The Metabolic Analysis And Regulation Of Brewer’s Yeast Under Very High Gravity Brewing

This dissertation chose the metabolism of carbon sources, cofactors and environment in an industry brewer’s yeast (Saccharomyces pastorianus) FBY0095 and its variant (S. pastorianus) L6 with improved very high gravity (VHG) fermentation performances as the model system to study the principal physiological and metabolic characteristics. The effect of VHG condition on fermentation rate and physiological activity of yeast at the metabolic level were investigated in detail. The main results were described as follows:(1) The brewer’s yeast FBY0095 (S. pastorianus) was used as starting strain to select variants for efficient VHG brewing by UV irradiation and ethyl methanesulfonate mutagenization combined with the domestication of ultra-high concentration of maltose and ethanol. Two variants (L3 and L6) screened displayed the better growth profile and the improvedα-glucosides transport and alcohol dehydrogenase activity on the plates with 15% (w/v) maltose and 15% (w/v) ethanol. Moreover, compared with FBY0095, both L3 and L6 variants showed faster fermentation rates, and their fermentation times were decreased in 21.43%. Furthermore, there were increases in apparent attenuation and ethanol concentration and improvement on flavor of resulting beer by L3 and L6 at the end of VHG fermentation (28 d).(2) The effect of concentration of glucose and maltriose on fermentation performances of S. pastorianus FBY0095 and S. pastorianus L6 with improved VHG fermentation performances under low temperature and anaerobic conditions were investigated. Furthermore, the metabolic flux and its control by glycolytic key enzymes (hexokinase, phosphofructokinase and pyruvate kinase) were quantitatively analyzed. The metabolic flux and key enzymes activities in S. pastorianus FBY0095 were decreased with the increase in wort gravity. The metabolic flux and key enzymes activities in S. pastorianus L6 were higher than those in S. pastorianus FBY0095 under VHG condition. Furthermore, the metabolic flux in S. pastorianus cells was intensely controlled by phosphofructokinase and pyruvate kinase, meaning that the catalytic reactions of the two key enzymes were regulation steps of glycolysis flux.(3) The effect of wort gravity on cofactors (adenosine phosphate and coenzyme I) in S. pastorianus FBY0095 and S. pastorianus L6 with improved VHG fermentation performances and the regulation of intracellular cofactors to fermentation rate were investigated. The ATP content and EC level in S. pastorianus FBY0095 were increased with the increase in wort gravity, which resulted in the decrease in wort sugars consumption. The increase in NADH/NAD~+ value in S. pastorianus FBY0095 companied with the increase in wort gravity, which led to the decrease in ethanol production rate and the changes of metabolic flux distribution in S. pastorianus FBY0095. In addition, the ATP content, EC level and NADH/NAD~+ value in S. pastorianus L6 were lower than those in S. pastorianus FBY0095, which resulted in the improved metabolic activity of S. pastorianus L6 and the increases in wort sugars consumption rate and ethanol production rate.(4) The effect of wort gravity on metabolic environment, including of the dynamic change process of intracellular H+ and the influence of ethanol stress on membrane, in S. pastorianus FBY0095 and S. pastorianus L6 with improved VHG fermentation performances were investigated. The activities of H+-ATPase on membrane of S. pastorianus FBY0095 decreased with the increase in wort gravity, which resulted in the decrease of H+ efflux rate, intracellular pH, difference of intra- and extracellular pH, and the acidification of intracellular metabolism environment and inactivation of metabolic reactions and carbon sources utilization. The metabolic activity of S. pastorianus cells was obviously inhibited by ethanol of 4% (w/v). In addition, compared to S. pastorianus FBY0095, S. pastorianus L6 displayed that higher unsaturation index of membrane, better ability of ethanol tolerance and improved metabolism environment.(5) The effect of soy peptides with different molecular weight on the growth and vitility S. pastorianus FBY0095. The results indicated that the soy peptide (Mw < 1kDa) could increase growth of yeast cells and the soy peptide (3kDa < Mw < 5kDa) could increase ethanol tolerance of yeast cells. The soy peptide (3kDa < Mw < 5kDa) adhere on membrane of S. pastorianus FBY0095 in YNB medium containing ethanol 12% (w/v), which resulted that decrease of permeability of membrane and increase of cells vitality. The effect of soy peptide (3kDa < Mw < 5kDa) on ability of ethanol tolerance of S. pastorianus FBY0095 and the reaction mechanism between soy peptide and yeast cells were investigated. Furthermore, The gene expression level on ethanol tolerance and metabolism (OLE1、ELO1、TPS1、PMA1 and MAL1) of S. pastorianus FBY0095 were increased by addition of 0.2% soy peptide, especially OLE1. S. pastorianus FBY0095 displayed that better fermentation performances, increased membrane unsaturation index, ethanol tolerance ability and H+-ATPase activity and improved metabolism environment by addition of 0.2% soy peptide during VHG brewing.