P-3
Evaluation of brewing yeast vitality through uptake and catabolism of a maltose analog
Presenter: Boris Stambuk, Departamento de Bioquimica, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900, Brazil
Co-Authors: Ricardo Herberts, Marcelo Dário, and Maria Luiza Cordioli, Departamento de Bioquimica, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900, Brazil

During brewing fermentation yeast cells are exposed to several environmental stresses (nutrient depletion, high temperatures, and ethanol concentrations) that may reduce the fermentation capacity of the cells, leading to stuck or incomplete fermentations after several recycles of the yeast slurry. As a consequence, brewers need to calculate yeast inoculation rates based on some measure of the viability (percentage of living cells) and/or vitality (fermentation capacity) of the yeast slurry that may be an indicator of the subsequent fermentative performance. However, all available methods (e.g. staining with methylene blue and/or the acidification power test) are generally based on the measurement of indirect yeast components and/or metabolic activities, not directly related to the fermentation of the sugars present in the wort. Since the most abundant fermentable sugars in brewer's wort are maltose and maltotriose, and since transport of these sugars across the plasma membrane is the rate-limiting step in their metabolism, we have developed a vitality test based on the uptake and intracellular hydrolysis of the maltose analog p-nitrophenyl-alpha-D-glucopyranoside (pNPaG). The 4-nitrophenol produced by the yeast slurry after pNPaG uptake and hydrolysis can be easily determined colorimetrically at 400 nm, reflecting the rates of alpha-glucoside fermentation by the cells. This assay was successfully used to test the vitality of different brewing yeast strains containing increasing concentrations of dead cells, and the fermentation profile of maltose-glucose mixtures with these yeasts was in good agreement with the results obtained by our colorimetric vitality assay. Our results also indicate that when yeast cells are exposed to sub-lethal stresses (e.g. 52°C during short incubation times), the present assay showed the best correlation with both the viability and vitality of the cells, while other assays commonly used by the brewing industry give false positive results. Hence, our results show that the colorimetric assay of pNPaG transport can be successfully used to measure and predict the fermentation capacity of brewer's yeast. The present vitality test is simple, rapid, and reliable, and can be easily implemented in any brewing plant as it doesn't require any sophisticated equipment.

Boris Stambuk graduated with a B.Sc. degree in biological sciences at Universidade de São Paulo, completed a M.Sc. degree in microbiology from Escola Paulista de Medicina, and holds a Ph.D. degree in biochemistry from Universidade de São Paulo, Brazil. In 1994, he move to Universidade Federal de Santa Catarina in Florianopolis, Brazil, where he became a lecturer and senior lecturer in biochemistry. Since 1997, Boris holds a research fellowship in applied microbiology from CNPq (Brazil) investigating yeast physiology, sugar fermentation, and biotechnology of industrial yeasts. In 2001, he also performed studies on pentose fermentation by yeasts as senior research associate at the National Renewable Energy Laboratory in Golden, CO.