Disease Resistant Bouquet Vine Varieties: Assessment of the Phenolic, Aromatic, and Sensory Potential of Their Wines.

The search for grape varieties resistant to diseases and to climatic changes notably concerns the wine industry. Nine monovarietal wines from new red grape varieties resistant to cryptogamic diseases (downy and powdery mildews) were evaluated in terms of their total phenolic, anthocyanin and proanthocyanidin contents, anthocyanin profile, volatile composition, and sensory attributes. Thus, the question remains, will these hybrid grapes (≥97.5% of Vitisvinifera genome) lead to wines with organoleptic properties similar to those of Vitisvinifera wines that consumers are used to? Total phenolic (1547-3418 mg GA/L), anthocyanin (186-561 mg malvidin/L), and proanthocyanidin (1.4-4.5 g tannins/L) contents were in broad agreement with those previously described in the literature for monovarietal wines produced with well-known red grape varieties (Cabernet Sauvignon, Merlot, Syrah). With regard to fruity aroma, ethyl esters of straight-chain fatty acids (530-929 µg/L) stood out clearly as the major volatile components for all hybrid wines considered. Sensory analysis revealed significant differences (p < 0.05) for visual aspect, aroma, flavor, global balance, astringency, and body. Overall, these new hybrid grape varieties are not only resistant to cryptogamic diseases, but also present enough potential to become quality wines, since their phenolic and volatile attributes are close to those of common red monovarietal wines.

Comparative evolution of oxygen, carbon dioxide, nitrogen, and sulfites during storage of a rosé wine bottled in PET and glass.

The management of dissolved and headspace gases during bottling and the choice of packaging are both key factors for the shelf life of wine. Two kinds of 75 cL polyethylene terephthalate (PET) bottles (with or without recycled PET) were compared to glass bottles filled with a rosé wine, closed with the same screwcaps and stored upright at 20 °C in light or in the dark. Analytical monitoring (aphrometric pressure, headspace volume, O2, N2, CO2, and SO2) was carried out for 372 days. After the consumption of O2 trapped during bottling, the total O2 content in glass bottles remained stable. A substantial decrease of CO2 and SO2 concentration and an increase of O2 concentration were observed in the PET bottles after 6 months because of the considerable gas permeability of monolayer PET. Light accelerated O2 consumption during the early months. Finally, the kinetic monitoring of partial pressures in gas and liquid phases in bottles showed contrasting behavior of O2 and N2 in comparison with CO2.

New precursor of 3-mercaptohexan-1-ol in grape juice: thiol-forming potential and kinetics during early stages of must fermentation.

Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are key aroma impact compounds in many young white wines, especially of the variety Sauvignon blanc (SB). Although great effort has been invested to identify their precursors in recent years, the origin of the majority of 3MH and 3MHA generated during wine fermentation still cannot be explained. Here we demonstrate that supplying an external source of hydrogen sulfide to grape juice hugely increases its thiol-forming potential. We further describe the discovery of (E)-2-hexen-1-ol as an additional new thiol precursor and demonstrate that it possesses, together with (E)-2-hexenal, an immense thiol-forming potential during fermentation. Both C6-compounds are extremely rapidly metabolized by yeast during the first hours after inoculation, even under commercial conditions, and can be interconverted during this phase depending on their initial concentration in the grape juice. Spiking grape juice with additional acetaldehyde greatly enhanced the (E)-2-hexen-1-ol to (E)-2-hexenal conversion rate. Delaying the metabolization of the two unsaturated C6-thiol precursors by yeast, at the same time as increasing hydrogen sulfide production early in fermentation, opens up a great opportunity to tap into this enormous potential 3MH and 3MHA source in grape juice and extends the possibility of thiol production to other non-grape-based alcoholic beverages as well.

Pathogenesis of Junonia coenia densovirus in Spodoptera frugiperda: a route of infection that leads to hypoxia.

To evaluate densovirus potential against lepidopteran pests and their capacity to invade new hosts, we have characterised in vivo the infection and pathogenesis of the Junonia coenia densovirus (JcDNV) in the noctuid pest Spodoptera frugiperda. Here we show that infection starts with the ingestion of viral particles that cross the midgut epithelium without replicating. By quantitative PCR we established the kinetic and the route of infection, from virus ingestion to replication in visceral tracheae and hemocytes. JcDNV has a high particle-to-infection ratio mostly due to the barrier function of the midgut. Pathology and cytopathology suggested that infection of tracheal cells impairs oxygen delivery to demanding tissues leading to cytopathic effects in all the tissues. Finally, larval death results from several physiological shocks, including molting arrest and anoxia.

