Activités glycosidases chez Œnococcus œni (importance dans la libération de la vanilline à partir de bois de chêne)

Les bactéries lactiques (BL) réalisant la fermentation malolactique (FML) ont une influence significative sur la stabilité et la qualité organoleptique du vin. La FML peut être réalisée en barriques et l'influence de cette pratique sur la modification des teneurs en composés volatils du bois de chêne est étudiée dans ce travail. Nous avons montré qu'Oenococcus oeni était capable d'augmenter les teneurs en compoosés volatils du bois de chêne lors de la FML. Cette libération est importante, en particulier pour la vanilline, lorsque le métabolisme bactérien est le plus actif. L'enrichissement persiste au cours de l'élevage et participe ainsi à la complexité aromatique du vin fini. Cette libération indique la présence de précurseurs dans le bois de chêne et l'aptitude enzymatique pour OE. oeni de les métaboliser en molécules volatiles. Plusieurs hypothèses ont été envisagées et la présence de précurseurs glycosylés a été retenue, aussi bien pour la vanilline que pour d'autres composés issus du bois comme certains aldéhydes phénoliques mais aussi l'eugénol, l'isoeugénol ou la whiskylactone. L'action de glycosidases sur la vanilline libérée a été étudiée et cet aldéhyde est présent dans les extraits de bois sous formes monoglycosidiques où les groupements glycones majoritaires sont l'arabinose et le xylose. Les BL possèdent des activités glycosidases largement répandues au niveau de l''espèce OE. oeni mais avec une grande variabilité intraspécifique. Cette activité serait à l'origine des teneurs en vanilline libérées pendant la FML. Enfin, la purification de l'a-L-arabinosidase impliquée dans l'hydrolyse de la vanilline chez cette espèce a été initiée.Lactic acid bacteria (LAB) conducting the malolactic fermentation (MLF) have a significant influence on the stability and organoleptic quality of wine. The MLF can be performed in barrels and the influence of this practice on the modification of the levels of oak wood volatile compounds is also studied in this work. We showed that Oenococcus oeni was able to increase the levels of oak wood volatile compounds during the malolactic fermentation. This release is important, particularly for vanillin when LAB metabolism is the most active. Enrichment persists during the ageing and contributes to the aromatic complexity of wine. This release indicates the presence of precursors in oak wood and the production of enzymes by this species that affect vanillin formation. Several mechanisms were considered and the presence of glycosylated precursors were investigated, as well as vanillin for other oak wood compounds such as phenolic aldehydes and also eugenol, isoeugenol or the whiskylactone. The effect of glycosidases on the released vanillin was studied and this aldehyde is present in wood extracts in monoglycosidic forms where the major glycones are the arabinose and the xylose. The LAB have widespread glycosidase activities within the species O. oeni but with a great variability. This activity causes the release of vanillin during MLF. Lastly, the purification of the a-L-arabinosidase involved in the hydrolysis of vanillin in this species was initiated.BORDEAUX2-BU Santé (330632101) / SudocBORDEAUX1-BU Sciences-Talence (335222101) / SudocVILLENAVE D'ORNON-Bib. ISVV (335502201) / SudocSudocFranceF

Metabolic impact of redox cofactor perturbations on the formation of aroma compounds in Saccharomyces cerevisiae.

Redox homeostasis is a fundamental requirement to the sustainment of metabolism, energy generation and growth in Saccharomyces cerevisiae. Redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved here thanks to a dedicated biological tool overexpressing native NADH- or engineered NADPH-dependent 2,3-butanediol dehydrogenase in presence of acetoin. We report that the targeted perturbation of the cofactors balances (NAD(+)/NADH or, in a lesser extent, NADP(+)/NADPH) significantly affects the production of volatile compounds. In most cases, variations of the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox on the availability of intermediates of the central carbon metabolism. This is the case for α-keto acids and acetyl-CoA, which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affect the synthesis of some volatile molecules, as methionol, phenylethanol, and propanoic acid, reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings point out that both the availability of precursors from the central carbon metabolism and the accessibility to reduced cofactors contribute to the modulation of the formation of volatile compounds by the cell redox status

Isotopic Tracers Unveil Distinct Fates for Nitrogen Sources during Wine Fermentation with Two Non-Saccharomyces Strains

© 2020 by the authors.Non-Saccharomyces yeast strains have become increasingly prevalent in the food industry, particularly in winemaking, because of their properties of interest both in biological control and in complexifying flavour profiles in end-products. However, unleashing the full potential of these species would require solid knowledge of their physiology and metabolism, which is, however, very limited to date. In this study, a quantitative analysis using 15N-labelled NH4Cl, arginine, and glutamine, and 13C-labelled leucine and valine revealed the specificities of the nitrogen metabolism pattern of two non-Saccharomyces species, Torulaspora delbrueckii and Metschnikowia pulcherrima. In T. delbrueckii, consumed nitrogen sources were mainly directed towards the de novo synthesis of proteinogenic amino acids, at the expense of volatile compounds production. This redistribution pattern was in line with the high biomass-producer phenotype of this species. Conversely, in M. pulcherrima, which displayed weaker growth capacities, a larger proportion of consumed amino acids was catabolised for the production of higher alcohols through the Ehrlich pathway. Overall, this comprehensive overview of nitrogen redistribution in T. delbrueckii and M. pulcherrima provides valuable information for a better management of co- or sequential fermentation combining these species with Saccharomyces cerevisiae.Pauline Seguinot was funded by the Lallemand Inc. and Ying Su was supported a fellowship granted by the Generalitat Valencia.Peer reviewe

