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Yeast cell immobilization causes changes in several cellular physiological properties, resulting in severely unbalanced flavor profiles of beers produced in immobilized-cell systems. To explore the reasons for the altered production of flavor-active metabolites, such as volatile esters, the transcription rates of several genes were measured in free and immobilized cells. Expression profiles of the stress-related genes HSP12 and SSA3 revealed that cell immobilization exerted certain stresses on the yeast cells, possibly nutrient starvation and osmotic stress. However, yeast cells may also benefit from the protecting effect of the immobilization matrix, since without a protecting matrix, the free cells in the standard free-cell system showed a stronger induction of HSP12, a general stress marker, compared with the immobilized cells. In accordance with the lower stress gene expression levels, the immobilized cells accumulated trehalose as low as 26% of that of the standard free cells. The expression level of ATF1, encoding alcohol acetyl transferase I, was significantly raised in the immobilized cells, resulting in a twofold increase of iso-amyl acetate formation. The activity of ATF1 is regulated by the protein kinases Sch9p and PKA, involved in stress responses and nutrient-sensing signaling pathways. The particular microenvironment created by cell immobilization thus possibly activates the cAMP/PKA/Sch9 pathway, resulting in an induction of ATF1 expression leading to enhanced ester concentrations in the final fermentation product.

Previous results have indicated that supplementation of stationary-phase cropped brewer's yeast with the unsaturated fatty acid linoleic acid is a good alternative to wort aeration with respect to its fermentation performance in a subsequent fermentation cycle. The experiments were carried out with a well-defined fermentation medium. Here, we show that, also with beer wort, the cropped yeast that was suspended in fermented wort and under nongrowing conditions rapidly incorporated the exogenous y acid. Linoleic acid is also taken up by spheroplasts, which excludes the possibility that it is adsorbed onto the cell walls. The intracellular localization of linoleic acid was studied. It was rapidly recovered from the free fatty acids pool, diglycerides, triglycerides, and phospholipids. The highest amounts were recovered from the neutral lipids and the free fatty acids fractions. The results obtained with yeast first grown under stirred conditions were the same as those obtained with yeast grown under nonstirred conditions. The results with the stationary-phase cells are discussed with respect to results obtained with cropped yeast suspended in fresh wort under growing conditions. Some differences in uptake were also found when the cropped yeast was suspended and supplemented in water rather than in fermented wort. Further studies are required to investigate the fermentation performance of the supplemented yeast in a subsequent fermentation cycle.

A novel bioreactor with immobilization matrices was constructed to study physiological differences between immobilized and free cells and to compare the fermentation behavior of the immobilized-cel...

Most breweries collect yeast from a previous fermentation cycle for further use in a subsequent cycle. However, the cropped cells are deficient in membrane sterols and unsaturated fatty acids (UFAs) which are required for good fermentation performance in the next cycle. Consequently, the cellular levels of these compounds must be restored to obtain an optimal fermentation performance. There are currently three possibilities to satisfy this requirement. The common practice is aeration of the wort before pitching, thus providing oxygen needed for lipid synthesis during the first stages of fermentation. Oxygenation (aeration) of cropped yeast slurries is a second alternative. Finally, the addition of the required lipids to wort is sometimes suggested as an alternative to aeration. We examined a fourth possibility, namely the supplementation with UFA of cropped cells. Previously, we reported that the supplementation of stationary phase cropped brewer's yeast with linoleic acid is a good alternative to wort aeration. This conclusion resulted from results obtained with a well-defined stirred synthetic fermentation medium. We also showed that cells cropped from non-stirred tall-tube fermented malt wort incorporated linoleic acid into different cellular lipid fractions, when suspended and supplemented in fermented wort. Now, we report that such yeast, pitched in malt wort in non-stirred tall-tubes, showed growth and attenuation profiles comparable to unsupplemented yeast in pre-aerated wort. Moreover, the synthesis of acetate esters, which is known to be affected when UFAs are added directly to the wort, was not significantly affected. We hypothesize that the active uptake of linoleic acid during fermentation and its activation by coenzyme A (CoA) and phospholipid synthesis are responsible for the effects on ester synthesis, through repression of the alcohol acetyltransferase-encoding gene, ATF1. In supplemented cropped yeast, these reactions occur prior to fermentation, thus avoiding interferences with acetate ester synthesis. In serial repitching experiments with repeated linoleic acid supplementation of the cropped yeast, the fermentation performances of the yeast remained comparable to those of non-supplemented yeast in pre-aerated wort. However, due to a progressive increase of cellular UFA, negative effects on acetate ester synthesis appeared. Nevertheless, the supplementation of cropped yeast with UFAs can be considered as an interesting alternative to wort oxygenation to restore optimal membrane functions.

Immobilised-cell fermentors offer great benefits compared to traditional free-cell systems. However, a major problem is unbalanced flavour production when these fermentors are used for the production of alcoholic beverages. One of the keys to obtaining better control over flavour formation may be the concentration of dissolved CO2, which has inhibitory effects on yeast growth and metabolism. This article demonstrates that the presence of immobilisation matrices facilitates the removal of CO2 from the liquid medium, which results in a low level of dissolved CO2 during fermentation. Moreover, the formation of volatile higher alcohols and esters was greatly enhanced in the immobilised-cell system when compared to the free cell system. By sparging a CO2 flow (45 ml/min) into the immobilised-cell system, cell growth was reduced by 10–30% during the active fermentation stage, while the fermentation rate was unaffected. The uptake of branched-chain amino acids was reduced by 8–22%, and the formation of higher alcohols and esters was reduced on average by 15% and 18%, respectively. The results of this study suggest that mismatched flavour profiles with immobilised-cell systems can be adjusted by controlling the level of dissolved CO2 during fermentation with immobilised yeast.