A comparison of laboratory and pilot-scale fermentations in winemaking conditions.

We investigated the influence of the fermenter size on alcoholic fermentation. Experiments were carried out at pilot scale, in 100-L fermenters, and at laboratory scale, in stirred and static 1-L fermenters. Two musts, Grenache blanc and Sauvignon, were fermented with and without the addition of solid particles from grape musts. Highly clarified must fermentation kinetics was strongly affected by the scale of the experiment, with slower fermentation occurring in the 100-L fermenter. Alcohol, ester, and thiol synthesis in clarified sauvignon must fermentation was also strongly correlated with the fermentation scale. Addition of solid particles from grape tended to reduce the effects on kinetics associated with increasing the scale of the fermentation, by increasing the maximum rate of CO(2) production, and by shortening the duration of fermentation. The addition of such particles also decreased the effects of scaling up the fermentation on the concentration of some volatile compounds, i.e., isoamyl acetate, ethyl octanoate, but did not decrease this effect for other compounds, such as isobutyl acetate, isobutanol, and 3-mercaptohexanol.

Very early acetaldehyde production by industrial Saccharomyces cerevisiae strains: a new intrinsic character.

During a general survey of the acetaldehyde-producing properties of commercially available wine yeast strains, we discovered that, although final acetaldehyde production cannot be used as a discriminating factor between yeast strains, initial specific acetaldehyde production rates were of highly interest for classifying yeast strains. This parameter is very closely related to the growth- and fermentation-lag phase durations. We also found that this acetaldehyde early production occurs with very different extent between commercial active dry yeast strains during the rehydration phase and could partially explain the known variable resistance of yeast strains to sulfites. Acetaldehyde production appeared, therefore, as very precocious, strain-dependent, and biomass-independent character. These various findings suggest that this new intrinsic characteristic of industrial fermenting yeast may be likely considered as an early marker of the general fermenting activity of industrial fermenting yeasts. This phenomenon could be particularly important for understanding the ecology of colonization of complex fermentation media by Saccharomyces cerevisiae.

Plasma membrane sterols are involved in yeast's ability to adsorb polyphenolic compounds resulting from wine model solution browning.

The aim of this work was to demonstrate the direct interaction between membrane sterols of yeast lees and some polymerized phenolic compounds resulting from wine model solution browning. For this purpose, we first demonstrated by measurement of steady-state fluorescence anisotropy of the cationic fluorescent TMA-DPH probe the effect of polymerized compounds from the model reactions of (+)-catechin/acetaldehyde and (+)-catechin/glyoxylic acid on the plasma membrane order of Saccharomyces cerevisiae yeast lees enriched with different sterols. In a second set of experiments, we used S. cerevisiae plasma membrane vesicles spiked with different sources of sterol (ergosterol, cholesterol or a mix of grape phytosterols) to assess the effect of the same polymerized compounds on both vesicle integrity and membrane leakiness to protons by ACMA fluorescence. All the obtained results prove that yeast membrane sterols are able to strongly interact with some polymerized compounds resulting from the browning of model solutions, likely explaining the yeast ability to adsorb polyphenolic compounds and mainly the colorless intermediate compounds of the browning reactions.

Effect of different yeast strains and their culture conditions on the prevention of wine model solution browning by yeast lees.

The purpose of this work was to examine the possible involvement of yeast membrane components in the adsorption of browning compounds from oxidized white wine. For this purpose, different yeast strains and growth conditions (aerobiosis and anaerobiosis) were tested for their ability to prevent browning of two model solutions consisting of (+)-catechin/acetaldehyde and (+)-catechin/glyoxylic acid. The obtained results showed that the effects of yeast lees are different according to the type of the studied model solution and the growth conditions that affect both the quantity and the quality of membrane sterols of the yeasts. Moreover, in vitro experiments proved that yeast membrane sterols could be likely involved in the yeast's ability to adsorb polyphenolic compounds and mainly the colorless intermediate compounds of the browning reactions.

New insights on 3-mercaptohexanol (3MH) biogenesis in Sauvignon Blanc wines: Cys-3MH and (E)-hexen-2-al are not the major precursors.

