Marina Bely

Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture on high-sugar fermentation.

Conventional wine yeasts produce high concentrations of volatile acidity, mainly acetic acid, during high-sugar fermentation. This alcoholic fermentation by-product is highly detrimental to wine quality and, in some cases, levels may even exceed legal limits. In this study, a non-conventional species, Torulaspora delbrueckii, was used, in pure cultures and mixed with Saccharomyces cerevisiae yeast, to ferment botrytized musts. Fermentation rate, biomass growth, and the formation of volatile acidity, acetaldehyde, and glycerol were considered. This study demonstrated that T. delbrueckii, often described as a low acetic producer under standard conditions, retained this quality even in a high-sugar medium. Unlike S. cerevisiae, this species did not respond to the hyper-osmotic medium by increasing acetic production as soon as it is inoculated into the must. Nevertheless, this yeast produced low ethanol and biomass yields, and the fermentation was sluggish. As a result, T. delbrueckii fermentations do not reach the required ethanol content (14%vol.), although this species can survive at this concentration. A mixed culture of T. delbrueckii and S. cerevisiae was the best combination for improving the analytical profile of sweet wine, particularly volatile acidity and acetaldehyde production. A mixed T. delbrueckii/S. cerevisiae culture at a 20:1 ratio produced 53% less in volatile acidity and 60% less acetaldehyde than a pure culture of S. cerevisiae. Inoculating S. cerevisiae after 5 days fermentation by T. delbrueckii had less effect on volatile acidity and acetaldehyde production and resulted in stuck fermentation. These results contribute to a better understanding of the behaviour of non-Saccharomyces and their potential application in wine industry.

Genetic characterization and phenotypic variability in Torulaspora delbrueckii species: Potential applications in the wine industry.

In this study, several strains of Torulaspora delbrueckii yeast species were evaluated in the laboratory for their enological properties. In a preliminary step, the ability of different molecular methods to discriminate among T.delbrueckii strains was compared. A combination of 7 PCR methods was able to separate 21 strains into 18 groups, while an REA-PFGE method allowed, in one experiment, the separation into 19 groups. The T.delbrueckii strains used presented a wide phenotypic variability in fermentation behaviour, e.g. Lag Phase (LP) duration, T50 parameter (time necessary to ferment half the sugar), and ethanol production. These 3 parameters have to be considered for industrial selection, particularly the LP duration. The majority of T.delbrueckii strains produced 8 to 11% and 7 to 10% ethanol vol. at 17 degrees C and 24 degrees C, respectively, with a maximum ethanol concentration of 12.35 at 17 degrees C and 10.90% vol. at 24 degrees C. The phenotypic variability of this species was also reflected in volatile acidity, glycerol, and aroma production. These experiments confirmed the low volatile acidity and glycerol production of this species and revealed a difference in osmotic stress response, compared to Saccharomyces cerevisiae. T.delbrueckii presented high fermentation purity and produced low levels of undesirable volatile compounds, such as hydrogen sulphide and volatile phenols.

Reassessment of phenotypic traits for Saccharomyces bayanus var. uvarum wine yeast strains.

Among Saccharomyces yeast, S. cerevisiae and S. bayanus var. uvarum are related species, sharing the same ecosystem in sympatry. The physiological and technological properties of a large collection of genetically-identified S. bayanus var. uvarum wine strains were investigated in a biometric study and their fermentation behavior was compared at 24 degrees C and 13 degrees C. The variability of the phenotypic traits was considered at both intraspecific and interspecific levels. Low ethanol tolerance at 24 degrees C and production of high levels of 2-phenylethanol and its acetate were clearly revealed as discriminative technological traits, distinguishing the S. bayanus var. uvarum strains from S. cerevisiae. Although some S. bayanus var. uvarum strains produced very small amounts of acetic acid, this was not a species-specific trait, as the distribution of values was similar in both species. Fermentation kinetics at 24 degrees C showed that S. bayanus var. uvarum maintained a high fermentation rate after Vmax, with low nitrogen requirements, but stuck fermentations were observed at later stages. In contrast, a shorter lag phase compared with S.cerevisiae, higher cell viability, and the ability to complete alcoholic fermentation at 13 degrees C confirmed the low-temperature adaptation trait of S.bayanus var. uvarum. This study produced a phenotypic characterization data set for a collection of S. bayanus var. uvarum strains, thus paving the way for industrial developments using this species as a new genetic resource.

