| Pleurotus ostreatus is a Tricholomataceae fungus belonging to the Agaricales of Basidiomycetes and also known as white oyster mushroom and Pleurotus. The fruit body contains a lot of carbohydrates and polysaccharide is the main active substance has immunoloregulation and anti-oxidation activities. In this paper, the extraction process and purification of Pleurotus ostreatus polysaccharide (POP) was optimized, the levels of sub-polysaccharide antioxidant activity in vitro were studied, and the preliminary study of structure and modification of the pure polysaccharides was performed. The main experimental results are as follows:(1) Optimization of polysaccharides extraction process from Pleurotus Ostreatus by Response Surface Methodology, ultrasonic-assisted extraction process by Uniform Design and alkali extraction by Orthogonal Design. The result of Response Surface Methodology were the ratio of material to water1:44.6(g/mL),extraction temperature90℃,and extraction time4.3h. Under the optimal conditions, the yield of POP was3.25%. The optimal extraction conditions of Uniform Design were material to solvent1:20(g/mL), extraction time85min, ultrasonic extraction temperature75℃and ultrasonic power120W, the yield of POP was9.76%. The result of orthogonal experiment of alkali extraction were extraction temperature90℃,material to water1:45(g/mL), sodium hydroxide concentration0.5mol/L and extraction time2.5h, the yield of POP was9.54%.(2) Separation and purification of Pleurotus ostreatus polysaccharide was remove protein at first. The method of Sevage, Trichloroacetic acid (TCA), neutral protein enzyme and Sevage associate with neutral protein enzyme was chosen. Under process of Sevage, the removal rate of protein was96.59%, and retention of polysaccharides was46.15%. When the mass fraction of Trichloroacetic acid was9%, the removal rate of protein was65.98%, and retention of polysaccharides was51.36%. The neutral protein enzyme condition was lmg, enzymatic hydrolysis3h, the removal rate of protein was82.81%, and retention of polysaccharides was90.25%. The result of Sevage associate with neutral protein enzyme was95.44%and80.43%respectively. We chose the last method to remove the protein. The polysaccharide was purified by DEAE-cellulose-52ion exchange column chromatography, POP-Ⅰ, POP-Ⅱ and POP-Ⅲ were obtained and purified respectively by Sephadex G-100column chromatography again. After that four homogenous pure polysaccharides were obtained and were named POP-Ⅰ-1, POP-Ⅱ-1, POP-Ⅲ-1and POP-Ⅲ-2. Neither protein nor nuclear acid characteristic absorption peaks were shown in the UV-visible spectra of POP-Ⅰ-land POP-Ⅱ-1.(3) The anti-oxidation activities of at all levels of Pleurotus ostreatus polysaccharides. The polysaccharide got through hot water and alkali extraction, and POP-Ⅰ-1, POP-Ⅱ-1were chose to investigate their scavenging effect of DPPH·,·OH, O2·-and reducing power. The highest value of four polysaccharides’ scavenging activity to DPPH·was47.70%,41.9%,14.29%and11.22%respectively. The result of OH was100.00%,73.31%,8.41%and23.63%respectively. The optimum of scavenging activity to O2·-was20.21%,31.00%,17.23%and22.91%respectively. The reducing power was0.430,0.464,0.059and0.188respectively. Besides that, the polysaccharide got through hot water showed antioxidant properties on both lard oil and sesame oil.(4) The preliminary study of the structure and modification of Pleurotus ostreatus polysaccharide POP-Ⅰ-1and POP-Ⅱ-1.The result of their Thin-layer chromatography (TLC), Infrared Spectroscopy (IR) and Nuclear Magnetic Resonance (NMR) indicate that the polysaccharide was mainly composed of glucose, and a and β-glycosidic bond, most was a glycosidic bond. The sulfated modification on POP-Ⅰ-1and carboxymethylation on POP-Ⅱ-1and then named SPOP-Ⅰ-1, CPOP-Ⅱ-1. The result of their scavenging activity to·OH was18.16%and44.22%respectively. The optimum of scavenging activity to02·-was30.04%and40.23%respectively. Their anti-oxidation activities both were much better than POP-I-1and POP-Ⅱ-1. |
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