Efficient use of DNA molecular markers to construct industrial yeast strains.

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure

Single QTL mapping and nucleotide-level resolution of a physiologic trait in wine Saccharomyces cerevisiae strains.

International audienceNatural Saccharomyces cerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. However, the link between phenotype variation and genetic determinism is still difficult to identify, especially in wild populations. Using genome hybridization on DNA microarrays, it is now possible to identify single-feature polymorphisms among divergent yeast strains. This tool offers the possibility of applying quantitative genetics to wild yeast strains. In this instance, we studied the genetic basis for variations in acetic acid production using progeny derived from two strains from grape must isolates. The trait was quantified during alcoholic fermentation of the two strains and 108 segregants derived from their crossing. A genetic map of 2212 markers was generated using oligonucleotide microarrays, and a major quantitative trait locus (QTL) was mapped with high significance. Further investigations showed that this QTL was due to a nonsynonymous single-nucleotide polymorphism that targeted the catalytic core of asparaginase type I (ASP1) and abolished its activity. This QTL was only effective when asparagine was used as a major nitrogen source. Our results link nitrogen assimilation and CO(2) production rate to acetic acid production, as well as, on a broader scale, illustrating the specific problem of quantitative genetics when working with nonlaboratory microorganisms

Detection automatique et correction des carences en azote assimilable des fermentations alcooliques en conditions oenologiques: Etude cinetique et approche physiologique

SIGLEINIST T 77397 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Influence of physiological state of inoculum on volatile acidity production by <em>Saccharomyces cerevisiae</em> during high sugar fermentation

An approach consisting of controlling yeast inoculum to minimize volatile acidity production by Saccharomyces cerevisiae during the alcoholic fermentation of botrytized must was investigated. Direct inoculation of rehydrated active dry yeasts produced the most volatile acidity, while a yeast preparation pre-cultured for 24 hours reduced the final production by up to 23 %. Using yeasts collected from a fermenting wine as a starter must also reduced volatile acidity production. The conditions for preparing the inoculum affected the fermentation capacity of the first generation yeasts: fermentation duration, sugar to ethanol ratio, and wine composition. A pre-culture medium with a low sugar concentration (< 220 g/L) is essential to limit volatile acidity production in high sugar fermentations

Appl. microbiol. biotechnol.

The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48 h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: influence of microbial/physical interactions on wine characteristics

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine

Appl. microbiol. biotechnol.

Non-Saccharomyces yeast species, naturally found in grape must, may impact wine quality positively or negatively. In this study, a mixture of five non-Saccharomyces species (Torulaspora delbrueckii, Metschnikowia spp., Starmerella bacillaris (formerly called Candida zemplinina), Hanseniaspora uvarum, Pichia kluyveri), mimicking the composition of the natural non-Saccharomyces community found in grape must, was used for alcoholic fermentation. The impact of CO2 saturation of the grape juice was studied first on this mixture alone, and then in the presence of Saccharomyces cerevisiae. Two isogenic strains of this species were used: the first with a short and the second a long fermentation lag phase. This study demonstrated that saturating grape juice with CO2 had interesting potential as an oenological technique, inhibiting undesirable species (S. bacillaris and H. uvarum) and stimulating non-Saccharomyces of interest (T. delbrueckii and P. kluyveri). This stimulating effect was particularly marked when CO2 saturation was associated with the presence of S. cerevisiae with long fermentation lag phase. The direct consequence of this association was an enhancement of 3-SH levels in the resulting wine

La fermentation malolactique dans les vins de base de cognac : Une étape peu suivie mais très fréquente

Article de revue professionnelleLes données sur la fermentation malolactique (FML) des vins de base de cognac (VDB) sont aujourd’hui encore très fragmentaires et rares. Son effet sur la qualité des eaux-de-vie est flou. Le seul fait bien établi est la diminution de la teneur en éthanal. On ignore sa fréquence, car les vins ne sont pas analysés pour la contrôler ; mais généralement on admet qu’elle est inhabituelle. Pourtant elle peut se dérouler rapidement après la fermentation alcoolique, ou jamais. Dans ce travail, les populations de levures et de bactéries indigènes ont été étudiées depuis le début de la vinification jusqu’à la distillation du VDB. Les dynamiques sont différentes selon les chais et les pratiques mises en œuvre pour les fermentations alcooliques. Le fait marquant et inattendu est que la FML s’est achevée dans la majorité des cas, malgré les conditions très variables des chais. Par ailleurs, cette étude fournit des données sur les cinétiques fermentaires, sur les espèces microbiennes (levures et bactéries). Des souches d’O. oeni ont été typées. Ces résultats démontrent l’intérêt d’une étude approfondie de la FML et de son impact sur le cognac

Int J Food Microbiol.

The aim of this work was to study ester formation and the aromatic impact of Torulaspora delbrueckii when used in association with Saccharomyces cerevisiae during the alcoholic fermentation of must. In order to evaluate the influence of the inoculation procedure, sequential and simultaneous mixed cultures were carried out and compared to pure cultures of T. delbrueckii and S. cerevisiae. Our results showed that mixed inoculations allowed the increase, in comparison to S. cerevisiae pure culture, of some esters specifically produced by T. delbrueckii and significantly correlated to the maximal T. delbrueckii population reached in mixed cultures. Thus, ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate were considered as activity markers of T. delbrueckii. On the other hand, isobutyl acetate and isoamyl acetate concentrations were systematically increased during mixed inoculations although not correlated with the development of either species but were rather due to positive interactions between these species. Favoring T. delbrueckii development when performing sequential inoculation enhanced the concentration of esters linked to T. delbrueckii activity. On the contrary, simultaneous inoculation restricted the growth of T. delbrueckii, limiting the production of its activity markers, but involved a very important production of numerous esters due to more important positive interactions between species. These results suggest that the ester concentrations enhancement via interactions during mixed modalities was due to S. cerevisiae production in response to the presence of T. delbrueckii. Finally, sensory analyses showed that mixed inoculations between T. delbrueckii and S. cerevisiae allowed to enhance the complexity and fruity notes of wine in comparison to S. cerevisiae pure culture. Furthermore, the higher levels of ethyl propanoate, ethyl isobutanoate, ethyl dihydrocinnamate and isobutyl acetate in mixed wines were found responsible for the increase of fruitiness and complexity