An expeditious method of yeast age estimation was developed based on selective bud scar staining (Alexa Fluor 488-labelled wheat-germ agglutinin) and subsequent fluorescence intensity measurement by flow cytometry. The calibration curve resulting from the cytometric determination of average bud scar fluorescence intensities vs. microscopically counted average bud scar numbers of the same cell populations showed a good correlation and allowed routine cell age estimation by flow cytometry. The developed method was applied for yeast age control in traditional batch and continuous beer fermentations. At the pitching rates used in industrial beer fermentations, our results support former findings by locating a gradient of increasing yeast age from the top to the bottom zone of the fermenter cone. The results also indicate that in continuous beer fermentation, the increasing bud scar fluorescence of immobilized cells could help to schedule the replacement of aged biomass, prior to loss of viability or deterioration of process performance and product quality.
Lehnert R., Novák P., Macieira F., Kuřec M., Teixeira J.A., Branyik T. (2009): Optimisation of labscale continuous alcohol-free beer production. Czech J. Food Sci., 27: 267-275.In order to study the formation and conversion of the most important flavour compounds, the real wort used in alcohol-free beer fermentation was mimicked by a complex model medium containing glucose, yeast extract, and selected aldehydes. The fermentation experiments were carried out in a continuously operating gas-lift reactor with brewing yeast immobilised on spent grains (brewing by-product). During the continuous experiment, parameters such as oxygen supply, residence time (Rt), and temperature (T) were varied to find the optimal conditions for the alcohol-free beer production. The formation of ethanol, higher alcohols (HA), esters (ES), as well as the reduction of aldehydes and consumption of glucose were observed. The results suggest that the process parameters represent a powerful tool in controlling the degree of fermentation and flavour formation brought about by immobilised biocatalyst. Subsequently, the optimised process parameters were used to produce real alcohol-free beer during continuous fermentation. The final product was compared with batch fermented alcohol-free beers using the methods of instrumental and sensorial analysis.
The influence of oxygen supply on the formation and conversion of the most important flavor compounds during continuous, alcohol-free beer production was studied in a complex model medium. The medium contained inorganic salts, nutrients, and aldehydes (hexanal, 2-methyl propanal, 3-methyl butanal, and furfural) and mimicked real brewery wort, with the advantage of a constant composition. Fermentation experiments were carried out in a continuously operating gas-lift reactor, with brewing yeast immobilized on spent grains. The formation (ethanol, higher alcohols, esters, vicinal diketones, and acetaldehyde) and reduction (aldehydes) of flavor-active compounds at different aeration rates were observed. The results suggest that the oxygen supply represents an influential tool for controlling the degree of fermentation and flavor formation carried out by an immobilized biocatalyst. Under optimal oxygen supply conditions in the continuously operating gas-lift reactor, it was possible to obtain a fermented model medium with a composition approaching that of commercial alcohol-free beers produced by batch process.
This work presents a yeast-cell vitality-assessment method based on on-line intracellular fluorescence measurement. The intracellular NAD(P)H fluorescence of a cell suspension is recorded during transition from aerobic to anaerobic conditions and the output signal is evaluated as a measure of yeast vitality (quality). This fluorescence method showed a highly satisfactory correlation with even low dead cell numbers where the acidification power test could not be applied. Abbreviations NAD(P)nicotinamide adenine dinucleotide (phosphate) FI fluorescence intensity NAD(P)H reduced form of nicotinamide adenine dinucleotide (phosphate)FI rel relative increase of NAD(P)H fluorescence AP acidification power μ max maximum specific growth rateThe high quality of microbial catalyst, expressed as "vitality", strongly influences the course of several processes in food industry, such as beer and wine fermentation, dough leavening or cheese making. The term vitality describes a group of characteristics and capabilities of microbial cells related to their metabolic activity and stress resistance (Sigler et al. 2006). Several quantitative methods have been developed to evaluate the vitality of cells including measurements based on intracellular content of different components (Majara et al. 1996;Hutter 2002) or manifestation of metabolic activity (Peddie et al. 1991;Imai et al. 1994) or gravimetric analysis of the course of fermentation (Košin et al. 2008). In fact, none of these methods has found widespread application in industrial biotechnologies.One of the areas where an expeditious and reliable method of yeast vitality estimation would be appreciated is brewing industry. The brewing yeast vitality is an important factor determining the fermentation performance and the formation of sensorially active metabolic by-products (Guido et al. 2004;Brányik et al. 2005). Nevertheless, the methods of yeast vitality estimation developed so far have not met the requirements for routine applicability (Mochaba et al. 1998;Košin et al. 2007).Sensors using intracellular fluorophores (NAD(P)H) have been developed for different application in biotechnology and particularly for bioprocess monitoring (Marose et al. 1998;Podrazký and Kuncová 2005). However, the number of practical applications of fluorometry in biotechnologies has been very limited due to the many factors that affect culture fluorescence (Li et al. 1990).This paper presents a novel application of NAD(P)H-dependent fluorescence monitoring for determination of yeast vitality. The method is based on the monitoring of intensity of NAD(P)H fluorescence during a forced transition from aerobic to anaerobic conditions (AA transition). The relative fluorescence increase (FI rel ) change and its rate (dFI/dt) during AA transition was evaluated as a measure of yeast vitality and it was compared with acidification power (AP) test, specific growth rate (μ) measurement and dead cell count. MATERIALS AND METHODSYeast strain. The industrial bottom-fermenting brewing yeast strain Saccharomyces cer...
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