Preparation And Immune Activity Study Of Anser Cygnoides Blood Peptide

Xianghai Anset cygnoides is an endemic species in Jilin Province and lives all year round in Xianghai Nature Reserve of Jilin.In 2015,Xianghai Anset cygnoides was recognized as a new species by the National Center for Biotechnology Information(Genbank ID: 1856198).According to Qian Jin Shi Zhi written by Sun Simiao in the Tang Dynasty,Anset cygnoides has the function of regulating viscera and nourishing spleen and stomach.In this paper,the components of Xianghai Anset cygnoides blood protein peptide were obtained by enzymatic hydrolysis,membrane separation technology,gel filtration chromatography technology and other bioengineering technologies.The immune activity of each component was investigated as well.In order to develop Xianghai Anset cygnoides immune active products.The research contents were showed as following:The product of Xianghai Anset cygnoides was extracted by enzymatic hydrolysis.The compound protease was selected as the tool enzyme.The compound protease is composed of neutral protease,alkaline protease and papain with a mass ratio of 1: 1: 1.Based on single factor experiment,the conditions for extracting Anset cygnoides blood protein were optimized by response surface method.The molecular weight of Anset cygnoides blood protein was investigated by SDS-PAGE electrophoresis before and after digestion with proteases.The results showed that 0.10% trisodium citrate is used as anticoagulant.The best process parameter of zymolytic was 47 ?,p H 7,4500 U/g enzyme and 5 h zymolytic time and under this condition the degree of hydrolysis was 29.29%.The molecular weight of Xianghai Anset cygnoides blood protein(PEC)prepared based on the enzymatic of complex enzymes was mainly distributed at 10 k Da.Through membrane separation technology,Xianghai Anset cygnoides blood peptides were purified.By single factor experiment,the best process conditions of ultrafiltration and the best method of membrane cleaning were determined.The Anset cygnoides blood peptides were analyzed by ultraviolet spectrum and amino acid analyzer.Gel filtration chromatography was used to separate and purify Anset cygnoides blood peptides.The molecular weight of the separated components of Anset cygnoides blood peptides were investigated by SDS-PAGE electrophoresis.The results showed that the optimal process condition for ultrafiltration was that the concentration of the feed liquid is 25 mg/m L,the temperature of the feed liquid was 40 ?,the operating time was 50 min,the operating pressure was 0.20 MPa and the p H was 6.5.The best membrane cleaning method was circulation with distilled water for 30 min,washed with 250 ppm Na Cl O alkaline solution at 35?40 ? for 60 min,then washed with 0.2% SDS alkaline solution at 35?40 ? for 60 min,finally washed with distilled water for 30 min to neutral p H.At this time,the membrane flux recovery rate was 88.5%.With UV spectra,Xianghai Anset cygnoides Blood Peptide(PMS)got by membrane separation method had two peaks at 220 nm and280 nm,respectively.And it contained 17 kinds of amino acids: the component of glutamic acid was about 11.79%,glycine was 9.84%,arginine was 7.28%,valine was 5.47%.which implied that Anset cygnoides blood polypeptide has immune activity.After separation and purification,three components with molecular weight of approximately 9 k Da,7 k Da,and5 k Da were obtained,which were named as ACP1,ACP2,and ACP3,respectively.The effects of PEC?PMS?ACP1?ACP2 and ACP3 on immune function were studied by spleen lymphocyte proliferation experiment,ELISA method used to detect cytokine secretion experiment,mouse bodies weight change experiment,mouse organ index change experiment,delayed type hypersensitivity experiment,serum hemolysin level determination experiment,macrophage phagocytosis of chicken erythrocyte experiment.The results showed that all components of Xianghai Anset cygnoides blood could promote immune function,and PMS has the best effect on the proliferation ability of splenic lymphocytes at the concentration of 200 ?g/m L(P<0.05).ACP2 has the best effect on cytokine secretion at the concentration of 200 ?g/m L(P<0.05).The high-dose PMS group had the best effect on the changes of body weight,organ index,delayed allergy and serum hemolysis levels in mice(P<0.05).ACP2 high-dose group had the best effect on macrophage phagocytosis(P<0.05).According to the above experimental results,Xianghai Anset cygnoides blood protein peptide components have immunological activity.Among them,PMS and ACP2 have better immune enhancement effect,and PMS has the best immune activity.This may be due to the synergistic effect of PMS from ACP1,ACP2,and ACP3.It shows that polypeptide of Xianghai Anset cygnoides blood had certain promoting effects on the body's immune functions.