Molecular mechanism of flocculation self-recognition in yeast and its role in mating and survival.

UNLABELLED: We studied the flocculation mechanism at the molecular level by determining the atomic structures of N-Flo1p and N-Lg-Flo1p in complex with their ligands. We show that they have similar ligand binding mechanisms but distinct carbohydrate specificities and affinities, which are determined by the compactness of the binding site. We characterized the glycans of Flo1p and their role in this binding process and demonstrate that glycan-glycan interactions significantly contribute to the cell-cell adhesion mechanism. Therefore, the extended flocculation mechanism is based on the self-interaction of Flo proteins and this interaction is established in two stages, involving both glycan-glycan and protein-glycan interactions. The crucial role of calcium in both types of interaction was demonstrated: Ca(2+) takes part in the binding of the carbohydrate to the protein, and the glycans aggregate only in the presence of Ca(2+). These results unify the generally accepted lectin hypothesis with the historically first-proposed "Ca(2+)-bridge" hypothesis. Additionally, a new role of cell flocculation is demonstrated; i.e., flocculation is linked to cell conjugation and mating, and survival chances consequently increase significantly by spore formation and by introduction of genetic variability. The role of Flo1p in mating was demonstrated by showing that mating efficiency is increased when cells flocculate and by differential transcriptome analysis of flocculating versus nonflocculating cells in a low-shear environment (microgravity). The results show that a multicellular clump (floc) provides a uniquely organized multicellular ultrastructure that provides a suitable microenvironment to induce and perform cell conjugation and mating. IMPORTANCE: Yeast cells can form multicellular clumps under adverse growth conditions that protect cells from harsh environmental stresses. The floc formation is based on the self-interaction of Flo proteins via an N-terminal PA14 lectin domain. We have focused on the flocculation mechanism and its role. We found that carbohydrate specificity and affinity are determined by the accessibility of the binding site of the Flo proteins where the external loops in the ligand-binding domains are involved in glycan recognition specificity. We demonstrated that, in addition to the Flo lectin-glycan interaction, glycan-glycan interactions also contribute significantly to cell-cell recognition and interaction. Additionally, we show that flocculation provides a uniquely organized multicellular ultrastructure that is suitable to induce and accomplish cell mating. Therefore, flocculation is an important mechanism to enhance long-term yeast survival.

A universal fixation method based on quaternary ammonium salts (RNAlater) for omics-technologies: Saccharomyces cerevisiae as a case study.

Genomics, transcriptomics, proteomics and fluxomics are powerful omics-technologies that play a major role in today's research. For each of these techniques good sample quality is crucial. Major factors contributing to the quality of a sample is the actual sampling procedure itself and the way the sample is stored directly after sampling. It has already been described that RNAlater can be used to store tissues and cells in a way that the RNA quality and quantity are preserved. In this paper, we demonstrate that quaternary ammonium salts (RNAlater) are also suitable to preserve and store samples from Saccharomyces cerevisiae for later use with the four major omics-technologies. Moreover, it is shown that RNAlater also preserves the cell morphology and the potential to recover growth, permitting microscopic analysis and yeast cell culturing at a later stage.

The influence of microgravity on invasive growth in Saccharomyces cerevisiae.

This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium.

Contribution of monophenols to beer flavour based on flavour thresholds, interactions and recombination experiments.

Although monophenols are known to contribute to the flavour of many foods and beverages, little is known about their influence on beer flavour. Therefore, the contribution of 11 monophenols to the overall beer flavour was studied by determining their flavour thresholds. Large differences in sensitivity were observed between individual tasters. Next, flavour interactions between monophenols were examined in nine binary mixtures, which showed that strong interactions like synergy and antagonism occur. Based on these results, the flavour contribution of the monophenols was estimated by calculating flavour units. These proved to be rather low for most of the studied monophenols. However, recombination experiments demonstrated that monophenols enriched beer flavour with spicy, smokey and vanilla flavour aspects. This showed how monophenols might influence overall flavour, even at sub-threshold concentrations.

Flocculation gene variability in industrial brewer's yeast strains.

