Isolation, Purification And Activity Evaluation Of Enzymatic Hydrolysate Of Enteromorpha Prolifra Polysaccharide

Enteromorpha prolifra is a kind of economic type macroscopic green alga in the southeast coast of China. It can be edible and contains many kinds of bioactive substances. According to reports in the published literatures, Enteromorpha polysaccharide had many physiological functions such as improving immune function in mammals’, decreasing fat in blood, acting as anti-inflammatory and antioxidant. However, environment changes could cause the explosive reproduction of Enteromorpha prolifra, thereafter resulting in occurrence of green tide. In general, they are buried. However, the problem has not been solved fundamentally. Therefore, studying on the polysaccharide from Enteromorpha prolifra, can not only can improve the utilization rate of Enteromorpha prolifra, but also lay the foundation for its deep processing into functional food. In this thesis, using Enteromorpha prolifra as raw material, and depredated water soluble polysaccharide from Enteromorpha prolifera was enzymatic hydrolyzed. The hydrolysaltes were evaluated for the antioxidant activity. The component with best activity was further isolated and purified. Structure and activity of the obtained homogeneous polysaccharide were preliminary investigated. The results in this thesis provide an experimental basis for the development and utilization of polysaccharides and degradation of polysaccharide from Enteromorpha prolifra.The results were as follows:1. Five enzymes including saccharifying enzyme, pectinase, cellulose, a-amylase and lipase were tested for hydrolysis of polysacchride, the resuslts showed that accharifying enzyme and pectinase can effectively catalyze the hydrolysis of the water extracted polysaccharide from Enteromorpha prolifra. Response surface method was employed to optimize enzymplysis conditions of the polysaccharide from Enteromorpha prolifra. The optimum enzymolysis conditions of saccharifying enzyme were obtained as follows:enzyme concentration is14.20U/mL, reaction temperature is48.84℃, pH is4.48, and under these conditions, hydrolysis was carried out for5h, degradation rate of polysaccharide was determined to be12.65%; pectinase:amount of enzyme added is7.80%of polysaccharide, reaction temperature is55.56℃, pH is4.45, and under these conditions, hydrolysis was carried out for5h, the degradation rate of polysaccharide was determined to be12.71%.2. Enteromorpha polysaccharide was hydrolyzed by saccharifying enzyme and pectinase, respectively, for O h,1h,2h,3h,4h and5h. It was found that enzymatic polysaccharide exhibited better free radical scavenging ability on three radicals (DPPH、·OH、O2-) than non enzymatic polysaccharide. Scavenging ability of pectinase enzymolysis polysaccharide is better than that of saccharifying enzyme for the three kinds of free radical. When the polysaccharide was enzymolysised for3hours by pectinase, the resulting hydrolysate exhibited best radical scavebging effect, which was named EPCE. At a concentriation of5mg/mL, the removal rates of DPPH,·OH and O2·were58.32%,44.68%and78.31%, respectively. The molecular weight range of EPCE was determined to be9.5×103～1.7×105by GPC. The results of GC-MS analysis showed that EPCE was mainly composed of rhamnose and xylose, and contains a small amount of mannose, glucose and galactose. The molar percentage of each monosaccharide was71.72%:10.6%:6.33%:8.85%:2.5%.3. In this study, EPCE was isolated and purified with the cellulose DEAE-52and Sephadex G-100. Three fractions were isolated, one neutral polysaccharide being named EPCE1and two acidic polysaccharide fractions being named EPCE2and EPCE3, respectively. UV detection inferred that EPCE1and EPCE2had no absorption peaks at260～280nm, indicating that the two samples do not contained nucleic acid and protein. While the absorption of EPCE3at the280nm is very weak, indicating that this polysaccharide may have a small amount of bilding glycoprotein. HPLC analysis showed that EPCE1, EPCE2and EPCE3had only one peak, suggesting that they are homogeneous components. They were investigated for in vitro antioxidant activity, EPCE2was found to possess the best activity among the three polysaccharide fractions. At a concentration of1mg/mL, the scavenging rates of EPCE2against DPPH,·OH and·O2-were46.22%,10.15%and78.82%, respectively. The physical and chemical analysis of EPCE2showed that there was no amino acid, protein and starch; according to Phenol-sulfuric acid method, the total sugar contents of EPCE2was93.27%, and uronic content is21.75%molecular weight of EPCE2was determined as4.54×104by GPC; monosaccharide composition of EPCE2was analyzed by GC-MS, glucose is main composition, it also contains a small amount of mannose, xylose and galactose. The molar percentage of four monosaccharides was70.74%:16.89%:10.23%:2.14%.