Filip Delvaux

Parameters Affecting Ethyl Ester Production by Saccharomyces cerevisiae during Fermentation

ABSTRACT Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection.

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.

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.

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.

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.

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.

The role of oxygen in yeast metabolism during high cell density brewery fermentations.

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 fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.

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.

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.

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.