The brewer's yeast genome encodes a 'Flo' flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmental-friendly technique to separate yeast cells from the final beer. FLO genes have the notable capacity to evolve and diverge many times faster than other genes. In actual practice, this genetic variability may directly alter the flocculin structure, which in turn may affect the flocculation onset and/or strength in an uncontrolled manner. Here, 16 ale and lager yeast strains from different breweries, one laboratory Saccharomyces cerevisiae and one reference Saccharomyces pastorianus strain, with divergent flocculation strengths, were selected and screened for characteristic FLO gene sequences. Most of the strains could be distinguished by a typical pattern of these FLO gene markers. The FLO1 and FLO10 markers were only present in five out of the 18 yeast strains, while the FLO9 marker was ubiquitous in all the tested strains. Surprisingly, three strongly flocculating ale yeast strains in this screening also share a typical 'lager' yeast FLO gene marker. Further analysis revealed that a complete Lg-FLO1 allele was present in these ale yeasts. Taken together, this explicit genetic variation between flocculation genes hampers attempts to understand and control the flocculation behavior in industrial brewer's yeasts.

Determination of volatile monophenols in beer using acetylation and headspace solid-phase microextraction in combination with gas chromatography and mass spectrometry.

Monophenols are widely spread compounds contributing to the flavour of many foods and beverages. They are most likely present in beer, but so far, little is known about their influence on beer flavour. To quantify these monophenols in beer, we optimised a headspace solid-phase microextraction method coupled to gas chromatography-mass spectrometry. To improve their isolation from the beer matrix and their chromatographic properties, the monophenols were acetylated using acetic anhydride and KHCO(3) as derivatising agent and base catalyst, respectively. Derivatisation conditions were optimised with attention for the pH of the reaction medium. Additionally, different parameters affecting extraction efficiency were optimised, including fibre coating, extraction time and temperature and salt addition. Afterwards, we calibrated and validated the method successfully and applied it for the analysis of monophenols in beer samples.

Optimisation of specialty malt volatile analysis by headspace solid-phase microextraction in combination with gas chromatography and mass spectrometry.

The objective of this study was to develop a technique for analysing 14 flavour components, relevant for specialty malts. Therefore, a method was developed for the analysis of these components in dry ground malt using headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry. A procedure was optimised for the optimal amount of sample, fibre selection, extraction temperature and extraction time. Afterwards, the method was calibrated and validated by the quantification of the specialty malt flavour components in a colour, a caramel and a roasted malt.

Electronic tongue as a screening tool for rapid analysis of beer.

An electronic tongue (ET) comprising 18 potentiometric chemical sensors was applied to the quantitative analysis of beer. Fifty Belgian and Dutch beers of different types were measured using the ET. The same samples were analyzed using conventional analytical techniques with respect to the main physicochemical parameters. Only non-correlated physicochemical parameters were retained for further analysis, which were real extract, real fermentation degree, alcohol content, pH, bitterness, color, polyphenol and CO(2) content. Relationship between the ET and physicochemical datasets was studied using Canonical Correlation Analysis (CCA). Four significant canonical variates were extracted using CCA. Correlation was observed between 6 physicochemical variables (real extract and fermentation degree, bitterness, pH, alcohol and polyphenols' content) and 14 sensors of the ET. The feasibility of the ET for the quantification of bitterness in beer was evaluated in the aqueous solutions of isomerized hop extract and in the set of 11 beers with bitterness varying between 14 and 38 EBU (European Bitterness Units). Sensors displayed good sensitivity to isomerized hop extract and good prediction of the bitterness in beer was obtained. Calibration models with respect to the physicochemical parameters using ET measurements in 50 Belgian and Dutch beer samples were calculated by Partial Least Square regression. The ET was capable of predicting such parameters as real extract, alcohol and polyphenol content and bitterness, the latter with Root Mean Square Error of Prediction (RMSEP) of 2.5.

Decrease of aged beer aroma by the reducing activity of brewing yeast.

