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The goal of this study was to evaluate how grape composition modifications linked to maturity level could affect the wine ester composition and aromatic expression. An experimental design has been developed from grapes of

Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast

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.

The yeast species Starmerella bacillaris (synonym Candida zemplinina) is widely associated with oenological ecosystems and is frequently isolated from grape and grape must. Previous work showed that the genetic diversity of this species is high in wine environments and it is shaped by geographic location. Most analysed C. zemplinina strains, however, have been isolated from Vitis vinifera, disregarding the existence of other worldwide-distributed Vitis species used in winemaking. In this work, we address the impact of the Vitis species and geographic location on the genetic diversity of C. zemplinina. Microsatellite genotyping analysis was applied to two remarkable populations of C. zemplinina from Argentina and Portugal (Azores Archipelago), isolated from neighbouring V. vinifera and Vitis labrusca vineyards. The study also included a large population of previously characterized worldwide-isolated C. zemplinina strains. Genetic analyses confirmed that geographic localization significantly shaped the genetic diversity of C. zemplinina. No genetic differentiation on the basis of the Vitis species was recorded, indicating that C. zemplinina populations from neighbouring V. vinifera and V. labrusca vineyards are genetically homogeneous. In addition, no impact of the vintage was found on the C. zemplinina populations being both highly diversified and homogeneous during initial stages of alcoholic fermentation. Altogether, these results confirmed that winemaking-related factors (i.e., vintage, Vitis species, and alcoholic fermentation) do not impact the genetic diversity of C. zemplinina and that only geographic localization significantly shapes this yeast species.

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.

International audience; 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.

MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial.; Results: MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+ and Ni2+ . Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Km and Vmax ) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions.; Conclusion: Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.; © 2017 Society of Chemical Industry.

Starmerella bacillaris is an ascomycetous yeast ubiquitously present in grapes and fermenting grape musts. In this report, we present the draft genome sequence of the S. bacillaris type strain CBS 9494, isolated from sweet botrytized wines, which will contribute to the study of this genetically heterogeneous wine yeast species.

The yeast Lachancea thermotolerans (previously Kluyveromyces thermotolerans) is a species of large, yet underexplored, oenological potential. This study delivers comprehensive oenological phenomes of 94 L. thermotolerans strains obtained from diverse ecological niches worldwide, classified in nine genetic groups based on their pre-determined microsatellite genotypes. The strains and the genetic groups were compared for their alcoholic fermentation performance, production of primary and secondary metabolites and pH modulation in Chardonnay grape juice fermentations. The common oenological features of L. thermotolerans strains were their glucophilic character, relatively extensive fermentation ability, low production of acetic acid and the formation of lactic acid, which significantly affected the pH of the wines. An untargeted analysis of volatile compounds, used for the first time in a population-scale phenotyping of a non-Saccharomyces yeast, revealed that 58 out of 90 volatiles were affected at an L. thermotolerans strain level. Besides the remarkable extent of intra-specific diversity, our results confirmed the distinct phenotypic performance of L. thermotolerans genetic groups. Together, these observations provide further support for the occurrence of domestication events and allopatric differentiation in L. thermotolerans population.

Volume 7, no. 3, e00872-18, 2018, [https://doi.org/10.1128/MRA.00872-18][1]. Page 1: The article title should read as given above. Page 1, line 1: “ Starmerella bacillaris (syn., Candida zemplinina )” should read “ Candida zemplinina (syn., Starmerella bacillaris ).” Pages 1 and 2: “ S.

Starmerella bacillaris is an ascomycetous yeast ubiquitously present in grapes and fermenting grape musts. In this report, we present the draft genome sequence of the S. bacillaris type strain CBS 9494, isolated from sweet botrytized wines, which will contribute to the study of this genetically heterogeneous wine yeast species.

International audience; Aims To characterize Oenococcus oeni strains isolated from North-Apulian wines where malic acid degradation is usually achieved by spontaneous fermentations, and to determine the influence of bacterial inoculation time on the malolactic performances in ‘Nero di Troia’ wine using a complete autochthonous microbial regime. Methods and Results Oenococcus oeni strains from wines produced with the autochthonous (Apulia Region, southern Italy) grape variety ‘Uva di Troia’ were isolated, selected and characterized. Multilocus sequence typing and variable number tandem repeat analysis were used to investigate intraspecific diversity. Oenococcus oeni strains were tested in co-inoculation and in sequential inoculation, with two autochthonous yeast strains previously isolated from ‘Nero di Troia’ wine. After a preliminary screening using co-inoculation regime, the O. oeni strains were grouped in reason of the different behaviour in malic acid performances. Results suggested that the efficient degradation of malic acid in co-inoculation is a strain-dependent characteristic. Conclusions Autochthonous yeast/bacterium combinations were identified as starter culture, and used in a co-inoculation approach, for vinification of regional wines. Significance and Impact of the Study The ‘microbial terroir’ of typical fermented food and beverage production represents a dynamic sector of applied research in food microbiology. In this work, we propose the use of autochthonous bacteria and yeast for wine production from an indigenous grape variety.

