The Research Of Brewing Yeast Tolerance Under The High Gravity Condition

High gravity brewing can not only reduce energy consumption and cost, but also can improve the degree of the "light" beer. So high gravity brewing has become an important research direction. This paper showed brewing yeast tolerance under high gravity( 20°P) from four aspects: high consistency brewing strain screening, different gravity of fermentation, second fermentation and proteomics analysis.First of all, the fermentation performance of six brewing yeast strains under high gravity has been determined: dry cell weight( DCW), weight loss of CO2, apparent fermentation degree, main fermentative sugar content(glucose, maltose, malt trisaccharide), flavor substances and ethanol content. We found that different strains had different fermentation performance. Bw34/70 strain had better fermentation performance, higher fermentation degree( 95.96%) and more completely glucose metabolism. The whole genome sequencing of this strain has been completed, in order to facilitate the subsequent proteomics analysis experiment, we chose Bw34/70 strain as the follow-up experiments strain.The original wort gravity was set to 12°P, 12+8°P, 16+4°P and 20°P. The fermentation performance was determined respectively. We found that high gravity of wort would extend yeast growth and had certain inhibitory effect on the fermentation performance. It also delayed the time of sugar consumption, especially the maltose, and some flavor substances content was lower. The fermentation performance was different in different nitrogen source content, 16+4°P had the best fermentation performance:dry cell weight was 10.907g/L, weight loss of CO2 was 10.47 g, apparent fermentation degree could exceed 90%.By adding carbon source or nitrogen source to secondary fermentation to improve the performance of fermentation, we found secondary adding carbon source can improve the biomass and the degree of fermentation. CO2 weightlessness was the same with high gravity fermentation, and some flavor content was also improved. However it had a little effects on the use of the main sugars in the wort. Overall yeast fermentation performance impaction of secondary adding nitrogen source was less than the impaction of secondary adding carbon source. Histidine had the best results( slow utilization type), glutamic acid( rapid utilization type) and phenylalanine( later utilization type) had little influence in this experiment.At last, the intracellular proteins at logarithmic phase and stationary phase were analyzed by two-dimensional electrophoresis technology. Then the representative protein was selected to sequence. First protein extraction method was optimized. We used zirconia bead, added the protease inhibitors PMSF to reduce the loss of protein, and took the kit method for protein extraction. Finally, we found that high gravity fermentation protein at both logarithmic phase and stability phase pointed to be reduced or even disappeared. Three representative proteins were detected by mass spectrum, their function were related to the metabolic pathway and cell death. Those explained highly enriched environment could inhibit some intracellular proteins, especially the synthesis of some enzymes in metabolic pathways, which caused the change of fermentation performance.