Study On Structure,Activity And Degradation Products Of Exopolysaccharide From Rhodotorula Mucilaginosa

In recent years,microbial polysaccharides have received more and more attention due to their numerous biological activities.Microorganisms exopolysaccharides are easy to be isolated and purified,and can be fermented continuously,without the restriction of seasons and regions,compared with animal and plant polysaccharides.Yeast exopolysaccharides as a major categories of microbial polysaccharide also has various physiological activities such as anti-oxidation,anti-tumor,anti-virus,etc.It can be developed as a drug or health product,and has a broad market prospect.In this dissertation,Exopolysaccharides?EPS?was used as the research object to explore its biological activity in vivo and in vitro and its mechanism of action.The structures of polysaccharides were analyzed by infrared spectroscopy,nuclear magnetic resonance,methylation and GC-MS.In order to break the limitation of application of polysaccharides due to the large molecular weight and so on,the method of degrading polysaccharides by the hydrochloric acid method was optimized by single factor experiments combined with response surface analysis to compare the in vitro antioxidant activity and micromorphological changes before and after polysaccharide degradation.The main findings are as follows:?1?Preparation,Chemical Composition and in Vivo Activity of EPSThe white flocculated exopolysaccharide was isolated from the fermentation broth of Rhodotorula mucilaginosa,and its yield was 205.3mg/mL.Its polysaccharide content was 52.7%,protein content was 1.4%,and sulfate ester content was 34%,and sulfuric acid content was 5.1%.The glucose tolerance test?OGTT?showed that exopolysaccharides of Rhodotorula mucilaginosa could significantly inhibit glucose-induced elevation of blood glucose,decrease the area under the blood glucose curve?AUC?,and significantly increase the level of glucose tolerance in normal mice compared to the blank group.Subcutaneous injection and intraperitoneal injection of exopolysaccharide had analgesic effect on mice,the pain threshold increased by99.9%and 124.9%respectively,the analgesic effect was significantly better than the positive control.Anti-fatigue experiments showed that compared with the control group,EPS can significantly prolong the weight-bearing swimming time of the mice,and the high-dose,middle-dose,and low-dose groups exercise weight-bearing swimming on the mice.The increase rates of time were 95.55%,61.78%and 50.04%respectively,which significantly increased the serum LDH activity,decreased serum BUN,BLA accumulation,improved the liver SOD,CAT,GSH-Px activity,reduced the accumulation of MDA in the liver,with the anti-fatigue effect.?2?Preliminary Purification and in Vitro Antioxidant Activity of EPSThe EPS was separated on a DEAE-52 cellulose column to obtain five components:EPS1,EPS2,EPS3,EPS4 and EPS5.The yields were 20.0%,14.7%,2.6%,48.2%and 14.5%,respectively.Antioxidation test in vitro showed that all five polysaccharides had antioxidant capacity.When the concentration was 0.06 mg/mL the scavenging rates of OH radicals for each component and Vc were 7.3%,13.8%,18.2%,21.8%,29.4%,and 21.2%,respectively.The scavenging effect of EPS4 and EPS5 on·OH radicals was better than that of Vc at the same concentration.When the concentration was 0.05 mg/mL,the clearance rates of O^2-·radicals by each component and Vc were 24.3%,25.1%,27.6%,26.8%,27.9%,and 16.1%,respectively.The scavenging ability of each component to O^2-·radicals was greater than that of Vc.?3?Purification and Structural Analysis of EPS4The EPS4 fraction with the highest yield and better antioxidant activity was further purified using a Sephacryl S-400 gel column to obtain a single polysaccharide fraction EPS4-1.The molecular weight of 1.23 x 10⁶Da was measured by high performance gel permeation chromatography.The monosaccharide composition analysis showed that EPS4-1 was composed of mannose,glucose,galactose,and xylose in a ratio of 2.20:1.38:1.00:0.23.Based one-dimensional NMR analysis,EPS4-1 was a pyran-type polysaccharide,and only the alpha configuration of glycosidic bonds.The oxidation experiment of periodate experiments and methylation and GC-MS analysis showed that the linkage of EPS4-1 polysaccharide chains was composed of[?3)-Galp-?1?],[?2?-Manp-?1?],[?2?-Glcp-?1?]and[?2,3,4?-Xylp-(1?]and[Glcp-(1?]non-reducing terminal residues.Congo red experiments showed that the polysaccharide EPS4-1 did not show triple helix structure.?4?Degradation and Antioxidant Activity and Morphology of EPS4The EPS4 components were degraded by the hydrochloric acid method,and the single-factor experiment and response surface combined with optimization of the acid hydrolysis of EPS4 were the degradation temperature of 100°C,the degradation time of 7 h,and the hydrochloric acid concentration of 0.4 mol/L.The reducing sugar content was 55.18%.Antioxidant experiments showed that the antioxidant capacity of polysaccharide was improved after degradation.The reducing power of polysaccharides after degradation was 54.6%higher than that before degradation,and the DPPH radical,·OH radical and O^2-·radical scavenging capacities were increased by 18.6%,24.3%and 35.8%,respectively.Scanning electron microscopy showed that the polysaccharide EPS4 was irregular and large,indicating smooth,smooth edges and compact structure.The surface of the polysaccharide SEPS4 obtained after degradation became loose and porous,and the smooth edge appeared broken.Atomic force microscopy showed that the aggregation behavior of the polysaccharides after acid hydrolysis changed significantly,and the lumps disappeared into spherical and rod-like structures.It is presumed that hydrochloric acid destroyed the intramolecular hydrogen bonds,reduced the self association behavior of polysaccharides,disrupted the glycoside bonds of linked branched chains,reduced the branching degree of polysaccharides,and resulted in the disintegration of polysaccharides into rod like and round spheres.