspora). Saccharomyces yeasts were isolated from vineyard soil only after grape harvest, to thereafter become undetectable. The analysis of sterile-must fermentations inoculated with soil samples showed that soil was not the origin of the most abundant fermentative yeasts in spontaneous grape fermentations (Saccharomyces and Hanseniaspora), regardless of the soil management system applied. In contrast, other fermentative wine yeasts such as Lanchacea and Torulaspora seemed to be permanently resident in the vineyard soil, especially in the NV vineyard. Hence, contamination of the grapes with vineyard soil may increase yeast biodiversity during spontaneous fermentation, which could affect the wine's organoleptic quality.

Mannan defective (mnn) mutants have constituted a fundamental tool in the study of the structure and biosynthesis of mannoproteins in Saccharomyces cerevisiae. They were isolated by the group of Dr. C.E. Ballou by random mutagenesis, and a selection method using specific antibodies obtained against the wild-type strain. Initially, the mutants were characterized biochemically, and in subsequent years the genes in which they were mutated were identified. All of them encode membrane proteins that catalyze the transfer of mannoses to N-oligosaccharides, sometimes isolated or as part of complexes made up of several proteins. However, the specific mutation of each of these mutants has only been identified in the case of mnn3. In this work, we have completed the characterization of the mutants by sequencing the mutated genes in each of them. As expected, they are point mutations that involve the change of one amino acid for another in the mutated protein, or for a stop signal, resulting in a truncated protein.

For still wines, killer strains of Torulaspora delbrueckii can be used instead of non-killer strains to improve this species' domination during must fermentation, with an ensured, reliable impact on the final wine quality. The present work analysed the usefulness of these killer yeasts for sparkling-wine making. After the first fermentation, the foaming capacity of T. delbrueckii base wines was very low compared to Saccharomyces cerevisiae base wines. Significant positive correlations of foaming parameters were found with the amounts of C4–C16 ethyl esters and proteins, and negative with some anti-foaming alcohols produced by each yeast species. There were, however, no evident positive effects of polysaccharides on those parameters. The organoleptic quality of the T. delbrueckii base wines was judged inappropriate for sparkling-wine making, so that the following second-fermentation experiments only used a single assemblage of S. cerevisiae base-wines. While second fermentation was completed with inoculation of S. cerevisiae (both alone and mixed with T. delbrueckii) to yield dry sparkling wines with high CO2 pressure, single inoculation with T. delbrueckii did not complete this fermentation, leaving sweet wines with poor CO2 pressure. Yeast death due to CO2 pressure was much greater in T. delbrueckii than in S. cerevisiae, making any killer effect of S. cerevisiae over T. delbrueckii irrelevant because no autolysed cells were found during the first days of mixed-inoculated second fermentation. Nonetheless, the organoleptic quality of the mixed-inoculated sparkling wines was better than that of wines single-inoculated with S. cerevisiae, and showed no deterioration in foam quality. This seemed mainly to be because T. delbrueckii increased the amounts of ethyl propanoate and some acids (e.g., isobutyric and butanoic), alcohols (e.g., 3‑ethoxy‑1‑propanol), and phenols (e.g., 4‑vinylguaiacol). For these sparkling wines, no significant correlations between foaming parameters and aroma compounds were found, probably because the differences in foaming parameter values among these wines were fairly small. This is unlike the case for the base wines for which there were large differences in these parameters, which facilitated the analysis of the influence of aroma compounds on base-wine foamability.

Torulaspora delbrueckii is probably the non-Saccharomyces yeast that is currently most used for winemaking. Multiple advantages have been claimed for it relative to conventional S. cerevisiae strains. However, many of these claimed advantages are based on results in different research studies that are contradictory or non-reproducible. The easiest way to explain these discrepancies is to attribute them to the possible differences in the behaviour of the different strains of this yeast that have been used in different investigations. There is much less knowledge of the physiology, genetics, and biotechnological properties of this yeast than of the conventional yeast S. cerevisiae. Therefore, it is possible that the different results that have been found in the literature are due to the variable or unpredictable behaviour of T. delbrueckii, which may depend on the environmental conditions during wine fermentation. The present review focusses on the analysis of this variable behaviour of T. delbrueckii in the elaboration of different wine types, with special emphasis on the latest proposals for industrial uses of this yeast.

