Yeast physiology and flavour formation during production of alcohol-free beer

Production of alcohol-free beer is performed with immobilized cells of Saccharomyces cerevisiae var. uvarum . In the reactor, combined stress factors such as low temperature (0-4°C) and anaerobic conditions limit cell metabolism.Cells of S. cerevisiae are able to grow as low as -2°C. Although sugar metabolism is instantaneously and similar in cells grown at high or low temperature, respiration differs significantly. Latter cells do not show substrate-induced respiration in spite of the presence of active mitochondria and biomass yield was substantially reduced.We noticed an increase in reductive capacity of yeasts grown under anaerobic conditions and we subsequently purified and characterized a novel NADP-dependent branched-chain ADH. Based on the high reductive activity with 3-methylbutanal at physiological pH and higher ionic strength, the enzyme will have an important function in reduction of Strecker aldehydes during alcoholic fermentation.The activity of the bcADH and the influence of immobilization on cell physiology were further monitored during alcohol-free beer production. Higher activities of glycolytic enzymes, and the bcADH were observed compared to those in batch grown cells. In addition, glucose flux was increased. The shifts in enzyme activities and glucose flux correlate with a higher in vivo reduction capacity of the immobilized cells.Formation of ester and diacetyl by immobilized yeast cells were investigated. Due to the anaerobic conditions, acetate ester production and in vitro enzyme activity increase simultaneous with the decrease in unsaturated fatty acids. Sterol metabolism is blocked; consequently, squalene accumulates. Despite the presence of active acetohydroxy acid synthase, no diacetyl-precursor is formed at low temperatures.S. cerevisiae W34 flocculates at the end of the exponential growth phase. Grown at low temperatures, cells have a higher flocculation capacity and a higher cell wall hydrophobicity. The immobilization of cells to DEAE-cellulose is influenced by both charge differences between cell wall and carrier and flocculation characteristics.In conclusion, immobilized cells show some unique physiological features at low temperatures. These contribute positively to the limited fermentation of wort to alcohol-free beer.