Altered fermentation performances, growth, and metabolic footprints reveal competition for nutrients between yeast species inoculated in synthetic grape juice-like medium

The sequential inoculation of non-Saccharomyces yeasts and Saccharomyces cerevisiae in grape juice is becoming an increasingly popular practice to diversify wine styles and/or to obtain more complex wines with a peculiar microbial footprint. One of the main interactions is competition for nutrients, especially nitrogen sources, that directly impacts not only fermentation performance but also the production of aroma compounds. In order to better understand the interactions taking place between non-Saccharomyces yeasts and S. cerevisiae during alcoholic fermentation, sequential inoculations of three yeast species (Pichia burtonii, Kluyveromyces marxianus, Zygoascus meyerae) with S. cerevisiae were performed individually in a synthetic medium. Different species-dependent interactions were evidenced. Indeed, the three sequential inoculations resulted in three different behaviors in terms of growth. P. burtonii and Z. meyerae declined after the inoculation of S. cerevisiae which promptly outcompeted the other two species. However, while the presence of P. burtonii did not impact the fermentation kinetics of S. cerevisiae, that of Z. meyerae rendered the overall kinetics very slow and with no clear exponential phase. K. marxianus and S. cerevisiae both declined and became undetectable before fermentation completion. The results also demonstrated that yeasts differed in their preference for nitrogen sources. Unlike Z. meyerae and P. burtonii, K. marxianus appeared to be a competitor for S. cerevisiae (as evidenced by the uptake of ammonium and amino acids), thereby explaining the resulting stuck fermentation. Nevertheless, the results suggested that competition for other nutrients (probably vitamins) occurred during the sequential inoculation of Z. meyerae with S. cerevisiae. The metabolic footprint of the non-Saccharomyces yeasts determined after 48 h of fermentation remained until the end of fermentation and combined with that of S. cerevisiae. For instance, fermentations performed with K. marxianus were characterized by the formation of phenylethanol and phenylethyl acetate, while those performed with P. burtonii or Z. meyerae displayed higher production of isoamyl alcohol and ethyl esters. When considering sequential inoculation of yeasts, the nutritional requirements of the yeasts used should be carefully considered and adjusted accordingly. Finally, our chemical data suggests that the organoleptic properties of the wine are altered in a species specific manner

New Insights into the Origin of Volatile Sulfur Compounds during Wine Fermentation and Their Evolution during Aging

Volatile sulfur compounds (VSCs) are associated with unpleasant reductive aromas and are responsible for an important reduction in wine quality, causing major economic losses. Understanding the origin of these compounds in wine remains a challenge, as their formation and further evolution during winemaking can involve both chemical and biological reactions. Comparing the VSCs profile (i) of fermenting synthetic grape juices supplemented with a selected VSC (eight compounds tested) and incubated in presence or absence of yeast, and (ii) during storage of wines under an accelerated aging procedure, allowed us to elucidate the chemical and metabolic connections between VSCs during fermentation and aging. Yeast metabolism, through the Ehrlich pathway and acetylation reactions, makes an important contribution to the formation of compounds such as methionol, 3-methylthiopropionate, 3-methylthiopropylacetate, 3-mercaptopropanol, 2-mercaptoethanol and thioesters. By contrast, chemical reactions are responsible for interconversions between thiols and disulfides, the formation of thiols from thioesters or, more surprisingly, the formation of ethylthiopropanol from methionol during fermentation. During aging, variations in heavy VSC concentrations, such as an increase in 3-methylthiopropylacetate and a decrease in ethyl-3-methylthiopropionate formation, were evidenced. Overall, this study highlights that it is essential to consider both yeast metabolism and the high chemical reactivity of VSCs to understand their formation and evolution during winemaking

New Insights into the Origin of Volatile Sulfur Compounds during Wine Fermentation and Their Evolution during Aging

Volatile sulfur compounds (VSCs) are associated with unpleasant reductive aromas and are responsible for an important reduction in wine quality, causing major economic losses. Understanding the origin of these compounds in wine remains a challenge, as their formation and further evolution during winemaking can involve both chemical and biological reactions. Comparing the VSCs profile (i) of fermenting synthetic grape juices supplemented with a selected VSC (eight compounds tested) and incubated in presence or absence of yeast, and (ii) during storage of wines under an accelerated aging procedure, allowed us to elucidate the chemical and metabolic connections between VSCs during fermentation and aging. Yeast metabolism, through the Ehrlich pathway and acetylation reactions, makes an important contribution to the formation of compounds such as methionol, 3-methylthiopropionate, 3-methylthiopropylacetate, 3-mercaptopropanol, 2-mercaptoethanol and thioesters. By contrast, chemical reactions are responsible for interconversions between thiols and disulfides, the formation of thiols from thioesters or, more surprisingly, the formation of ethylthiopropanol from methionol during fermentation. During aging, variations in heavy VSC concentrations, such as an increase in 3-methylthiopropylacetate and a decrease in ethyl-3-methylthiopropionate formation, were evidenced. Overall, this study highlights that it is essential to consider both yeast metabolism and the high chemical reactivity of VSCs to understand their formation and evolution during winemaking