Soil's microbiological settlement in a Zea mays parcel under long-term agricultural practices aiming to minimize the disruption of the soil's structure, composition and natural biodiversity was analyzed by culture-dependent and culture-independent processes. Of the different processes, morphological-type differentiation of cultured microflora produced the best results and, while Polymerase Chain Reaction (PCR)-agarose electrophoresis has also provided us with reliable ones, soil PCR-DGGE (Denaturing Gradient Gel Electrophoresis) did not, which may occur because of the dependence of the method on the practice. Over a three-year period, this soil seemed very stable as its C/N ratio remained roughly constant and available for microbial growth. Because no soil overturning occurred, we were able to maintain most of the cultured microbial population whose fluctuations depended only on edaphoclimatic conditions. The number of cultured bacteria, molds, total microorganisms, and the biodiversity indices were usually lower in the driest season (fall) than in the rest of the year, except for Acinetobacter and Stenotrophomonas, which showed the opposite behavior. Coincident with the rise in temperature during the summer, the relative abundance of Gram+ bacteria increased, mostly reflecting an increase in the spore-forming bacteria Streptomyces and Bacillus. Despite these variations, the evenness index and the quantity of distinct microbiological life remained practically unaltered, recovering their maximum levels when the proper edaphoclimatic conditions were present, which indicates the long-term stability of the microbial community in this soil. The performed study put forward important insights for assessing the sustainability of maize production under long-term conservation agriculture management systems, highlighting that adequate management might prevent the degradation of soil quality, thus contributing to promote sustainable agriculture.

Viral M-dsRNAs encoding yeast killer toxins share similar genomic organization, but no overall sequence identity. The dsRNA full-length sequences of several known M-viruses either have yet to be completed, or they were shorter than estimated by agarose gel electrophoresis. High-throughput sequencing was used to analyze some M-dsRNAs previously sequenced by traditional techniques, and new dsRNAs from atypical killer strains of Saccharomyces cerevisiae and Torulaspora delbrueckii. All dsRNAs expected to be present in a given yeast strain were reliably detected and sequenced, and the previously-known sequences were confirmed. The few discrepancies between viral variants were mostly located around the central poly(A) region. A continuous sequence of the ScV-M2 genome was obtained for the first time. M1 virus was found for the first time in wine yeasts, coexisting with Mbarr-1 virus in T. delbrueckii. Extra 5′- and 3′-sequences were found in all M-genomes. The presence of repeated short sequences in the non-coding 3′-region of most M-genomes indicates that they have a common phylogenetic origin. High identity between amino acid sequences of killer toxins and some unclassified proteins of yeast, bacteria, and wine grapes suggests that killer viruses recruited some sequences from the genome of these organisms, or vice versa, during evolution.

espanolSe pretende acelerar la muerte celular y la autolisis de las levaduras durante la segunda fermentacion y crianza del cava inoculando con levaduras killer S. cerevisiae en la fase de tiraje. Partimos de la base de que varias levaduras killer matan a otras levaduras vinicas (killer o sensibles) en condiciones de laboratorio similares a las utilizadas en la elaboracion de cava (bajo pH y temperatura). Afortunadamente, algunas de estas estirpes de levaduras tambien mostraron un fenotipo killer intenso durante la segunda fermentacion en botella bajo presion de anhidrido carbonico. Ademas, el efecto killer en los cavas inoculados con cultivos mixtos de levaduras (killer + sensibles) mejoro la calidad de la espuma y la sensacion en boca respecto a los cavas elaborados sin efecto killer inoculados con cada levadura por separado. Esta mejora fue muy evidente cuando el efecto killer fue mas intenso y se produjo de forma rapida pocos dias despues del tiraje, y menos destacada cuando este efecto fue menos intenso y mas lento. Solo en los cavas en los que se produjo efecto killer se detectaron levaduras destrozadas consecuencia de la autolisis celular durante los primeros dias de fermentacion, que fueron reabsorbiendose en el vino progresivamente hasta desaparecer. La calidad del cava no correlaciono siempre con la concentracion de polisacaridos, proteinas, manano, o compuestos aromaticos. Esto sugiere que la sensacion en boca y la calidad de la espuma de estos vinos son caracteristicas complejas dependientes de los distintos compuestos liberados por las levaduras autolisadas y de las interacciones entre ellos. EnglishIt is intended to accelerate cell death and autolysis of yeasts during the second fermentation and aging of traditional sparkling-wine by inoculating S. cerevisiae killer yeast for second fermentation. We already knew that several killer yeasts kill other yeasts (killer or sensitive) in laboratory conditions similar to those used in the elaboration of sparkling-wine (low pH and temperature). Fortunately, some of these yeast strains also showed an intense killer phenotype during the second fermentation under carbon dioxide pressure. In addition, the killer effect in the sparkling-wine inoculated with mixed cultures of yeasts (killer + sensitive) improved the quality of the foam and the mouthfeel with respect to those sparkling- wines elaborated without killer effect, that were inoculated with each yeast strain separately. This improvement was very evident when the killer effect was intense and it occurred quickly few days after the yeast inoculation, and less evident when this effect was poor and slower. Destroyed yeasts resulting from cellular autolysis were detected during the first days of fermentation only in the sparkling-wines in which the killer effect occurred, and these yeasts were gradually reabsorbed in the wine until disappearing. Wine quality improvement did not correlate with the polysaccharide, protein, mannan, or aromatic compound concentrations, suggesting that the mouthfeel and foaming quality of sparkling wine are very complex properties influenced by different wine compounds coming from autolysed yeasts and their interactions.