Arlete Mendes-Faia

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

Aims:  To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae.

Transcriptional Response of Saccharomyces cerevisiae to Different Nitrogen Concentrations during Alcoholic Fermentation

ABSTRACT Gene expression profiles of a wine strain of Saccharomyces cerevisiae PYCC4072 were monitored during alcoholic fermentations with three different nitrogen supplies: (i) control fermentation (with enough nitrogen to complete sugar fermentation), (ii) nitrogen-limiting fermentation, and (iii) the addition of nitrogen to the nitrogen-limiting fermentation (refed fermentation). Approximately 70% of the yeast transcriptome was altered in at least one of the fermentation stages studied, revealing the continuous adjustment of yeast cells to stressful conditions. Nitrogen concentration had a decisive effect on gene expression during fermentation. The largest changes in transcription profiles were observed when the early time points of the N-limiting and control fermentations were compared. Despite the high levels of glucose present in the media, the early responses of yeast cells to low nitrogen were characterized by the induction of genes involved in oxidative glucose metabolism, including a significant number of mitochondrial associated genes resembling the yeast cell response to glucose starvation. As the N-limiting fermentation progressed, a general downregulation of genes associated with catabolism was observed. Surprisingly, genes encoding ribosomal proteins and involved in ribosome biogenesis showed a slight increase during N starvation; besides, genes that comprise the RiBi regulon behaved distinctively under the different experimental conditions. Here, for the first time, the global response of nitrogen-depleted cells to nitrogen addition under enological conditions is described. An important gene expression reprogramming occurred after nitrogen addition; this reprogramming affected genes involved in glycolysis, thiamine metabolism, and energy pathways, which enabled the yeast strain to overcome the previous nitrogen starvation stress and restart alcoholic fermentation.

Survey of hydrogen sulphide production by wine yeasts.

Twenty-one strains of commercial wine yeasts and 17 non-Saccharomyces species of different provenance were surveyed for their ability to produce hydrogen sulphide in synthetic grape juice medium indicator agar with different nitrogen sources, as well as in natural grape juice. Bacto Biggy agar, a commercially available bismuth-containing agar, was used to compare our results with others previously reported in the literature. Under identical physiological conditions, the strains used in this study displayed similar growth patterns but varied in colony color intensity in all media, suggesting significant differences in sulphite reductase activity. Sulphite reductase activity was absent for only one strain of Saccharomyces cerevisiae. All other strains produced an off-odor to different extents, depending significantly (P <0.05) on medium composition. Within the same species of some non-Saccharomyces yeasts, strain variation existed as it did for Saccharomyces. In natural musts, strains fell into three major groups: (i) nonproducers, (ii) must-composition-dependent producers, and (iii) invariable producers. In synthetic media, the formation of sulphide by strains of S. cerevisiae results from the reduction of sulphate. Therefore, this rapid screening methodology promises to be a very useful tool for winemakers for determining the risk of hydrogen sulphide formation by a given yeast strain in a specific grape juice.

Nitrogen addition influences formation of aroma compounds, volatile acidity and ethanol in nitrogen deficient media fermented by Saccharomyces cerevisiae wine strains.

The effects of nitrogen addition into nitrogen deficient/depleted media on the release of aroma compounds post-fermentation were investigated in three commercial yeast strains of Saccharomyces cerevisiae which highlight the yeast strain effect as well as nitrogen effects. By comparing the two timings of nitrogen addition, prior to fermentation or later at stationary phase (72 h), it was shown that nitrogen addition at stationary phase significantly decreases ethanol and acetic acid formation and significantly increases the following compounds: 2-phenylethanol, ethyl isobutyrate, 2-phenylethyl acetate, ethyl 2-methylbutyrate and ethyl propionate in the three strains, and also isovaleric acid, isoamyl alcohol and ethyl isovalerate in both PYCC4072 and UCD522. The strain EC1118 produced significantly less medium chain fatty acids, hexanoic, octanoic and decanoic acids and their respective esters after nitrogen addition. Therefore, timing of nitrogen addition to a ferment media can vary the concentration of certain aroma compound and might provide a means for varying wine composition.

The nature of the nitrogen source added to nitrogen depleted vinifications conducted by a Saccharomyces cerevisiae strain in synthetic must affects gene expression and the levels of several volatile compounds

Nitrogen starvation may lead to stuck and sluggish fermentations. These undesirable situations result in wines with high residual sugar, longer vinification times, and risks of microbial contamination. The typical oenological method to prevent these problems is the early addition of ammonium salts to the grape juice, although excessive levels of these compounds may lead to negative consequences for the final product. This addition reduces the overall fermentation time, regardless of the time of addition, but the effect is more significant when nitrogen is added during the yeast exponential phase. In this work we analysed the effect of adding different nitrogen sources (ammonia, amino acids or a combination of both) under nitrogen depletion in order to understand yeast metabolic changes that lead to the adaptation to the new conditions. These studies were carried out in a synthetic must that mimics the composition of the natural must. Furthermore, we studied how this addition affects fermentative behaviour, the levels of several yeast volatile compounds in the final product, arginase activity, and the expression of several genes involved in stress response and nitrogen metabolism during vinification. We found that the nature of the nitrogen source added during yeast late exponential growth phase introduces changes to the volatile compounds profile and to the gene expression. On the other hand, arginase activity and the expression of the stress response gene ACA1 are useful to monitor nitrogen depletion/addition during growth of the wine yeast considered under our vinification conditions.

