Study Of Purification And Characterzation Of Pleurotus Eryngii Protein

Pleurotus eryngii has high nutritional value and is rich in polysaccharides,proteins and other nutrients.In this paper,using Pleurotus eryngii as raw material,prepared and purified the protein of Pleurotus eryngii,and its initial structure was characterized.The physicochemical and functional properties of protein isolate（PEIP）and albumin（PEA）from Pleurotus eryngii were compared and analyzed;then construction of simulated gastric juice intestinal fluid system to study the antioxidant effects of PEIP and PEA during gastrointestinal digestion.The results are as follows:（1）The protein content in dry powder of Pleurotus eryngii is 17.57%,and the mainly protein fraction was albumin,accounting for 81.12%of the total isolated protein.The optimum extraction conditions of PEA were as follows:the ratio of liquid to material is 20m L/g,the extraction time is 60 min,the extraction temperature is 25℃.Under this condition,the extraction rate of protein was 24.23%.The isoelectric point of PEA was 4.0.The method of isoelectric point and salting-out precipitation had the best effect,and the precipitation rate of protein could reach 99.83%.（2）PEIP and PEA contain 18 kinds of amino acids and the essential amino acid accounts for 40.80%and 40.51%,respectively.Compared to PEIP,the surface hydrophobicity of PEA（265.25）was significantly higher than that of PEIP（164.27）（P<0.01）,while the content of total sulfhydryl group and disulfide bonds were smaller,which were 61.53μmol/g and 10.39μmol/g,respectively.And the thermal denaturation temperature of PEA（100.98°C）was lower than that of PEIP（108.27°C）.The water holding capacity（1.64 m L/g）and the oil holding capacity（5.59 m L/g）of PEA were significantly lower than those of PEIP（3.58 m L/g and 8.36 m L/g）（P<0.01）.The solubility,foaming,foam stability,emulsifying property and emulsifying stability of PEIP and PEA showed a similar trend in p H,and were the lowest at p I.Fourier transform infrared spectroscopy（FTIR）showed that the secondary structure of the PEIP and PEA were mainlyβ-fold andβ-turn.Scanning electron microscopy showed that PEIP possessed a honeycomb structure.Compared to PEA,the PEIP exhibited better functional properties.（3）The hydrolysis degree of PEIP and PEA increased with the prolongation of time in simulated gastric juice intestinal fluid system,and the hydrolysis degree of PEIP was higher than that of PEA in a concentration-dependent manner.The antioxidant activities of PEIP and PEA increased first and then decreased during the simulated gastric digestion.The scavenging rate of DPPH·clearance rate、O₂^-·clearance rate、OH·clearance rate and reducing force reached the maximum value at 3 h after gastric digestion.At the stage of simulated intestinal digestion,there was a positive correlation between antioxidant activity and substrate concentration.The highest O₂^-·clearance rate of 5 mg/m L PEIP was found at 1 h after intestinal digestion,and the antioxidant activity of PEA and PEIP were stronger at 5 h after intestinal digestion.When the substrate concentration was 5 mg/m L,PEIP and PEA had high antioxidant activity in the digestion stage of simulated gastric juice intestinal fluid system.（4）PEIP and PEA were purified by DEAE-52 cellulose anion exchange chromatography column Sephadex G-75 gel filtration column,and the final purified products PEIPF₅S₂and PEAF₄S₁were obtained,and SDS-PAGE analysis showed that the relative molecular weight of the proteins were 17.3 KDa and 20.7 KDa,respectively.PEIPF₅S₂and PEAF₄S₁have UV absorption peaks near the wavelength of 268 nm and 258 nm,respectively,and weak absorption at 280 nm.Theλ_maxof the fluorescence spectrum is located at 340 nm and 347 nm,respectively.The trend of FTIR spectrogram is similar,but there are several characteristic peaks.The main secondary structure of protein wasβ-folding.Compared with PEAF₄S₁,the UV absorption peak position of PEIPF₅S₂is red shifted,and theλ_maxof fluorescence spectrum is blue shifted;the content of ordered structure of PEIPF₅S₂was 65.71%,which indicated that its the protein aggregation was less,tryptophan residue was exposed and conformation was unfolded.