Effect of grape must polyphenols on yeast metabolism during alcoholic fermentation.

In red winemaking, polyphenols from grape berry pericarp and seed are extracted during fermentation and their interactions with yeast have been widely demonstrated. However, information concerning the impact of extracted polyphenols on yeast metabolism during fermentation is missing. The aim of this study was to further explore interactions between yeasts and polyphenols and to identify their effects on yeast metabolism and fermentation kinetics. This impact was studied in synthetic musts for four commercial Saccharomyces cerevisiae wine strains, using polyphenols purified from a thermovinification must, in both stressed (phytosterol deficient medium) and non-stressed conditions. Interactions between grape polyphenols and yeast cells were substantiated from the early stage of fermentation by means of epifluorescence and confocal microscopy. If these interactions were limited to yeast cell walls in non-stressed conditions, the passage of polyphenols through yeast envelope and their accumulation in the intracellular space of living cells was shown in phytosterol-deficient medium. Whatever the conditions used (stressed and non-stressed conditions) and for all strains, the presence of polyphenols led to a significant decrease of cell growth (50%), CO2 production rate (60 to 80%) and nitrogen consumption (3 to 4 times less), resulting in increased fermentation lengths. The perturbation of yeast growth and metabolism due to polyphenol compounds was likely mostly linked to their interactions with the yeast plasma membrane. From the mid-stationary phase to the end of the fermentation, an adaptive response was exhibited by yeast, resulting in lower mortality. This work evidenced a strong impact of polyphenols on yeast fermentative capacity and highlighted the importance of a better knowledge of the mechanisms involved to improve the management of fermentations in the context of red winemaking.

Dynamics and quantitative analysis of the synthesis of fermentative aromas by an evolved wine strain of Saccharomyces cerevisiae.

We performed a dynamic and quantitative analysis of the synthesis of fermentative aromas by an aromatic wine yeast, ECA5, obtained by adaptive evolution. During fermentation at pilot scale on synthetic and natural musts, ECA5 produced volatile compounds (higher alcohols and their acetates, ethyl esters) at higher rates than the ancestral strain, with the exception of propanol. Marked differences in the chronology of synthesis of several compounds were observed between the two strains. Overproduction of phenyl ethanol occurred mainly during the growth phase for ECA5, consistent with its higher flux through the pentose phosphate pathway, which plays a key role in biosynthetic processes. The kinetics of production of isobutanol and isoamyl alcohol were differently affected by different media (synthetic or natural must) and, in particular, according to the nature of the sterols in the media (ergosterol or phytosterols). We also observed differences in the chronology of synthesis of ethyl acetate and isoamyl acetate or ethyl esters, suggesting that the regulation of the synthesis of these compounds in the evolved strain differs from that in the ancestral strain. This study shows that a dynamic analysis of volatile compounds, using high acquisition frequency online gas chromatography, can provide novel insights into the synthesis and regulation of aromas and is thus a potentially powerful tool for strain characterization.

Nitrogen-backboned modeling of wine-making in standard and nitrogen-added fermentations.

Nitrogen has a strong impact on the key bio-mechanisms involved during the grape-must fermentation but also on the synthesis of flavour markers determining the aromatic profile of the wine. This paper first presents a consistent dynamical mass balance model describing the main physiological phenomena implied in standard batch fermentations, i.e. consumption of sugar and nitrogen and synthesis of ethanol. It also includes nitrogen compounds such as hexose transporters. Moreover, a common practice in wine-making is the addition of nitrogen during the fermentation in order to boost and shorten the process duration. A tractable representation of this boost effect has therefore been developed as an extension of the first model. It is apparent that yeast makes a different use of nitrogen depending on the fermentation stage at which the addition is effected, balancing the regrowth of biomass and the synthesis of supplementary hexose transporters. These models have been validated in line with experimental evidence deduced from extensive experimental studies.