The molar conversion yield of Cys-3MH into 3MH, during alcoholic fermentation, was traced using a deuterated isotope of the precursor added to different Sauvignon Blanc musts. This yield is close to that found in synthetic media supplemented with synthetic Cys-3MH, that is, below 1%. Yet, this represents only 3-7% of the total 3MH production in wine. This clearly shows that Cys-3MH is a precursor of 3MH, but not the main one in the different musts tested. The contribution of ( E)-hex-2-enal, which has been suggested as another potential precursor of 3MH, was discarded as well, as shown using also a deuterated analogue. The third suggested precursor of 3MH is a glutathionyl-3MH (G-3MH), which upon proteolytic degradation could release Cys-3MH. The knockout of the OPT1 gene, which encodes the major glutathione transporter, reduces 3MH accumulation by a 2-fold factor in grape must as compared to the wild type strain. Consequently, it is deduced that major 3MH precursor(s) are transported into yeast via Opt1p, which is in favor of G-3MH being a 3MH precursor. This work opens the search for the major natural precursor(s) of 3MH in Sauvignon Blanc must.

Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport.

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.

Acetaldehyde addition throughout the growth phase alleviates the phenotypic effect of zinc deficiency in Saccharomyces cerevisiae.

During experiments to determine the effects of exogenously added acetaldehyde on pure cultures of various yeast strains, we discovered that an early acetaldehyde perfusion during the growth phase allowed several yeasts to partially overcome the phenotypic effects of zinc depletion during alcoholic fermentation. We, therefore, performed genome-wide expression and proteomic analysis on an industrial Saccharomyces cerevisiae yeast strain (VL1) growing in zinc-replete or zinc-depleted conditions in the presence of perfused acetaldehyde to identify molecular markers of this effect. Zinc depletion severely affects ethanol production and therefore nicotinamide adenine dinucleotide (NAD) regeneration, although we observed partial compensation by the upregulation of the poorly efficient Fe-dependent Adh4p in our conditions. A coordinate metabolic response was indeed observed in response to the early acetaldehyde perfusion, and particularly of the lower part of glycolysis, leading to the cellular replenishment of NAD cofactor. These various findings suggest that acetaldehyde exchange between strains may inhibit the growth of some yeast strains while encouraging the growth of others. This phenomenon could be particularly important for understanding the ecology of colonization of complex fermentation media by S. cerevisiae after elimination of non-Saccharomyces yeasts.

Interactions between yeast lees and wine polyphenols during simulation of wine aging. II. Analysis of desorbed polyphenol compounds from yeast lees.

In the first part of this work, the analysis of the polyphenolic compounds remaining in the wine after different contact times with yeast lees during simulation of red wine aging was undertaken. To achieve a more precise view of the wine polyphenols adsorbed on lees during red wine aging and to establish a clear balance between adsorbed and remnant polyphenol compounds, the specific analysis of the chemical composition of the adsorbed polyphenolic compounds (condensed tannins and anthocyanins) after their partial desorbtion from yeast lees by denaturation treatments was realized in the second part of the study. The total recovery of polyphenol compounds from yeast lees was not complete, since a rather important part of the initial wine colored polyphenols, especially those with a dominant blue color component, remained strongly adsorbed on yeast lees, as monitored by color tristimulus and reflectance spectra measurements. All anthocyanins were recovered at a rather high percentage (about 62%), and it was demonstrated that they were not adsorbed in relation with their sole polarity. Very few monomeric phenolic compounds were extracted from yeast lees. With the use of drastic denaturing treatments, the total recovery of condensed tannins reached 83%. Such tannins extracted from yeast lees exhibited very high polymeric size and a rather high percentage of galloylated residues by comparison with initial wine tannins, indicating that nonpolar tannins were preferentially desorbed from yeast lees by the extraction treatments.

Formation of micella containing solubilized sterols during rehydration of active dry yeasts improves their fermenting capacity.

During their rehydration in aqueous media, active dry yeasts (ADY) may be supplemented with inactive yeasts, yeast derivatives, or other optional complementary nutrients to improve their fermentation capacity. We found that yeast sterols solubilized in situ during ADY rehydration were particularly efficient for stimulating the fermenting capacity of ADY. Spontaneous solubilization of sterols during rehydration occurred by the formation of micelles by membrane phospholipids and specific cell wall polysaccharides and sterols, both compounds being provided by inactive dry yeasts (IDY). These micelles contained a specific distribution of the initial sterols from the inactive yeasts. Above a concentration of 100 mg L(-1) in the rehydration medium, these micelles acted as emulsifiers. Their critical micellar concentration (cmc) was found to be about 4 g L(-1). During rehydration, purified micelles, at a concentration near the cmc, were able to interact quickly with yeast cell membranes by modifying the yeast plasma membrane order [monitored by steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) probe] and by increasing the sterol contents of ADY. Such an enrichment of ADY by very low concentrations of solubilized sterols was very efficient for the completion of fermentations. This is useful when musts are limited in available phytosterols or when micro-oxygenation is not desirable during fermentation.