BACKGROUND MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial.RESULTS MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+ and Ni2+. Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Km and Vmax) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions.CONCLUSION Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking.

Aim: In the context of climate change, adaptation of enological practices and implementation of novel techniques are major challenges for winemakers. The potential interventions are linked in particular with the alcohol content and the global acidity of wine. Here, we review current microbiological and technological strategies to overcome such issues.Methods and results: Reducing ethanol concentration poses a number of technical and scientific challenges, in particular looking for specific yeast strains with lower alcohol yield. Several non-genetically modified organism (GMO) strains – S. cerevisiae or interspecific hybrids of the Saccharomyces genus – have yet been developed using different strategies, and some of them allow decreasing the final ethanol concentration by up to 1%. Several membrane-based technologies have also been developed not only to reduce the ethanol content of wines but also to increase the acidity and more generally to control the wine pH. New strategies are also proposed to improve the control of winemaking, especially the management of alcoholic fermentation of sugar-rich musts and the control of oxidation during the process.Conclusion: Reducing ethanol of wines and increasing their acidity are good examples of novel techniques of interest in the context of climate change. Other strategies are still under study to adapt winemaking practices to changes in grape composition.Significance and impact of the study: [Membrane-based technologies can be used to reduce the ethanol content of wines or to increase the acidity. Microbiological strategies will also be soon available for winemakers.

International audience; 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.

Enzymes can be post-translationally modified, leading to isoforms with different properties. The phenotypic consequences of the quantitative variability of isoforms have never been studied. We used quantitative proteomics to dissect the relationships between the abundances of the enzymes and isoforms of alcoholic fermentation, metabolic traits, and growth-related traits in Saccharomyces cerevisiae. Although the enzymatic pool allocated to the fermentation proteome was constant over the culture media and the strains considered, there was variation in abundance of individual enzymes and sometimes much more of their isoforms, which suggests the existence of selective constraints on total protein abundance and trade-offs between isoforms. Variations in abundance of some isoforms were significantly associated to metabolic traits and growth-related traits. In particular, cell size and maximum population size were highly correlated to the degree of N-terminal acetylation of the alcohol dehydrogenase. The fermentation proteome was found to be shaped by human selection, through the differential targeting of a few isoforms for each food-processing origin of strains. These results highlight the importance of post-translational modifications in the diversity of metabolic and life-history traits.

Corrigendum to “A new method for monitoring the extracellular proteolytic activity of wine yeasts during alcoholic fermentation of grape must” [J. Microbiol. Methods 119 (2015) 176–179]

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.

Campinas State University (UNICAMP) – Faculty of Food Engineering – Department of Food Science. P.O. Box 6121, CEP 13083-862 Campinas-SP, Brazil. E-mail: dr.danirod@gmail.com. Federal University of Mato Grosso (UFMT) – Food Engineering. Rodovia BR-070, Km 5, CEP 78600-000 Barra do Garcas-MT, Brazil. Park University, 8700 NW River Park Drive, Parkville, MO 64152, United States. Bordeaux II University – Science Institute of Grape and Wine (ISVV). 210 Chemin de Leysotte, CS 50008, 33882 Villenave D’Ornon, France. Federal University of Santa Maria (UFSM) – Department of Food Science and Technology. Roraima Avenue 1000, Cidade Universitaria, CEP 97105-900 Santa Maria-RS, Brazil.

International audience; 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.

International audience; The yeast Candida zemplinina (Starmerella bacillaris) is frequently isolated from grape and wine environments. Its enological use in mixed fermentation with Saccharomyces cerevisiae has been extensively investigated these last few years, and several interesting features including low ethanol production, fructophily, glycerol and other metabolites production, have been described. In addition, molecular tools allowing the characterization of yeast populations have been developed, both at the inter- and intraspecific levels. However, most of these fingerprinting methods are not compatible with population genetics or ecological studies. In this work, we developed 10 microsatellite markers for the C. zemplinina species that were used for the genotyping of 163 strains from nature or various enological regions (28 vineyards/wineries from seven countries). We show that the genetic diversity of C. zemplinina is shaped by geographical localization. Populations isolated from winemaking environments are quite diverse at the genetic level: neither clonal-like behaviour nor specific genetic signature were associated with the different vineyards/wineries. Altogether, these results suggest that C. zemplinina is not under selective pressure in winemaking environments.

International audience; The existing methods for testing proteolytic activity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolytic activity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolytic activity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins.

