Evaluation Of High Gravity Brewing Yeast And Study On Its Stress Resistance Mechanism

Beer is the third most popular drink in the world,second only to water and tea.China accounts for 20%of the global beer production,among which Lager beer produced by industry accounts for over 90%of the total output.Domestic beer industry has been committed to improving product quality and reducing production energy consumption and cost.High gravity brewing technology is of great significance to beer industry.Applying High gravity brewing technology in industrial beer production can reduce costs,reduce emissions and save water resources.However,there are many shortcomings in high gravity brewing,in which the fermentation performance of yeast will decrease significantly,while beer flavor substances will not increase proportionally.At present,the research on high gravity brewing is mostly limited to fermentation technology research and yeast breeding promotion.Exploring high gravity brewing from yeast gene level can further reveal the molecular mechanism of high gravity brewing and provide guidance for yeast breeding.In this study,by analyzing and comparing the fermentation performance and flavor phenotype of several industrial beer yeasts preserved in the laboratory under the condition of high gravity brewing,an excellent strain and its parent strain were selected for fermentation process analysis,and the strain was determined to be an excellent yeast for high gravity brewing through various indexes evaluation.Through metabolomics and transcriptomics analysis,the metabolic pathway and differential regulatory genes which have a key impact on the stress resistance of beer yeast were obtained,and the gene function was further verified,and the important intracellular metabolites were detected and analyzed.It lays a foundation for the subsequent improvement of high gravity brewing yeast.The main research results of this paper are as follows:1.Analysis and evaluation of high gravity brewing performance:under the high gravity brewing conditions,fermentation performance of brewer's yeast declines and flavor substances change.The stress test and fermentation process analysis of M14 and Tsing2 strains showed that M14 strain was superior to Tsing2 strain in all directions and was an excellent strain for high gravity brewing.2.Transcriptomics and metabolomics analysis:The differential genes and pathways of M14strain in the initial fermentation stage were analyzed,and the transcriptome and metabolomics of M14 and Tsing2 strains in the late fermentation stage were analyzed.It was determined that the important metabolic pathways regulated in high gravity brewing were carbon metabolism and amino acid metabolism.High gravity brewing can significantly inhibit the important genes of EMP pathway and prevent yeast from utilizing glycogen.Stress under high gravity brewing also led to significant changes in amino acid pathway,and the content and gene expression of various amino acids which can relieve stress changed significantly.According to the phenotypic differences between M14 and Tsing2,nine key differential regulatory genes(FDH1,MAN2,PCL1,TPK1,MET30,ATH1,PFK26,TOP3 and RPM2)were obtained by screening the significant differential genes of the two strains.3.Verification of gene function of stress-resistance related genes:select the above genes to construct overexpression and knockdown strains,carry out high gravity fermentation experiment on genetically engineered bacteria and detect the ratio of intracellular ATP and NADH/NAD~+.MAN2,PCL1 and PFK26 can significantly improve the fermentation performance of Tsing2 strain,and the influence mechanism was preliminarily analyzed through the dynamic changes of ATP.Moreover,there is little difference between the flavor substances of genetically engineered bacteria and the original strains.