Interactions between yeast lees and wine polyphenols during simulation of wine aging: I. Analysis of remnant polyphenolic compounds in the resulting wines.

Wine aging on yeast lees is a traditional enological practice used during the manufacture of wines. This technique has increased in popularity in recent years for the aging of red wines. Although wine polyphenols interact with yeast lees to a limited extent, such interactions have a large effect on the reactivity toward oxygen of wine polyphenolic compounds and yeast lees. Various domains of the yeast cell wall are protected by wine polyphenols from the action of extracellular hydrolytic enzymatic activities. Polysaccharides released during autolysis are thought to exert a significant effect on the sensory qualities of wine. We studied the chemical composition of polyphenolic compounds remaining in solution or adsorbed on yeast lees after various contact times during the simulation of wine aging. The analysis of the remnant polyphenols in the wine indicated that wine polyphenols adsorption on yeast lees follows biphasic kinetics. An initial and rapid fixation is followed by a slow, constant, and saturating fixation that reaches its maximum after about 1 week. Only very few monomeric phenolic compounds remained adsorbed on yeast lees, and no preferential adsorption of low or high polymeric size tannins occurred. The remnant condensed tannins in the wine contained fewer epigallocatechin units than the initial tannins, indicating that polar condensed tannins were preferentially adsorbed on yeast lees. Conversely, the efficiency of anthocyanin adsorption on yeast lees was unrelated to its polarity.

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation.

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P(50))]. P(50) was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.

Redox interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in mixed culture under enological conditions.

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.

Isolation and properties of promitochondria from anaerobic stationary-phase yeast cells.

Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells.

Impact of oxygen consumption by yeast lees on the autolysis phenomenon during simulation of wine aging on lees.

Potential oxygen consumption by lees, more precisely by nonviable yeasts, during wine aging was recently described. Additionally, yeast autolysis is described as the main mechanism of degradation of lees during wine aging. Thus, to understand the effect of oxygen consumption by yeast lees during wine aging, an accelerated wine aging methodology was tested. Wine aging in the presence of yeast lees was studied both in the presence and in the absence of oxygen. Different markers of yeast autolysis were followed to find a relationship between oxygen consumption by yeast lees and changes in the final wine composition after aging. No differences for compounds tested were found in the wine and in the lees except among sterol compounds in lees: in the presence of oxygen, the concentration of ergosterol in lees was significantly lower than that in the absence of oxygen. It was hypothesized that ergosterol could be oxidized under the influence of oxygen, but none of the known products of ergosterol oxidation were recovered in the corresponding yeast lees. In addition, the decrease of ergosterol content in yeast lees cannot account for the total amount of oxygen consumed by yeast lees during such wine aging.

Oxygen consumption by anaerobic Saccharomyces cerevisiae under enological conditions: effect on fermentation kinetics.

The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.

Impact of oxygen addition during enological fermentation on sterol contents in yeast lees and their reactivity towards oxygen.

During enological fermentations, superfluous oxygen consumption by yeast cells is observed. The superfluous oxygen consumed by the yeast cells is mainly related to the operation of non-respiratory oxygen consumption pathways resulting in an overall decrease in the total sterol fraction in yeast. On the other hand, yeast lees remaining at the end of alcoholic fermentations exhibit specific oxygen utilization rates ranging from 1 to 4 micromol O2 h- 10(-10) cells from the second to the thirteenth month of wine aging. This oxygen consumption capacity of yeast lees was independent of residual cell viability. In this study, we investigated the potential relationship between the oxygen added to commercial yeast strains during enological fermentation and the capacity of the corresponding yeast lees to interact with oxygen. Additions of low (7 mg l(-)) and excess (37 mg l(-1)) amounts of oxygen at the end of the cell growth phase were compared in terms of repercussions on the oxygen consumption activity of the corresponding yeast lees. As expected, the superfluous oxygen consumption by yeast cells during fermentation had a positive influence on the fermentation kinetics and increased cell biomass formation. Oxygen consumption rates and the total capacity of oxygen consumption by the corresponding yeast lees clearly decreased when oxygen was added during fermentation. This marked decrease in yeast lees reactivity towards oxygen was concomitantly related to an increase in ergosterol synthesis and to oxygen-dependent sterol degradation. Such degradation occurred when oxygen was added in excess. Therefore, oxygenation control during fermentation appears to be a potential way to optimize both the fermentation kinetics and control yeast lees reactivity towards oxygen. For practical applications, oxygenation control during alcoholic fermentation may be considered as a general tool for decreasing the highly reductive effect of yeast lees during wine aging.