International audience; Heterosis is a universal phenomenon that has major implications in evolution and is of tremendous agro-economic value. To study the molecular manifestations of heterosis and to find factors that maximize its strength, we implemented a large-scale proteomic experiment in yeast. We analyzed the inheritance of 1,396 proteins in 55 inter- and intraspecific hybrids obtained from Saccharomyces cerevisiae and S. uvarum that were grown in grape juice at two temperatures. We showed that the proportion of heterotic proteins was highly variable depending on the parental strain and on the temperature considered. For intraspecific hybrids, this proportion was higher at nonoptimal temperature. Unexpectedly, heterosis for protein abundance was strongly biased toward positive values in interspecific hybrids but not in intraspecific hybrids. Computer modeling showed that this observation could be accounted for by assuming concave relationships between protein abundances and their controlling factors, in line with the metabolic model of heterosis. These results point to nonlinear processes that could play a central role in heterosis.

Variations in ethanol, volatile acidity, and aromatic compounds produced by different Saccharomyces cerevisiae strains were studied in controlled synthetic medium (CSM). Different amounts of assimilable nitrogen (in the form of ammonium sulfate) were added at two fermentation stages (i.e. the beginning of fermentation and the halfway point). There were significant differences in the amount of ethanol produced when ammonia was added to the CSM, although this depended on the yeast strain used. When assimilable nitrogen was added, ethanol production either increased (with Fermicru AR2 and Stellevin NT116 yeast strains) or decreased (with LW LVCB CT1+ yeast strain). The degree of variation also depended on the time that the ammonia was added, with differences of up to 0.7% (v/v). Adding ammonium to the CSM always resulted in lower volatile acidity in the fermentation product. Different yeast strains (P < 0.0001) and varying amounts of ammonium produced significant differences. Maximum impact - up to 70% less volatile acidity - was obtained using the Stellevin NT116 yeast strain and adding 280 mgN/L ammonium at the halfway point in fermentation. Acetoin production increased at higher concentrations of added ammonium, most markedly when it was added halfway through fermentation. The total amount of esters increased when 140 mgN/L was added to all three yeasts strains tested, irrespective of the fermentation stage. Smaller amounts of higher alcohols were produced following larger ammonium additions, especially at the beginning of fermentation.

In agricultural sciences, breeding strategies have historically been used to select new, optimized plant varieties or animal breeds. Similar strategies are possible for genetic improvement of wine yeasts. We optimized 11 relevant enological traits in a single clone using successive hybridization and segregation steps. A hybrid obtained by crossing two parent strains derived from commercial wine yeasts showed that some of the traits were readily optimized. Dominance/recessivity, heterosis and transgression were observed among 51 segregating progeny. On the basis of this information, all the optimal characters from both parents were combined in a single strain following two targeted sexual crosses. This article presents a powerful methodology for obtaining a single wine strain with numerous fermentative qualities that does not produce off-flavors.

We analyzed the variability of volatile acidity and glycerol production by Saccharomyces cerevisiae on a large sample of high sugar musts. The production of volatile acidity was inversely correlated with the maximum cell population and the assimilable nitrogen concentration. The higher the nitrogen concentration, the less volatile acidity was produced. An approach to minimize volatile acidity production during high sugar fermentations by adjustment of assimilable nitrogen in musts was investigated in terms of both quantity and addition time. It was found that the optimal nitrogen concentration in the must is 190 mgN· l −1 . The best moment for nitrogen addition was at the beginning of fermentation. Addition at the end of the growth phase had less effect on volatile acidity reduction. We suggest that by stimulating cell growth, nitrogen addition provides NADH in the redox-equilibrating process, which in turn reduces volatile acidity formation.

International audience; The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (~1900 years ago), and to the Neolithic era for bioprocesses (~4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.

International audience; Yeast species of Hanseniaspora and Candida genus are predominant during the early stages of winemaking, while species of Metschnikowia, Pichia, Zygoascus, Issatchenkia, Torulaspora and other genera are present at lower population levels. The impact of common oenological practices on yeast dynamics during the prefermentative stage and the early stage of alcoholic fermentation (AF) remains elusive. In this work, the effect of four prefermentative oenological practices (clarification degree, temperature, sulphite and starter yeast addition) on yeast dynamics was evaluated in a Chardonnay grape must. The growth curves of four genus or species, namely Saccharomyces spp., Hanseniaspora spp., Candida zemplinina and Torulaspora delbrueckii, were followed by quantitative PCR. The fermentation kinetics were also recorded, as well as the production of acetic acid. Variance analysis allowed determining the effect of each practice and their interaction factors, as well as their relative importance on yeast dynamics and fermentation kinetics. Our experimental design showed that the population dynamics of the four species were differently impacted by the oenological practices, with some species being more sensitive than others to the clarification degree (C. zemplinina), sulphite addition (Saccharomyces spp.), starter yeast inoculation (Hanseniaspora spp.) or prefermentation temperature (T. delbrueckii). Significant interaction effects between practices were revealed, highlighting the interest of experimental design allowing interaction analysis, as some factors may buffer the effect of other ones. Hanseniaspora genus showed atypical behaviour: growth dynamics showed a decrease during AF that we interpreted as early cellular lysis. In conclusion, this study provides new insights on the impact of common oenological practices on the dynamics of non-Saccharomyces yeast that will be useful for a better management of mixed fermentation between S. cerevisiae and non-Saccharomyces yeasts.

