Ana Mendes-Ferreira

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.

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.

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.

Saccharomyces cerevisiae Signature Genes for Predicting Nitrogen Deficiency during Alcoholic Fermentation

ABSTRACT Genome-wide analysis of the wine yeast strain Saccharomyces cerevisiae PYCC4072 identified 36 genes highly expressed under conditions of low or absent nitrogen in comparison with a nitrogen-replete condition. Reverse transcription-PCR analysis for four of these transcripts with this strain and its validation with another wine yeast strain underlines the usefulness of these signature genes for predicting nitrogen deficiency and therefore the diagnosis of wine stuck/sluggish fermentations.

The timing of diammonium phosphate supplementation of wine must affects subsequent H2S release during fermentation.

Aims: The aim of this study was to evaluate the impact of supplementation by diammonium phosphate (DAP) on hydrogen sulfide (H2S) production, when DAP given either prior to fermentation or during the early stationary growth phase of yeast.

H. guilliermondii impacts growth kinetics and metabolic activity of S. cerevisiae: The role of initial nitrogen concentration

Non-Saccharomyces yeasts include different species which comprise an ecologically and biochemically diverse group capable of altering fermentation dynamics and wine composition and flavour. In this study, single- and mixed-culture of Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used to ferment natural grape-juice, under two nitrogen regimes. In single-culture the strain H. guilliermondii failed to complete total sugar breakdown even though the nitrogen available has not been a limiting factor of its growth or fermentative activity. In mixed-culture, that strain negatively interfered with the growth and fermentative performance of S. cerevisiae, resulting in lower fermentation rate and longer fermentation length, irrespective of the initial nitrogen concentration. The impact of co-inoculation on the volatile compounds profile was more evident in the wines obtained from DAP-supplemented musts, characterised by increased levels of ethyl and acetate esters, associated with fruity and floral character of wines. Moreover, the levels of fatty acids and sulphur compounds which are responsible for unpleasant odours that depreciate wine sensory quality were significantly lower. Accordingly, data obtained suggests that the strain H. guilliermondii has potential to be used as adjunct of S. cerevisiae in wine industry, although possible interactions with S. cerevisiae still need to be elucidated.