The grape must non-Saccharomyces microbial community: impact on volatile thiol release.

Several studies have reported the beneficial influence of non-Saccharomyces yeasts and their potential applications in the wine industry, mainly in mixed-culture fermentation with S. cerevisiae. The potential impact of 15 non-Saccharomyces strains from 7 species on 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) release in model medium and Sauvignon Blanc must was evaluated after partial fermentation. Whereas the impact of non-Saccharomyces on 4MSP release in both media was low, some M. pulcherrima, T. delbrueckii and K. thermotolerans strains had a high capacity to release 3SH, despite their minimal fermentation activity. As previously demonstrated for Saccharomyces yeast, this contribution is strain dependant. Taking into account their dynamic and quantitative presence during the whole process, the real impact of non-Saccharomyces yeast on 4MSP and 3SH release was evaluated using a recreated community simulating the yeast ecosystem. Our results revealed a positive impact on 3SH release in Sauvignon Blanc wines by promoting non-Saccharomyces yeast activity and delaying the growth of S. cerevisiae. Some non-Saccharomyces yeast strains are capable of making a positive contribution to volatile thiol release in wines, essentially during the pre-fermentation stage in winemaking, when this microbiological sub-population is dominant.

Genetic improvement of thermo-tolerance in wine Saccharomyces cerevisiae strains by a backcross approach

During red wine fermentation, high temperatures may cause stuck fermentation by affecting the physiology of fermenting yeast. This deleterious effect is the result of the complex interaction of temperature with other physicochemical parameters of grape juice, such as sugar and lipid content. The genetic background of fermenting yeast also interacts with this complex matrix and some strains are more resistant to high temperatures than others. Here, the temperature tolerance of nine commercial starters was evaluated, demonstrating that, at high sugar concentrations, half of them are sensitive to temperature. Using a classical backcross approach, one thermo-sensitive commercial starter was genetically improved by introducing quantitative trait loci conferring resistance to temperature. With this breeding program it is possible to obtain a thermo-resistant strain sharing most of its genome with the initial commercial starter. The parental and improved strains were compared for population growth and fermentation ability in various conditions. Despite their common genetic background, these two strains showed slight physiological differences in response to environmental changes that enable identification of the key physiological parameters influencing stuck fermentation.

Evidence for autotetraploidy associated with reproductive isolation in Saccharomyces cerevisiae: towards a new domesticated species.

Partial or whole‐genome duplications have played a major role in the evolution of new species. We have investigated the variation of ploidy level in a panel of domesticated strains of Saccharomyces cerevisiae coming from different geographical origins. Segregation studies and crosses with tester strains of different ploidy levels showed that part of the strains were well‐balanced autotetraploids displaying tetrasomic inheritance. The presence of up to four different alleles for various loci is consistent with a polyploidization mechanism relying on the fusion of two nonreduced meiospores coming from two S. cerevisiae strains. Autotetraploidy was also in accordance with karyotype and flow cytometry analyses. Interestingly, most bakery strains were tetraploids, suggesting a link between ploidy level and human use. The null or drastically reduced fertility of the hybrids between tetraploid and diploid strains indicated that domesticated S. cerevisiae strains are composed of two groups isolated by post‐zygotic reproductive barriers.

Population Size Drives Industrial Saccharomyces cerevisiae Alcoholic Fermentation and Is under Genetic Control

ABSTRACT Alcoholic fermentation (AF) conducted by Saccharomyces cerevisiae has been exploited for millennia in three important human food processes: beer and wine production and bread leavening. Most of the efforts to understand and improve AF have been made separately for each process, with strains that are supposedly well adapted. In this work, we propose a first comparison of yeast AFs in three synthetic media mimicking the dough/wort/grape must found in baking, brewing, and wine making. The fermentative behaviors of nine food-processing strains were evaluated in these media, at the cellular, populational, and biotechnological levels. A large variation in the measured traits was observed, with medium effects usually being greater than the strain effects. The results suggest that human selection targeted the ability to complete fermentation for wine strains and trehalose content for beer strains. Apart from these features, the food origin of the strains did not significantly affect AF, suggesting that an improvement program for a specific food processing industry could exploit the variability of strains used in other industries. Glucose utilization was analyzed, revealing plastic but also genetic variation in fermentation products and indicating that artificial selection could be used to modify the production of glycerol, acetate, etc. The major result was that the overall maximum CO2 production rate (V max) was not related to the maximum CO2 production rate per cell. Instead, a highly significant correlation between V max and the maximum population size was observed in all three media, indicating that human selection targeted the efficiency of cellular reproduction rather than metabolic efficiency. This result opens the way to new strategies for yeast improvement.