Metabolic responses of Saccharomyces cerevisiae to valine and ammonium pulses during four-stage continuous wine fermentations.

Nitrogen supplementation, which is widely used in winemaking to improve fermentation kinetics, also affects the products of fermentation, including volatile compounds. However, the mechanisms underlying the metabolic response of yeast to nitrogen additions remain unclear. We studied the consequences for Saccharomyces cerevisiae metabolism of valine and ammonium pulses during the stationary phase of four-stage continuous fermentation (FSCF). This culture technique provides cells at steady state similar to that of the stationary phase of batch wine fermentation. Thus, the FSCF device is an appropriate and reliable tool for individual analysis of the metabolic rerouting associated with nutrient additions, in isolation from the continuous evolution of the environment in batch processes. Nitrogen additions, irrespective of the nitrogen-containing compound added, substantially modified the formation of fermentation metabolites, including glycerol, succinate, isoamyl alcohol, propanol, and ethyl esters. This flux redistribution, fulfilling the requirements for precursors of amino acids, was consistent with increased protein synthesis resulting from increased nitrogen availability. Valine pulses, less efficient than ammonium addition in increasing the fermentation rate, were followed by a massive conversion of this amino acid in isobutanol and isobutyl acetate through the Ehrlich pathway. However, additional routes were involved in valine assimilation when added in stationary phase. Overall, we found that particular metabolic changes may be triggered according to the nature of the amino acid supplied, in addition to the common response. Both these shared and specific modifications should be considered when designing strategies to modulate the production of volatile compounds, a current challenge for winemakers.

Use of a continuous multistage bioreactor to mimic winemaking fermentation.

Continuous fermentation set-ups are of great interest for studying the physiology of microorganisms. In winemaking conditions, yeasts go through a growth phase and a stationary phase during which more than half of the sugar is fermented. A comprehensive study of wine-yeast physiology must therefore include yeasts in a non-growing phase. This condition is impossible to achieve within a chemostat, which led us to design a multi-stage fermentation device. In this study, we evaluated the ability of such a device to reproduce, in a series of steady states, the conditions of batch fermentation. Two-stage and four-stage fermentations were carried out with two different strains of Saccharomyces cerevisiae. The main characteristics of the fermentation process (biomass growth, by-product content of the medium) were compared with those observed in batch mode at the same stage of fermentation, which was defined by glucose uptake. The four-stage configuration showed a better ability to reproduce batch fermentation characteristics than the two-stage set-up. It also allowed to uncouple the variations of environmental parameters and proved to be a promising tool to gain new insights into yeast metabolism during alcoholic fermentation.

Kinetic analysis of a trehalase-overexpressing strain grown on trehalose: a new tool for respiro-fermentative transition studies in Saccharomyces cerevisiae.

AIMS: The aim was to demonstrate the use of a trehalase-overexpressing Saccharomyces cerevisiae strain grown on trehalose as a valuable tool in the studies of respiro-fermentative transition at a reduced scale. METHODS AND RESULTS: A trehalase-overexpressing strain was cultivated in synthetic medium on trehalose under aerobic conditions. This strain grew at a maximum specific growth rate of 0.16 h(-1) and showed a pure oxidative metabolism. Glucose pulse experiments were carried out in this system in order to quantify the short-term Crabtree effect. These data were then compared with glucose pulse experiments carried out in the conventional way with the wild-type strain in glucose-limited chemostats. Glucose-pulse experiments in aerobic batch cultures grown on trehalose led to a metabolic respiro-fermentative transition similar to the one observed in glucose-limited chemostats. CONCLUSIONS: This cultivation system allowed us to quantitatively mimic at the flask scale the Crabtree effect observed in conventional chemostat studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is of primary interest in S. cerevisiae studies in which: (i) the implementation of oxidative growth is required (as with studies of the Crabtree effect and heterologous protein production); (ii) small-scale culture systems are required (e.g. high-throughput mutant screening and isotopic labelling experiments).