Screening, Identification Of Exopolysaccharide-Producing Yeasts And Studies On Exopolysaccharide Structure Elementarily

Microbial polysaccharides were produced by bacterial, fungi and algae. Microbial polysaccharides which included cell wall polysaccharide, intracellular polysaccharide and extrcellular polysaccharide get more and more attention from the reaschers for their superiorities such as being easy to separation, continuous fermentation, no affection of season, regional, diseases and insect pests.Yeast polysaccharide is a kind of microbial extrcellular polysaccharides which has many physiological activities, such as modulating human immune system, oxidation prevention, antivirus, antitumor and so on.The main contents and results of this thesis are as follows.(1) Three higher exopolysaccharide-producing yeast stains were obtained by screening from natural samples using spread-plate method, The results of 5.8S rDNA sequence and phylogenetic tree analysis showed that similarities of two yeasts isolated from "Zuoyouhong" grape (Z14 and Z20) were over 99% compared with Issatchenkia orientalis, meanwhile another yeast L25 isolated from topsoil of Larix gmelini was identified as Cryptococcus humicolus, with the similarity of 98.8%.(2) In order to increase the yield of EPS, fermentated conditions of Issatchenkia orientalis Z20 were tested. The optimal medium for strain Z20 for producing EPS was as followed:8% glucose,0.2% (NH4)SO4,0.1% KH2PO4,0.1% yeast extract powder and 0.01%CaCl2, yield of EPS was 2.046 mg/mL, which increased by 80%(compared with 1.137 mg/mL of original strain). On the basis of optimal medium, the optimum fermentation conditions were obtained as follows:initial pH 6.0,180 rpm of revolution speed,50 mL liquid medium in a 250-mL Erlenmeyer flask,30℃of incubation temperature,2% of inoculation proportion and 6 days of fermentation time, under the optimum conditions, yield of EPS could reach to 2.327 mg/mL, which was 104% higher than the control (1.137 mg/mL).(3) Exopolysaccharides of Z20 were separated from medium, only one purified polysaccharide component EPS-Ⅰwas obtained by DEAE-cellulose 52 chromatography. The structure of EPS-Ⅰwere determined by Infrared spectrum, result showed that EPS-Ⅰhad polysaccharides specific function group such as-OH,-CH,-C=O etc.. The hydrolyzate of EPS-Ⅰwas analyzed by Gas chromatography and the result indicated that the hydrolyzate of exocellular polysaccharide mainly consisted of D-mannose, D-xylose and L-rhamnose, the monosaccharide compositions of EPS-Ⅰwere in the proportion of 84.91%,11.47% and 3.62% w/w, respectively.(4) Preliminary reasch about the antioxidant activity and bacteriostasis in vitro of EPS of stain Z20 was done. Total antioxidant capacity (TAC), clearance rate of DPPH-, hydroxyl radical and superoxide anion were investigated as abilities of antioxidation in vitro. The results demonstrated that EPS-Ⅰhad favorable antioxidative property, at the concentration of 20 mg/mL, the DPPH·and O2- radicals-scavenging activities of EPS-Ⅰcould reach to 80% above. Inhibition of saturated solution of EPS-Ⅰagainst Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Penicillium, Lichen and Aspergillus niger was validated by the filter paper disk method, the results showed that EPS-Ⅰagainst growth and breed of 6 stains above had little inhibition.