Enhanced 3-Sulfanylhexan-1-ol Production in Sequential Mixed Fermentation with Torulaspora delbrueckii/Saccharomyces cerevisiae Reveals a Situation of Synergistic Interaction between Two Industrial Strains.

The aim of this work was to study the volatile thiol productions of two industrial strains of Torulaspora delbrueckii and Saccharomyces cerevisiae during alcoholic fermentation (AF) of Sauvignon Blanc 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. The results confirmed the inability of T. delbrueckii to release 4-methyl-4-sulfanylpentan-2-one (4MSP) and its low capacity to produce 3-sulfanylhexyl acetate (3SHA), as already reported in previous studies. A synergistic interaction was observed between the two species, resulting in higher levels of 3SH (3-sulfanylhexan-1-ol) and its acetate when S. cerevisiae was inoculated 24 h after T. delbrueckii, compared to the pure cultures. To elucidate the nature of the interactions between these two species, the yeast population kinetics were examined and monitored, as well as the production of 3SH, its acetate and their related non-odorous precursors: Glut-3SH (glutathionylated conjugate precursor) and Cys-3SH (cysteinylated conjugate precursor). For the first time, it was suggested that, unlike S. cerevisiae, which is able to metabolize the two precursor forms, T. delbrueckii was only able to metabolize the glutathionylated precursor. Consequently, the presence of T. delbrueckii during mixed fermentation led to an increase in Glut-3SH degradation and Cys-3SH production. This overproduction was dependent on the T. delbrueckii biomass. In sequential culture, thus favoring T. delbrueckii development, the higher availability of Cys-3SH throughout AF resulted in more abundant 3SH and 3SHA production by S. cerevisiae.

Hybridization within Saccharomyces Genus Results in Homoeostasis and Phenotypic Novelty in Winemaking Conditions

Despite its biotechnological interest, hybridization, which can result in hybrid vigor, has not commonly been studied or exploited in the yeast genus. From a diallel design including 55 intra- and interspecific hybrids between Saccharomyces cerevisiae and S. uvarum grown at two temperatures in enological conditions, we analyzed as many as 35 fermentation traits with original statistical and modeling tools. We first showed that, depending on the types of trait – kinetics parameters, life-history traits, enological parameters and aromas –, the sources of variation (strain, temperature and strain * temperature effects) differed in a large extent. Then we compared globally three groups of hybrids and their parents at two growth temperatures: intraspecific hybrids S. cerevisiae * S. cerevisiae, intraspecific hybrids S. uvarum * S. uvarum and interspecific hybrids S. cerevisiae * S. uvarum. We found that hybridization could generate multi-trait phenotypes with improved oenological performances and better homeostasis with respect to temperature. These results could explain why interspecific hybridization is so common in natural and domesticated yeast, and open the way to applications for wine-making.

The evolution of Lachancea thermotolerans is driven by geographical determination, anthropisation and flux between different ecosystems

The yeast Lachancea thermotolerans (formerly Kluyveromyces thermotolerans) is a species with remarkable, yet underexplored, biotechnological potential. This ubiquist occupies a range of natural and anthropic habitats covering a wide geographic span. To gain an insight into L. thermotolerans population diversity and structure, 172 isolates sourced from diverse habitats worldwide were analysed using a set of 14 microsatellite markers. The resultant clustering revealed that the evolution of L. thermotolerans has been driven by the geography and ecological niche of the isolation sources. Isolates originating from anthropic environments, in particular grapes and wine, were genetically close, thus suggesting domestication events within the species. The observed clustering was further validated by several means including, population structure analysis, F-statistics, Mantel’s test and the analysis of molecular variance (AMOVA). Phenotypic performance of isolates was tested using several growth substrates and physicochemical conditions, providing added support for the clustering. Altogether, this study sheds light on the genotypic and phenotypic diversity of L. thermotolerans, contributing to a better understanding of the population structure, ecology and evolution of this non-Saccharomyces yeast.