| Pleurotus djamor is rich in nutrients,such as protein,crude fiber and amino acid,which are beneficial to the human body.Its polysaccharide(PDP)is one of the main active components,which has a variety of biological activities,such as antioxidant activity and hypoglycemic activity.Studies on Pleurotus djamor at home and abroad are mainly concentrated on domestication and cultivation,nutrient analysis,submerged fermentation,mycelial polysaccharide,laccase activity and protoplast breeding of good strains.While the studies on the isolation,purification,structure analysis and biological activity of crude polysaccharide are few.In this paper,the extraction conditions of polysaccharides from Pleurotus djamor fruiting body were optimized,the crude polysaccharides were isolated and purified,and the structure and antioxidant activity of the purified polysaccharides were studied.Firstly,the extraction conditions of polysaccharides from Pleurotus djamor fruiting body were optimized.In the single factor experiment,four factors were set,including liquid-solid ratio,extraction temperature,extraction time and extraction times.The extraction rate of polysaccharides was determined at each factor level.Based on the results,the optimum extraction conditions were determined by response surface method as follows:liquid-solid ratio of 30.6:1 ml/g,extraction temperature of 95℃,extraction time of 3.9 h,extraction times of 3 times.The actual extraction rate was 13.44%,which was only 1.19%different from the theoretical value of 13.28%.The results showed that there is a high consistency between the experimental values and the predicted values of the model equation,which showed that the extraction conditions are feasible.And then separating and purifying the extracted crude polysaccharide.Macroporous resin method was used to decolorize polysaccharide.The optimum conditions were as follows:resin dosage 1.5 g,decolorization temperature 45℃,decolorization time 120 min and pH 6.The optimal decolorization rate and polysaccharide retention rate were 74.15%and 80.37%respectively.Under the conditions of flow rate of 1.5 BV/h and volume of 4 BV,the decolorization rate and polysaccharide retention rate were 81.37%and 82.21%respectively.The effect ofdynamic decolorization was better than that of static decolorization.Sevage method was used to remove protein,and the removal rate of protein was 61.39%,polysaccharide retention rate was 93.29%.At this time,the protein content was only 2.17%.After separating pigment and protein,further purification is needed,and three components can be obtained after DEAE-cellulose-52 purification,namely PDP-1 obtained by deionized water elution and PDP-2 and PDP-3 obtained by 0.1 mol/L NaCl elution respectively.The three components are further purified by Sephadex G-150 to obtain their single symmetrical peak,namely PDP-1-1,PDP-2-1 and PDP-3-1 respectively.The physicochemical properties of crude polysaccharide and purified polysaccharide(PDP-1-1,PDP-2-1,PDP-3-1)from Pleurotus djamor were determined.The contents of total sugar,protein,uronic acid and sulfate were 69.65%,1.56%,11.25%,3.95%;83.16%,0.48%,9.82%,1.63%;89.11%,0.79%,11.94%,1.60%;84.78%,0.64%,21.08%,1.69%,respectively.Compared with Pleurotus djamor polysaccharide,the total sugar content of purified components PDP-1-1,PDP-2-1 and PDP-3-1 increased significantly.The protein content was decreased,only 0.48%,0.79%and 0.64%respectively.The uronic acid and sulfate group contents of PDP-3-1 were higher than those of crude polysaccharide,PDP-1-1 and PDP-2-1.The structure of polysaccharides from Pleurotus djamor was identified by chemical and spectral methods.The purify components PDP-1-1,PDP-2-1 and PDP-3-1 are all single component,their relative molecular weights were 12.9 KD,10.6 KD and 2753.7 KD respectively.HPLC showed that the chromatographic peaks were single and symmetrical,indicating that the purified components had high purity.The monosaccharide composition and mass percentage of the three purified components were mannose:glucose:galactose:arabinose:fucose=4.79:28.68:47.59:13.37:5.57;mannose:glucose:galactose:xylose:arabinose:fucose=6.43:33.65:36.17:7.20:9.88:6.68;mannose:glucose:galactose:fucose=4.12:87.24:3.90:4.74.By UV spectrum scanning,the crude polysaccharide contained a small amount of protein and nucleic acid,while the protein and nucleic acid of the three purified components were not detected.The characteristic absorption peaks of polysaccharides were detected by infrared spectroscopy.The result of Congo red experiments showed that that purify components PDP-1-1 and PDP-3-1 had a three-strand helix,where as PDP-2-1 did not have a three-strand helix structure.By nuclear magnetic resonance analysis,PDP-1-1 is composed of five kinds ofa-pyranose and P-pyranose,which are-P-D-Galp-、-α-D-Glcp-、-α-L-Arap-、-α-L-Fucp-、-α-D-Manp-;PDP-2-1 is mainly composed of-β-D-Galp-、-α-D-Glcp-、-α-L-Arap-、-α-L-Fucp-、-α-D-Manp-、-α-Xylp-.The antioxidant activity of crude polysaccharide and its purified components showed that the reducing power of crude polysaccharide and its purified components was weak,but it had better scavenging ability on hydroxyl radical,superoxide anion radical and DPPH radical,and had a certain chelating ability of Fe2+.Among them,the antioxidant capacity of purified component PDP-2-1 was better than that of crude polysaccharide and other two purified components. |
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