Improvement Of Brewing Yeast Autolytic Ability By Genetically Constructing Fks1 Mutant

If the number of autolytic brewing yeast was more than 5 % of the total cell number, as an unavoidable phenomenon in the brewing process, the quality of beer would seriously worsen. Intracellular substances hydrolyzed into smaller matters and excreted outside by penetration when brewing yeast died and autolyzed, which would make concentration of diacetyl and aldehyde increased accelerating the rate of beer aging, while beer hazed and abiological stability descended that influenced beer appearance and taste quality because of a lot of intracellular substances excreted.Previous reports that yeast autolysis was only related to cellular membrane and intracellular substances without cell wall effort. Recently researches about yeast autolysis discovered that cell wall metabolism affected autolytic activities of yeast. Polysaccharide of cell wall, mainlyβ-glucan and mannose obviously hydrolyzed when autolytic activities of yeast happened, which influenced beer together.Gene fks1 is 1, 3-β-D-glucan synthase gene in Saccharomyces cerevisiae, which induces a compensatory mechanism leading to fks2 expression. The content of glucan was dependent on quantity of fks1 lacked.On the basis of homologous recombination, recombinant plasmid pKC was constructed by replacing the internal fragment of fks1 of pTK with a copy of copper resistance gene (cup1) from the industrial brewing yeast strain G-03. Then recombinant plasmid pKC was cut by restrict enzyme reaction to fragment fks1::cup1 integrated to the chromosomal DNA of G-03 strain, a recombinant strain G-03/C was obtained on bluestone YPD plates with good genetic stability. The 1, 3-β-D-glucan was extracted from the cell wall of recombinant and original strain by alkali method, content of 1, 3-β-D-glucan from the cell wall of recombinant strain was 3.773 mg/g and was reduced by 39 % compared with original strain.Experiments for several yeast strains kept in the laboratory and certain yeast strain available from brewhouse were made to study yeast autolysis, and nitrogen matters were determined in the yeast autolysis process. Results domenstrated that content of nitrogen matters in the autolysate differed with yeast strains, while the rate of△non-α-amino nitrogen and△α-amino nitrogen was in the range 7.91-2.24. With times prolonging, the value was descended. The autolytic ability was determined between rembinant strain and original strain by the above method, and autolytic ability of recombinant strain reduced by 13.71 %.It was shown that the physiology character of recombinant strain changed, while routine fermentation parameters were no obvious different which was produced by the recombinant strain G-03/C and original strain G-03. After main fermentation the aging index TBA of G-03/C was reduced to 72.8 % and the anti-staling SI was increased by 264 % compared with G-03. TBA of final beer was reduced to 65.6 %, SI and RSV value were increased by 192.7 %, 26.6 %, respectively.