Breeding Of Saccharomyces Cerevisiae Starins With Broad-spectrum Killer Capabilities

Wine making is a complex dynamic bio-transformation process involved interactions of multiple yeast species and strains.Some currupt microorganisms can affect the quality of wine seriously.Inhibiting the contamination of bacteria and fungus in wine making is a matter of concern for wine producers and wine-related researchers.The biological control method has wide application foreground in inhibiting the contamination of currupt microorganisms.The aim of this study is to solve the problem of stuck fermentation caused by contamination in the wine industry.Through the fusion technique of inactiviated protoplast,the selected industrial strain NX11424 and the broad-spectrum killer yeast NX1134 were fused and fusion with broad-spectrum killer activity were screened.The main results were as follows:(1)The growth age of strain,enzyme treatment condition,osmotic pressure stabilizer and pretreatment liquid variety were studied on the effect of yeast NX11424 and killer yeast NX1134 protoplast preparation,and the inactivation conditions of NX11424 and NX1134 were explored.The conclusions are as follows: the protoplast preparation affected by the growth cycle of Saccharomyces cerevisiae NX11424 and Saccharomyces cerevisiae NX1134,and its growth logarithmic period in 3-9 h.So the strains grew 3-6 h are prepared by protoplast.The preparation of yeast protoplast affected by osmotic stabilizers,pretreatment solution significantly.but the influence of the enzyme treatment process conditions is not significant.Preparation conditions and technology of protoplast of NX11424 strain were as follows: 0.8 mol/L sorbitol stabilized osmotic fluid,0.2% β-mercapto ethanol and 0.05% EDTA-Na2 pretreatment solution,and the enzymatic hydrolysis conditions were: 1.5% snail enzyme solution,enzyme hydrolysis temperature25℃,enzyme hydrolysis time 150 min.Preparation of enzyme hydrolysis conditions of protoplasts of killer yeast NX1134 were as follows: 1% snail enzyme solution,enzyme hydrolysis temperature 25℃,enzyme hydrolysis time 120 min.NX1134 protoplast inactivation conditions were as follows: Irradiated at 30 cm under 30 W of violet lamp.NX11424 protoplast inactivation conditions were as follows: 60℃ of dry bath 20 min.(2)In the process of fusion screening,45 strains of Saccharomyces cerevisiae obtained by protoplast fusion technique were screened by molecular marker interdelta fingerprinting.The fermentation ability of each fusion is different: the fusion F82 is better than NX11424 which is 32.12 g/100 m L.The alcohol degrees fermentated by different yeasts have significant differences,the Sugar utilization rate of different yeasts was not significant.F82 has genetic stability of killer phenotype and tolerance,the test showed F82 can grow in ethanol 18%vol,700 g/L concentrations of high sugar and p H 2.5normally,F82 growed at 4℃ started to fermentat 132 h,F82 can growed in the concentration of 700 mg/L SO2 and showed good tolerance.(3)The morphology of the cells of F82 was identified oval,and the growth curve was similar with the parents,The Killer spectrum of F82 was the same as NX1134,which could kill wild yeast Rhodosporidium kratochvilovae C272 、 Pichia kluyveri C727 、Saccharomyces cerevisiae F-3-8.The molecular weight of L-ds RNA and M-ds RNA was2.5-3 kb and 1.0 kb.F82 could kill wild yeast C272,F-3-8 and 1296 when inoculated according to 1:1 proportions.The decreased p H of culture medium could be related to killer toxin.The dry wine fermented by F82 showed good performance of residual sugar and volatile acid and F82 have potentiality of development and application.