Screening Of Thermosensitive Autolytic Mutant Brewer’s Yeast And Study On Yeast Autolysis

Yeast autolysates contain nucleotides,bioactive polysaccharides,antioxidant peptides and a variety of nutrients.They are widely used in the food industry,cosmetics,etc.Temperature is a main factor affecting yeast autolysis.It not only affects the degree of yeast autolysis,but also affects the content of active components.Heat stress triggers cellular stress responses during yeast autolysis,which lead to the regulation of protein function and cell metabolism.Therefore,screening of thermosensitive autolytic mutant yeast contributed to accelerating the autolysis of yeast under lower temperature,improving the biological activities and application value of yeast autolysates.Meanwhile,the study of yeast autolysis under heat stress provided theoretical basis for the selection of yeast strains and yeast autolysis.In this study,a screening method of thermosensitive autolytic yeast was established.A mutant strain P-510,which rapidly autolyzed at 37℃ and the autolysates contained more active components,was selected from brewer’s yeast Pilsner with strong autolytic ability.Besides,by using transcriptomics and comparative genomics,the metabolic pathways and the key genes related to heat stress response were analyzed.The main results are as follows:(1)Screening of thermosensitive autolytic mutant yeast and the establishment of screening method: ARTP mutagenesis plus BCIP screening were employed to screen thermosensitive autolytic mutants(referred to as P-510)from parental strain Pilsner.The growth of P-510 was inhibited at 37℃.The mortality rate of P-510 increased reached 87.8% after 72 h culture at 37℃.The mutant rapidly autolyzed at 37℃ and the releasement of nucleic acid,protein and amino acid nitrogen of the autolysates were 142.8%,60.0% and 10.9% higher than those of Pilsner respectively.The mutants baker yeast A-14 and wine yeast D-12 were successfully screened by using this method.(2)Study on the characteristics of autolysates of thermosensitive autolytic mutant P-510: The content of flavor enhancing nucleotides in autolysates were 134.9% and 193.4% higher than that of Pilsner after 72 h and 144 h at 37℃,respectively.After 36,84 and 132 h of autolysis,the β-glucan in autolysates were 7.9%,30.0% and 40.3% higher than that of Pilsner,respectively.Meanwhile,the DPPH scavenging rate of P-510 autolysates at 37℃ reached the highest value at 60 h,which was 13.3% and 22.2% higher than that of Pilsner at 50℃ and 37℃,respectively.Compared with 50℃,autolysis at 37℃ could obtain more small molecular peptides.(3)Effect of heat stress response on yeast autolysis: The intracellular ATP,NADPH and ROS levels of Pilsner were higher than that of P-510.The gene expression differences caused by heat stress and gene mutation between P-510 and Pilsner were analyzed by transcriptomics.It was suggested that the gene expression of P-510 and Pilsner had different regulation under heat stress.Compared with Pilsner,P-510 showed down-regulation of TCA pathway,up regulation of glycolysis pathway,down regulation of pentose phosphate pathway and down regulation of hexose transport pathway.It was presumed that the inhibition of mitochondrial metabolism,the disorder of glycogen metabolism and the abnormal regulation of hexose transport in P-510 affected the resistance of cells in heat stress response,which resulted in the thermosensitive autolysis in P-510.The comparative genomics showed that 22 genes(GRE1、RKI1、ERG5、NDE2、HXT7)might be the key genes of yeast autolysis under heat stress.