The flavor profile of beer is subject to changes during storage. Since, possibly, yeast has an influence on flavor stability, the aim of this study was to examine if there is a direct impact of brewing yeast on aged aroma. This was achieved by refermentation of aged beers. It was shown that several aged aroma notes, such as cardboard, ribes, Maillard and Madeira, were removed almost entirely by brewing yeast, independently of the yeast or the beer type. This was explained by the reduction of aldehydes, mainly (E)-2-nonenal, Strecker aldehydes, 5-hydroxymethylfurfural and diacetyl, to their corresponding alcohols. Furthermore, it became evident that the reducing capacity of brewing yeast is high, but that yeast strain and compound specific residual concentrations remained in the refermented beer independently of the initial concentration. Finally, it appeared that aldehydes were not only reduced but also formed during refermentation.

Production and biological function of volatile esters in Saccharomyces cerevisiae.

The need to understand and control ester synthesis is driven by the fact that esters play a key role in the sensorial quality of fermented alcoholic beverages like beer, wine and sake. As esters are synthesized in yeast via several complex metabolic pathways, there is a need to gain a clear understanding of ester metabolism and its regulation. The individual genes involved, their functions and regulatory mechanisms have to be identified. In alcoholic beverages, there are two important groups of esters: the acetate esters and the medium-chain fatty acid (MCFA) ethyl esters. For acetate ester synthesis, the genes involved have already been cloned and characterized. Also the biochemical pathways and the regulation of acetate ester synthesis are well defined. With respect to the molecular basis of MCFA ethyl ester synthesis, however, significant progress has only recently been made. Next to the characterization of the biochemical pathways and regulation of ester synthesis, a new and more important question arises: what is the advantage for yeast to produce these esters? Several hypotheses have been proposed in the past, but none was satisfactorily. This paper reviews the current hypotheses of ester synthesis in yeast in relation to the complex regulation of the alcohol acetyl transferases and the different factors that allow ester formation to be controlled during fermentation.

Stability of high cell density brewery fermentations during serial repitching.

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the decreased yeast net growth observed in these high cell density brewery fermentations can adversely affect the physiological stability throughout subsequent yeast generations. Therefore, different O(2) conditions (wort aeration and yeast preoxygenation) were applied to high cell density fermentation and eight generations of fermentations were evaluated together with conventional fermentations. Freshly propagated high cell density populations adapted faster to the fermentative conditions than normal cell density populations. Preoxygenating the yeast was essential for the yeast physiological and beer flavor compound stability of high cell density fermentations during serial repitching. In contrast, the use of non-preoxygenated yeast resulted in inadequate growth which caused (1) insufficient yield of biomass to repitch all eight generations, (2) a 10% decrease in viability, (3) a moderate increase of yeast age, (4) and a dramatic increase of the unwanted flavor compounds acetaldehyde and total diacetyl during the sequence of fermentations. Therefore, to achieve sustainable high cell density fermentations throughout the economical valuable process of serial repitching, adequate yeast growth is essential.

Instrumental measurement of beer taste attributes using an electronic tongue.

The present study deals with the evaluation of the electronic tongue multisensor system as an analytical tool for the rapid assessment of taste and flavour of beer. Fifty samples of Belgian and Dutch beers of different types (lager beers, ales, wheat beers, etc.), which were characterized with respect to the sensory properties, were measured using the electronic tongue (ET) based on potentiometric chemical sensors developed in Laboratory of Chemical Sensors of St. Petersburg University. The analysis of the sensory data and the calculation of the compromise average scores was made using STATIS. The beer samples were discriminated using both sensory panel and ET data based on PCA, and both data sets were compared using Canonical Correlation Analysis. The ET data were related to the sensory beer attributes using Partial Least Square regression for each attribute separately. Validation was done based on a test set comprising one-third of all samples. The ET was capable of predicting with good precision 20 sensory attributes of beer including such as bitter, sweet, sour, fruity, caramel, artificial, burnt, intensity and body.

Determination of carbonyl compounds in beer by derivatisation and headspace solid-phase microextraction in combination with gas chromatography and mass spectrometry.