Optimization of honey-must preparation and alcoholic fermentation by Saccharomyces cerevisiae for mead production.

Mead fermentation is a time-consuming process, often taking several months to complete. Despite of the use of starter cultures several problems still persist such as lack of uniformity of the final products, slow or premature fermentation arrest and the production of off-flavors by yeast. Thus the aim of this study was to optimize mead production through the use of an appropriate honey-must formulation to improve yeast performance alcoholic fermentation and thereby obtain a high quality product. Honey-must was centrifuged to reduce insoluble solids, pasteurized at 65°C for 10 min, and then subjected to different conditions: nitrogen supplementation and addition of organic acids. Although the addition of diammonium phosphate (DAP) reduced fermentation length, it did not guarantee the completeness of the fermentation process, suggesting that other factors could account for the reduced yeast activity in honey-must fermentations. Sixteen yeast-derived aroma compounds which contribute to the sensorial quality of mead were identified and quantified. Global analysis of aromatic profiles revealed that the total concentration of aroma compounds in meads was higher in those fermentations where DAP was added. A positive correlation between nitrogen availability and the levels of ethyl and acetate esters, associated to the fruity character of fermented beverages, was observed whereas the presence of potassium tartrate and malic acid decreased, in general, their concentration. This study provides very useful information that can be used for improving mead quality.

Reduction of volatile acidity of wines by selected yeast strains

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose–low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose–high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.

The impact of acetate metabolism on yeast fermentative performance and wine quality: reduction of volatile acidity of grape musts and wines

Acetic acid is the main component of the volatile acidity of grape musts and wines. It can be formed as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, which can metabolize residual sugars to increase volatile acidity. Acetic acid has a negative impact on yeast fermentative performance and affects the quality of certain types of wine when present above a given concentration. In this mini-review, we present an overview of fermentation conditions and grape-must composition favoring acetic acid formation, as well the metabolic pathways leading to its formation and degradation by yeast. The negative effect of acetic acid on the fermentative performance of Saccharomyces cerevisiae will also be covered, including its role as a physiological inducer of apoptosis. Finally, currently available wine deacidification processes and new proposed solutions based on zymological deacidification by select S. cerevisiae strains will be discussed.

The nitrogen source impacts major volatile compounds released by Saccharomyces cerevisiae during alcoholic fermentation

Sulphur-containing amino acids, cysteine and methionine, are generally found in very low concentrations in grape-juice. The objective of this study was to identify the effects of methionine on aroma compounds formation. Nitrogen source effects on growth, fermentative behaviour and aroma compounds formation were evaluated in three strains of Saccharomyces cerevisiae cultivated in batch under moderate nitrogen concentration, 267mg YAN/L, supplied as di-ammonium phosphate (DAP), a mixture of amino acids with (AA) or without methionine (AA(wMet)), and a mixture of AA plus DAP. Fermentative vigour and final biomass yields were dependent on the nitrogen source, for each of the strains tested, in particular for EC1118. Additionally, despite the strain-dependent behaviour with respect to the basal level of H(2)S produced, the comparison of treatments AA and AA(wMet) showed that presence of methionine suppressed H(2)S production in all strains tested, and altered aroma compound formation, particularly some of those associated with fruity and floral characters which were consistently more produced in AA(wMet). Moreover, DAP supplementation resulted in a remarkable increase in H(2)S formation, but no correlation between sulphide produced and yeast fermentative vigour was observed. Results suggest that the use of different nitrogen sources results in the production of wines with divergent aroma profiles, most notably when EC1118 strain is used. Methionine determination and its management prior to fermentation are crucial for suppressing H(2)S and to endowing beverages with diverse sensory traits.

Accumulation of Non-Superoxide Anion Reactive Oxygen Species Mediates Nitrogen-Limited Alcoholic Fermentation by Saccharomyces cerevisiae

ABSTRACT Throughout alcoholic fermentation, nitrogen depletion is one of the most important environmental stresses that can negatively affect the yeast metabolic activity and ultimately leads to fermentation arrest. Thus, the identification of the underlying effects and biomarkers of nitrogen limitation is valuable for controlling, and therefore optimizing, alcoholic fermentation. In this study, reactive oxygen species (ROS), plasma membrane integrity, and cell cycle were evaluated in a wine strain of Saccharomyces cerevisiae during alcoholic fermentation in nitrogen-limiting medium under anaerobic conditions. The results indicated that nitrogen limitation leads to an increase in ROS and that the superoxide anion is a minor component of the ROS, but there is increased activity of both Sod2p and Cta1p. Associated with these effects was a decrease in plasma membrane integrity and a persistent cell cycle arrest at G0/G1 phases. Moreover, under these conditions it appears that autophagy, evaluated by ATG8 expression, is induced, suggesting that this mechanism is essential for cell survival but does not prevent the cell cycle arrest observed in slow fermentation. Conversely, nitrogen refeeding allowed cells to reenter cell cycle by decreasing ROS generation and autophagy. Altogether, the results provide new insights on the understanding of wine fermentations under nitrogen-limiting conditions and further indicate that ROS accumulation, evaluated by the MitoTracker Red dye CM-H2XRos, and plasma membrane integrity could be useful as predictive markers of fermentation problems.