Study On Breeding Deacidification Yeast Strain For Wine

F-20-7 is the inter-kingdom fusant of wine yeast 1450 (Saccharomyces cerevisiae) and SD-2a (Oenococcus oeni), which was obtained by College of Enology in 2003. Because the key enzyme genes are on the plasmid, so the ability of degrading malic acid is not stable. In this study, we used Oenococcus oeni and the fusant F-20-7 as the parents, to construct fine degrade malic acid strain by the method of electronic protoplast fusion. At the same time, we were seeing about the feasibility of protoplast fusion between inter-kingdoms.The main results are as follows:1. According to the result of deacidfication experiment on 9 strains of Oenococcus oeni, we found SD-2a maintain the character of fine deacidfication, may continue to use as the parent of fusion.2. The antibiotic experiment shows: 50μg/mL actidione could restrain wine yeast 1450 growth; 100μg/mL actidione could restrain fusant F-20-7 growth. According to this dosage we selected the second generation fusant.3. Through single factor experiment and orthogonal experiment, we definited optimum condition of parents strain protoplast preparation. Grape wine yeast 1450 protoplast prepare conditions are: Fungus age 12h, enzyme density 1.5%, enzymolysis time 90 minutes, protoplast preparation and regeneration rate achieves 12.40%; Oenococcus oeni SD-2a protoplast prepare conditions are: Fungus age 20h, penicillin G sodium density 0.5μg/mL, enzyme density 1.0 mg/mL, enzymolysis time 30 minutes, protoplast preparation and regenerationrate achieves 20.86%.4. The first protoplast fusion carried on between wine yeast 1450 and Oenococcus oeni SD-2a, the fusion condition is: PEG-4000 concentration 30%, fusion time 30minutes, fusion temperature 30℃, the regenerate culture medium is HYPD+50μg/mL actidione. The second protoplast fusion carried on between F-20-7 and Oenococcus oeni SD-2a, the fusion condition is: Arrangement frequency 800KHz, oscillation amplitude 29v, pulse time 30 ms, pulse frequency 5, pulse delay time 60×10ms. The regenerate culture medium is double-decked plate YPD medium, which is added to100μg/mL of actidione.