Headspace solid-phase microextraction (SPME) followed by gas chromatography and mass spectrometry was applied for quantification of 41 chemically diverse carbonyl compounds in beer. Therefore, in-solution derivatisation with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) combined with SPME was optimised for fibre selection, PFBHA concentration, extraction temperature and time and ionic strength. Afterwards, the method was calibrated and validated successfully and extraction efficiency was compared to sampling with on-fibre derivatisation. In-solution derivatisation enabled the detection of several compounds that were poorly extracted with on-fibre derivatisation such as 5-hydroxymethylfurfural, acrolein, hydroxyacetone, acetoin, glyoxal and methylglyoxal. Others, especially (E)-2-nonenal, were extracted better with on-fibre derivatisation.

The influence of yeast oxygenation prior to brewery fermentation on yeast metabolism and the oxidative stress response.

Yeast preoxygenation can confer important advantages to brewery fermentations by means of omitting the need to oxygenate the wort. However, the impact of yeast preoxygenation on yeast metabolism has never been assessed systematically. Therefore, expression analysis was performed of genes that are of importance in oxygen-dependent pathways, oxidative stress response and general stress response during 8 h of preoxygenation. The gene expressions of both the important transcription factors Hap1 and Rox1, involved in oxygen sensing, were mainly increased in the first 3 h, while YAP1 expression, which is involved in the oxidative stress response, increased drastically only in the first 45 min. The results also show that stress-responsive genes (HSP12, SSA3, PAU5, SOD1, SOD2, CTA1 and CTT1) were induced during the process, together with the accumulation of trehalose. The accumulation of ergosterol and unsaturated fatty acids was accompanied by the expression of ERG1, ERG11 and OLE1. Genes involved in respiration (QCR9, COX15, CYC1 and CYC7) also increased during preoxygenation. Yeast viability did not decrease during the process, and the fermentation performance of the yeast reached a maximum after 5 h of preoxygenation. These results suggest that yeast cells acquire a stress response along the preoxygenation period, which makes them more resistant against the stressful conditions of the preoxygenation process and the subsequent fermentation.

Phenotypic diversity of Flo protein family-mediated adhesion in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome encodes a Flo (flocculin) adhesin family responsible for cell-cell and cell-surface adherence. In commonly used laboratory strains, these FLO genes are transcriptionally silent, because of a nonsense mutation in the transcriptional activator FLO8, concealing the potential phenotypic diversity of fungal adhesion. Here, we analyse the distinct adhesion characteristics conferred by each of the five FLO genes in the S288C strain and compare these phenotypes with a strain containing a functional copy of FLO8. Our results show that four FLO genes confer flocculation, but with divergent characteristics such as binding strength, carbohydrate recognition and floc size. Adhesion to agar surfaces, on the other hand, largely depended on two adhesins, Flo10 and Flo11. Expression of any FLO gene caused a significant increase in cell wall hydrophobicity. Nevertheless, the capacity to adhere to plastic surfaces, which is believed to depend on hydrophobic interactions, differed strongly between the adhesins. Restoring Flo8 yielded both flocculation and cell-surface adherence, such as invasive growth, a phenotype not observed when any of the single FLO genes was overexpressed. Taken together, this study reveals how S. cerevisiae carries a small reservoir of FLO genes that allows cells to display a wide variety of adhesive properties.

Decrease of 4-vinylguaiacol during beer aging and formation of apocynol and vanillin in beer.

In this study the decrease of 4-vinylguaiacol (4VG) during beer aging was investigated and the products that arise from it were identified. Two compounds, vanillin and apocynol, were identified in beer model solutions after forced aging and in naturally aged beers by GC-MS and HPLC-ECD analyses. Both account for up to 85% of the decrease of 4VG. Only in the presence of substantial amounts of oxygen in the bottle headspace was vanillin detected. Apocynol [4-(1-hydroxyethyl)-2-methoxyphenol] was found to be the main degradation product, and its formation was shown to be highly dependent on the beer pH. Because both apocynol and vanillin have a clear vanilla-like aroma, the decrease of 4-vinylguaiacol during beer aging might impart a shift from a clove-like aroma in fresh specialty beers (such as wheat beers and other top-fermented blond or dark ales) to a sweeter, more vanilla-like flavor impression of aged specialty beers.

Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers.

The glycoside hydrolase activity of Saccharomyces cerevisiae and Brettanomyces custersii was examined on sour cherry (Prunus cerasus L.) glycosides with bound volatile compounds. Refermentations by the beta-glucosidase-negative S. cerevisiae strains LD25 and LD40 of sour cherry juice-supplemented beer demonstrated only a moderate increase of volatiles. In contrast, the beta-glucosidase-positive B. custersii strain LD72 showed a more pronounced activity towards glycosides with aliphatic alcohols, aromatic compounds and terpenoid alcohols. Important contributors to sour cherry aroma such as benzaldehyde, linalool and eugenol were released during refermentation as shown by analytical tools. A gradually increasing release was observed during refermentations by B. custersii when whole sour cherries, sour cherry pulp or juice were supplemented in the beer. Refermentations with whole sour cherries and with sour cherry stones demonstrated an increased formation of benzyl compounds. Thus, amygdalin was partially hydrolysed, and a large part of the benzaldehyde formed was mainly reduced to benzyl alcohol and some further esterified to benzyl acetate. These findings demonstrate the importance and interesting role of certain Brettanomyces species in the production of fruit lambic beers such as 'Kriek'.

Release and evaporation of volatiles during boiling of unhopped wort.

The release and evaporation of volatile compounds was studied during boiling of wort. The observed parameters were boiling time, boiling intensity, wort pH, and wort density. The effect of every parameter was discussed and approached chemically, with an eye on beer-aging processes. The results indicated that pH highly influenced the release of flavor compounds and that the formation of Strecker aldehydes was linear with boiling time. However, because of evaporation of volatiles, information about the applied thermal load on wort is lost when using a volatile heat load indicator. The thiobarbituric acid (TBA) method, which includes the nonvolatile precursors of volatile aging compounds, proved to be a more reliable method to determine all kinds of heat load on wort. Finally, it was discussed how the obtained insights could help to understand the mechanism of beer aging.

Optimisation of a complete method for the analysis of volatiles involved in the flavour stability of beer by solid-phase microextraction in combination with gas chromatography and mass spectrometry.

Headspace solid-phase microextraction combined with gas chromatography and mass spectrometry was used for the quantification of 32 volatiles which represent the typical chemical reactions that can occur during beer ageing. Detection was accomplished by employing on-fibre derivatisation using o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) and normal HS-SPME extraction. The procedures were optimised for SPME fibre selection, PFBHA loading temperature and time, extraction temperature and time, and effect of salt addition. Interference of matrix effects was overcome by calibrating according to the standard addition method and by using internal standards. Afterwards, the method was validated successfully and was applied to study the flavour stability of different beer types.

Influence of lipids in the generation of phenylacetaldehyde in wort-related model systems.

The effect of lipids on the formation of the Strecker aldehyde phenylacetaldehyde during wort boiling was studied to determine the role that small changes in the lipid content of the wort have in the production of significant flavor compounds in beer. Wort was treated with 0-2.77 mmol per liter of glucose, linoleic acid, or 2,4-decadienal and heated at 60-98 degrees C for 1 h. After this time, the amount of the Strecker aldehyde phenylacetaldehyde increased in the samples treated with linoleic acid or decadienal but not in the samples treated with glucose. Thus, the amount of phenylacetaldehyde produced in the presence of linoleic acid was 1.1-2.5 times the amount of the Strecker aldehyde produced in the control wort, and this amount increased to 3.6-4.6 times when decadienal was employed. The higher reactivity of decadienal than linoleic acid for this reaction decreased with temperature and was related to the oxidation of linoleic acid that occurred to a higher extent at higher temperatures. The above results suggest that lipids can contribute to the formation of Strecker aldehydes during wort boiling and that changes in the lipid content of the wort will produce significant changes in the formation of Strecker aldehydes in addition to other well-known consequences in beer quality and yeast metabolism. On the other hand, because of the high glucose content in wort, small changes in its content are not expected to affect the amount of Strecker aldehydes produced.