Disparity in pseudohyphal morphogenic switching response to the quorum sensing molecule 2-phenylethanol in commercial brewing strains of Saccharomyces cerevisiae.

Saccharomyces cerevisiae can undergo filamentous growth in response to specific environmental stressors, particularly nitrogen-limitation, whereby cells undergo pseudohyphal differentiation, a process where cells transition from a singular ellipsoidal appearance to multicellular filamentous chains from the incomplete scission of the mother-daughter cells. Previously, it was demonstrated that filamentous growth in S. cerevisiae is co-regulated by multiple signaling networks, including the glucose-sensing RAS/cAMP-PKA and SNF pathways, the nutrient-sensing TOR pathway, the filamentous growth MAPK pathway, and the Rim101 pathway, and can be induced by quorum-sensing aromatic alcohols, such as 2-phenylethanol. However, the prevalent research on the yeast-pseudohyphal transition and its induction by aromatic alcohols in S. cerevisiae has been primarily limited to the strain Σ1278b. Due to the prospective influence of quorum sensing on commercial fermentation, the native variation of yeast-to-filamentous phenotypic transition and its induction by 2-phenylethanol in commercial brewing strains was investigated. Image analysis software was exploited to enumerate the magnitude of whole colony filamentation in 16 commercial strains cultured on nitrogen-limiting SLAD medium; some supplemented with exogenous 2-phenylethanol. The results demonstrate that phenotypic switching is a generalized, highly varied response occurring only in select brewing strains. Nevertheless, strains exhibiting switching behavior altered their filamentation response to exogenous concentrations of 2-phenylethanol.

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 wort volatile analysis by headspace solid-phase microextraction in combination with gas chromatography and mass spectrometry.

The aim of this study was to create a simple, solventless technique without derivatisation in order to analyze a broad range of volatiles in beer wort. A method was developed using headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry. The procedure was optimised by selection of the appropriate fibre and optimisation of extraction temperature, extraction time, and salting-out. The detection limits were well below the actual wort concentrations of the selected volatiles, ranging from 12 ng/l for linalool to 0.53 microg/l for furfural. Moreover, the procedure showed a good linearity and was applied to the analysis of wort samples taken from a wort boiling process in an industrial brewery.

Characterization of volatiles in unhopped wort.

The volatile fraction of wort components was studied during boiling. Not less than 118 volatile compounds were identified when unhopped pilsner wort was boiled and samples of wort and condensed vapors were analyzed with headspace SPME-GC/MS, of which 54 were confirmed with reference compounds. The wort samples contained 61 identifiable compounds, while the vapor condensate yielded 108 different compounds. Almost 30 known compounds were found that have not been described before in unhopped pilsner wort. One previously unknown aldol reaction product was tentatively identified as 2-phenyl-2-octenal. The detection of branched 2-alkenals underlines the importance of the aldol condensation in Maillard-type reactions, while the tentative identification of alkyloxazoles and alkylthiazoles could once more accentuate the central role of alpha-dicarbonyl compounds, aldehydes, and amino acids in flavor generation. The condensation of wort vapors joined with the SPME-GC/MS technique has proven to be a useful tool in volatile analysis.

Influence of the brewing process on furfuryl ethyl ether formation during beer aging.

In beer, the development of a solvent-like stale flavor is associated with the formation of furfuryl ethyl ether. The synthesis rate of this important flavor compound is proportional to the concentration of furfuryl alcohol in beer. This study shows that furfuryl alcohol in beer is mainly formed by Maillard reactions initiated during wort boiling and malt production. A mechanism for its formation from alpha-(1,4)-oligoglucans and amino acids in wort and beer is proposed. During wort boiling, a quadratic relationship was found between the wort extract concentration, on the one hand, and the increase of furfuryl alcohol and furfural, on the other. The reduction of furfural by yeast during fermentation further increases the furfuryl alcohol content. In pale beers, the furfuryl alcohol concentration is essentially determined by the thermal load on wort during brewing operations. In dark beers, a considerable fraction of furfuryl alcohol may, however, come from the dark malts used. These results lead to important practical conclusions concerning the control over furfuryl ethyl ether in beer.

Furfuryl ethyl ether: important aging flavor and a new marker for the storage conditions of beer.

Recently, it was reported that furfuryl ethyl ether is an important flavor compound indicative of beer storage and aging conditions. A study of the reaction mechanism indicates that furfuryl ethyl ether is most likely formed by protonation of furfuryl alcohol or furfuryl acetate followed by S(N)2-substitution of the leaving group by the nucleophilic ethanol. For the reaction in beer, a pseudo-first-order reaction kinetics was derived. A close correlation was found between the values predicted by the kinetic model and the actual furfuryl ethyl ether concentration evolution during storage of beer. Furthermore, 10 commercial beers of different types, aged during 4 years in natural conditions, were analyzed, and it was found that the furfuryl ethyl ether flavor threshold was largely exceeded in each type of beer. In these natural aging conditions, lower pH, darker color, and higher alcohol content were factors that enhanced furfuryl ethyl ether formation. On the other hand, sulfite clearly reduced furfuryl ethyl ether formation. All results show that the furfuryl ethyl ether concentration is an excellent time-temperature integrator for beer storage.

Evolution of chemical and sensory properties during aging of top-fermented beer.

The aging and consequent changes in flavor molecules of a top-fermented beer were studied. Different aging conditions were imposed on freshly bottled beer. After 6 months of aging, the concentration changes were recorded for acetate esters, ethyl esters, carbonyls, Maillard compounds, dioxolanes, and furanic ethers. For some flavor compounds, the changes with time of storage were monitored at different temperatures, either with CO(2) or with air in the headspace of the bottles. For some molecules a relationship was determined between concentration changes and sensory evaluation results. A decrease in volatile esters was responsible for a reduced fruity flavor during aging. On the contrary, various carbonyl compounds, some ethyl esters, Maillard compounds, dioxolanes, and furanic ethers showed a marked increase, due to oxidative and nonoxidative reactions. A very high increase was found for furfural, 2-furanmethanol, and especially the furanic ether, 2-furfuryl ethyl ether (FEE). For FEE a flavor threshold in beer of 6 mug/L was determined. In the aged top-fermented beer, FEE concentrations multiple times the flavor threshold were observed. This was associated with the appearance of a typical solvent-like flavor. As the FEE concentration increased with time at an almost constant rate, with or without air in the headspace, FEE (and probably other furanic ethers) is proposed as a good candidate to evaluate the thermal stress imposed on beer.