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.

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.

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.

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.

Variability in the release of free and bound hydroxycinnamic acids from diverse malted barley (Hordeum vulgare L.) cultivars during wort production.

Volatile phenols have long been recognized as important flavor contributors to the aroma of various alcoholic beverages. The two main flavor-active volatile phenols in beer are 4-vinylguaiacol and 4-vinylphenol. They are the decarboxylation products of the precursors ferulic acid and p-coumaric acid, respectively, which are released during the brewing process, mainly from malt. In this study, the variability in the release of free and ester-bound hydroxycinnamic acids from nine malted barley ( Hordeum vulgare L.) varieties during wort production was investigated. A large variability between different barley malts and their corresponding worts was observed. Differences were also found between free ferulic acid levels from identical malt varieties originating from different malt houses. During mashing, free hydroxycinnamic acids in wort are both water-extracted and enzymatically released by cinnamoyl esterase activity. Esterase activities clearly differ between different barley malt varieties. Multiple linear regression analysis showed that the release of ferulic acid during mashing did not depend only on the barley malt esterase activity but also on the amount of ester-bound ferulic acid initially present in the wort and on its endoxylanase activity. The study demonstrates the importance of selecting a suitable malt variety as the first means of controlling the final volatile phenol levels in beer.

Determination of hydroxycinnamic acids and volatile phenols in wort and beer by isocratic high-performance liquid chromatography using electrochemical detection.

The suitability of a simple and rapid isocratic RP-HPLC method with amperometric electrochemical detection for the simultaneous detection and quantification of hydroxycinnamic acids and their corresponding aroma-active volatile phenols in wort and beer is reported. The technique gives good specificity and sensitivity, and can therefore be used for routine monitoring of hydroxycinnamic acids in wort and the development of volatile phenolic flavour compounds during the beer production process and subsequent conservation.

Ferulic acid release and 4-vinylguaiacol formation during brewing and fermentation: indications for feruloyl esterase activity in Saccharomyces cerevisiae.

The release of ferulic acid and the subsequent thermal or enzymatic decarboxylation to 4-vinylguaiacol are inherent to the beer production process. Phenolic, medicinal, or clove-like flavors originating from 4-vinylguaiacol frequently occur in beer made with wheat or wheat malt. To evaluate the release of ferulic acid and the transformation to 4-vinylguaiacol, beer was brewed with different proportions of barley malt, wheat, and wheat malt. Ferulic acid as well as 4-vinylguaiacol levels were determined by HPLC at several stages of the beer production process. During brewing, ferulic acid was released at the initial mashing phase, whereas moderate levels of 4-vinylguaiacol were formed by wort boiling. Higher levels of the phenolic flavor compound were produced during fermentations with brewery yeast strains of the Pof(+) phenotype. In beer made with barley malt, ferulic acid was mainly released during the brewing process. Conversely, 60-90% of ferulic acid in wheat or wheat malt beer was hydrolyzed during fermentation, causing higher 4-vinylguaiacol levels in these beers. As cereal enzymes are most likely inactivated during wort boiling, the additional release of ferulic acid during fermentation suggests the activity of feruloyl esterases produced by brewer's yeast.

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.

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.

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.

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.

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.

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.

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.

Immobilized yeast cell systems for continuous fermentation applications.

In several yeast-related industries, continuous fermentation systems offer important economical advantages in comparison with traditional systems. Fermentation rates are significantly improved, especially when continuous fermentation is combined with cell immobilization techniques to increase the yeast concentration in the fermentor. Hence the technique holds a great promise for the efficient production of fermented beverages, such as beer, wine and cider as well as bio-ethanol. However, there are some important pitfalls, and few industrial-scale continuous systems have been implemented. Here, we first review the various cell immobilization techniques and reactor setups. Then, the impact of immobilization on cell physiology and fermentation performance is discussed. In a last part, we focus on the practical use of continuous fermentation and cell immobilization systems for beer production.