Preparation Of Pleurotus Eryngii F-measure Oligopeptide Using Ultrasonic Assisted Enzymatic Hydrolysis

As a bioactive peptide with physiological activity,high F oligopeptide has attracted great attention from both food and medicine.To enhance the efficiency of protein extraction by alkali extraction and acid precipitation from fruit bodies of Pleurotus eryngii,cell wall disruption was used ultrasonic irradiation followed by snailase hydrolysis.Preparation of oligopeptides solution was used double enzymatic hydrolysis.To purify oligopeptides solution,a seriers of biological separation processing were used in combination,including activated carbon adsorpting and removing aromatic amino acids,ultrafiltration membrane filtration,macroporous resin for desalination,Sephadex G-15 gel filtration chromatography.The analysis of amino acid analyzer showed that the prepared peptide was a high F value oligopeptide.The main results are as follows:1.Established the processing technology and the parameters of protein extraction from fruitbody Pleurotus eryngii based on ultrasonic-snailase hydrolysis.The results showed that Ultrasonic conditions by single factor optimization were: ultrasonic power 300 W,ultrasonic time 26 min,ratio of water to material 1.95:5(mL/g);snailase optimum enzymolysis conditions were: temperature 40,pH 5,7%,enzyme dosage 1.5 h and fruitbody protein under this condition,the extraction rate reached 72.82%.Then using the BP neural network model simulation based on the orthogonal test to calculate more accurate enzymolysis conditions for a temperature of 42.8 DEG C,4.8 pH,7% enzyme dosage and the time is 1.5 h,the extraction rate was 75.92%,compared to the results of orthogonal test is increased by 4% compared with hot alkali increased by 33.35%.At the same time,the ultrasonic assisted enzymatic hydrolysis time was shortened by about 30 min and the concentration of alkaline solution was decreased.2.The enzymatic hydrolysis kinds of the double-enzymes were screened and the optimum hydrolysis conditions were optimized.The results showed that using the hydrolysis degree as the index,the best endopeptidase selected for double enzyme hydrolysis protein liquid was alkaline protease,and flavor protease was the best exopeptidase.The optimal conditions of alkaline protease hydrolysis were: enzyme dosage 4.21%,pH 11.08,temperature 51.1 C.Under this conditions,the degree of hydrolysis of protein liquid was 23.78%;The optimal conditions of lavorzyme hydrolysis were: enzyme dosage 4.27%,pH 7.04,temperature 50.95 C,under this condition,the protein hydrolysis degree was 39.67%;followed by double enzyme hydrolysis,multi peptide liquid pH was adjusted to 9,compared with two hydrolysis method,the composite the hydrolysis was reduced by nearly 60 minutes than the hydrolysis time used,but the hydrolysis degree redused about 10%.So,in order to prepare high F-measure oligopeptide better,the hydrolysis way should choose double-enzymes stepwise tochnology.3.The best activated carbon type was selected,and the optimum conditions for the adsorption of aromatic amino acids were optimized.The results showed that using branched chain amino acids and aromatic amino acid retention rate,removal rate and OD ratio as the indexes,the optimal activated carbon type was XHT-150.The optimized conditions for the removal of aromatic by Box-Benhnken(BBD)experimental design were: the time of 3.1/h,pH 4.25,solid material ratio 1:9.77.Under this condition,the test was carried out to obtain the OD ratio of 25.97.The ratio of OD predicted by the model was 26.51.The polypeptide liquid after removing aromatic amino acid was measured,the result showed that the content of aromatic amino acids accounted for only 0.15% of the total amino acid content,and the content of branched chain amino acid reached 30.27% of total amino acid content.The content of aromatic amino acids and branched chain amino acids were compared,and the F value was 30.34,which reached the requirement of high F value oligopeptide.4 The process parameters of macroporous resin were selected.Using membrane flux as the index,based on the ultrafiltration membrane with pore size of 6000 D,the optimal pressure of ultrafiltration was 2.5 Mpa.The selected macroporous resin was DA201-C.The static adsorption conditions for desalting by macroporous resin were : pH 4,60 min,eluent concentration of 75% ethanol,the elution time of 120 min.The optimal flow rate was 200 mL/h,the elution flow rate was 50 mL/h,the elution rate was about 120 min,and the recovery rate was 79.68%.5 The conditions of preparing peptide fluid in the range of oligopeptide molecular were obtained by gel chromatography separation.The result showed that the OD value of A280 nm for the oligopeptide between 100-120 min was significantly higher than that in A220 nm absorption.Thus,we can preliminary judge that the content BCAA in oligopeptide was higher than that of AAA.Based on the oligopeptide amino acid analysis result,and F-measure of the oligopeptide reached 34.78,raised 14.63% than before.It also showed that gel chromatography had enrichment effect for BCAA.