International audience; In eukaryotes, mitochondrial DNA (mtDNA) has high rate of nucleotide substitution leading to different mitochondrial haplotypes called mitotypes. However, the impact of mitochondrial genetic variant on phenotypic variation has been poorly considered in microorganisms because mtDNA encodes very few genes compared to nuclear DNA, and also because mitochondrial inheritance is not uniparental. Here we propose original material to unravel mitotype impact on phenotype: we produced interspecific hybrids between S. cerevisiae and S. uvarum species, using fully homozygous diploid parental strains. For two different interspecific crosses involving different parental strains, we recovered 10 independent hybrids per cross, and allowed mtDNA fixation after around 80 generations. We developed PCR-based markers for the rapid discrimination of S. cerevisiae and S. uvarum mitochondrial DNA. For both crosses, we were able to isolate fully isogenic hybrids at the nuclear level, yet possessing either S. cerevisiae mtDNA (Sc-mtDNA) or S. uvarum mtDNA (Su-mtDNA). Under fermentative conditions, the mitotype has no phenotypic impact on fermentation kinetics and products, which was expected since mtDNA are not necessary for fermentative metabolism. Alternatively, under respiratory conditions, hybrids with Sc-mtDNA have higher population growth performance, associated with higher respiratory rate. Indeed, far from the hypothesis that mtDNA variation is neutral, our work shows that mitochondrial polymorphism can have a strong impact on fitness components and hence on the evolutionary fate of the yeast populations. We hypothesize that under fermentative conditions, hybrids may fix stochastically one or the other mt-DNA, while respiratory environments may increase the probability to fix Sc-mtDNA.

International audience; Alcoholic fermentation of grape must is a complex process, involving several yeast genera and species. The early stages in fermentation are dominated by non-Saccharomyces yeasts that are gradually replaced by the Saccharomyces cerevisiae species, which takes over the fermentation. Quantitative studies have reported the influence of non-Saccharomyces yeast species on wine quality and evaluated their biotechnological interest. The industrial yeast market, which, until recently, exclusively focused on S. cerevisiae, now offers S. cerevisiae/non-Saccharomyces (including Torulaspora delbrueckii) multi-starters. The development of these new mixed industrial starters requires a better understanding of the interaction mechanisms between yeast populations in order to optimize the aromatic impact of the non-Saccharomyces yeast while ensuring complete alcoholic fermentation thanks to S. cerevisiae. For this purpose, a new double-compartment fermentor was designed with the following characteristics: (1) physical separation of two yeast populations, (2) homogeneity of the culture medium in both compartments, (3) fermentation kinetics monitored by weight loss due to CO2 release, and (4) independent monitoring of growth kinetics in the two compartments. This tool was used to compare mixed inoculations of S. cerevisiae/T. delbrueckii with and without physical separation. Our results revealed that physical contact/proximity between S. cerevisiae and T. delbrueckii induced rapid death of T. delbrueckii, a phenomenon previously described and attributed to a cell-cell contact mechanism. In contrast, when physically separated from S. cerevisiae, T. delbrueckii maintained its viability and its metabolic activity had a marked impact on S. cerevisiae growth and viability. The double fermentor is thus a powerful tool for studying yeast interactions. Our findings shed new light on interaction mechanisms described in microorganism populations.

International audience; 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.

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 CO 2 production rate ( V max ) was not related to the maximum CO 2 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.

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.

International audience; 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.

International audience; 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.

International audience; 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.

To better describe the importance of initial assimilable nitrogen content on the kinetics of alcoholic fermentation and the effectiveness of ammonium additions, a study was done using an automatic device for fermentation monitoring. A good correlation was found between the maximum CO2 production rate, obtained early during the fermentation, and the assimilable nitrogen content of the must. So it is possible to use this kinetic parameter to detect nitrogen deficiencies. For industrial applications, a threshold value of 1.3 gCO2/l·h at 24°C (corresponding to about 140 mg N/l) is proposed. In the case of deficient must, a nitrogen addition during the fermentation rapidly increased the CO2 production rate and reduced the fermentation duration. This reduction, which may be approximately predicted from (dCO2/dt)max, is the same, provided that the addition is made before the halfway point of the fermentation.

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.

International audience; Natural 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.