| This paper is focused on the construction of high malic acid-reducing yeast by protoplast fusion and the optimization of cultivation.The conditions that affected the protoplast formation rate and regeneration rate of Saccharomyces cerevisae were analysed. Five-factor and four- level orthogonal experiments were designed to study the single factor, including strain age, enzymolysis temperature and osmotants, analyzed the range of each factor and assess affecting degree of each factor. Finally the optimum conditions for Saccharomyces cerevisae and Schizosaccharomyces melidevorans protoplast formation and regeneration were determined.The relationship between UV-light irradiated dose and mortality of Schizosaccharomyces melidevorans protoplast was examined in this study. The results suggested that when UV-light irradiated dose was 20 watt, distance was 30cm and irradiated time was 5min, the lethality of protoplast was 100%. We got the UV-light irradiation time to lethality curve; For the Saccharomyces cerevisae,When heated up to 70°C, 10min, the lethality of the protoplast was 100%. Then the conditions above were seleted as the genetic maker.After two protoplasts were get, the heat-inactivated protoplast C1 was fused with UV-inactivated protoplast M1 by treating with 35% PEG ,pH6.0 for 30 min, the complementation frequency of fusion was 1.04X10-5 . Then the culture medium was chosen to identify the degradation of malic acid for primary screening. The blue degree of strain color was selected as criterion to evaluated the fusant's ability to degrade malic acid. 36 fusants of blue from plates were selected for secondary screening.Fusants from primary screening were incubated in grape juice medium and fermented in small tubes for 48h. The rate and amount of producing gas were chose as criterions for secondary screening. Finally 9 fusants of higher gas-producing rate and amount were obtained.Fusants from secondary screening were tested in several aspects including cell morphology, genetic stability and the amount of chromosomal DNA, the fermention ability and the flocculent capacity. Formation of fusant was confirmed by the increased amount of chromosomal DNA per cell indicated that multiple fusions had taken place.High performance capillary electrophoresis (HPCE) was used to determine the degradation of the malic acid. Through all these experiments, the fusion Z29 was screened, whose malic acid degradation rate was 31.7%, 30.4% higher than the Saccharomyces cerevisae.The genetic stability was valued after 15 generations, showed that the degration rate of malic acid and other features come from parental strains was stable.The fusion Z29 express fine properties came from parental strains in fermention speed and the flocculent capacity, including good torlerance of SO2, at the same time, giving the... |
