Study On The Preparation,Purification And Identification Of ACE Inhibitory Activity Peptides From Se-enriched Tea Protein

Selenium in animals,plants and microbial nutrition is significant for a kind of trace element,its role is mainly a drop in blood pressure by selenium protein polypeptide or selenium.Tea polypeptide has certain blood pressure activity,is between protein and amino acid of the peptide,and compared to other biological molecules,has good biological activity and diversity,and its security is higher,lower blood pressure activity is more significant.Combined with selenium tea protein polypeptide effective research and development of se-enriched tea polypeptide from protein will have higher application value.In this paper,the natural selenium-rich tea protein and ordinary not rich selenium tea protein extraction and purification,natural selenium-rich alkali soluble protein ACE inhibitory peptides and ordinary not rich selenium tea tea protein ACE inhibitory peptides related research was conducted on the preparation,separation,purification and structure,the main results were as follows:?1?Tea powder after dealing with the decoloring,polysaccharides and so on,in 0.1 M NaOH,4 mg/ml PVPP,temperature 60?,material liquid than 25 under the conditions of extracting tea protein,Se-TP get four components on the elution conditions,respectively,Se-TP??Tris-HCl?,Se-TP??0.2 M NaCl?,Se-TP??0.3 M NaCl?,Se-TP??0.6 M NaCl?,part of the Se-TP?ACE inhibitory activity is higher,at 41.3%.And TP in the elution conditions of three components,respectively,TP??Tris HCl?and TP??0.3 M NaCl?,TP??0.5 M NaCl?,the best TP?ACE inhibitory activity of components.heat pretreatment is beneficial to the exposure of the active group,it is easier to get active enzyme solution sequence,comprehensive consideration,choose the pretreatment conditions of 100?water bath for 5 min.Five kinds of protease?alkaline protease,neutral protease,papain,trypsin,pepsin?after enzymolysis,detect the enzymolysis liquid of ACE inhibitory activity and degree of hydrolysis,final selection of alkaline protease and neutral protease and compound enzyme solution.?2?composite enzyme solution of single factor experiment showed that five factors?pH,temperature,adding enzyme amount,reaction time,proportion of enzyme?,within a certain range,the activity of enzyme products along with the increasing of five factors,increase after decreases first.Five factors affect digestion process is mainly manifested in the protease enzyme activity and the influence of active amino acid exposure.Composite enzyme digestion process is:the best optimal enzymatic hydrolysis conditions:pH 8,temperature 60?,with enzyme amount is 3500 U,time of 5 h,proportion of enzyme?alkaline,neutral?for 2:5,ordinary not rich selenium alkali soluble tea protease activity of ACE inhibitory peptides mixture IC₅₀0 solution is25.95 mg/ml,natural selenium-rich alkali soluble tea protease activity of ACE inhibitory peptides mixture IC₅₀0 solution is 17.07 mg/ml.TPep by ultrafiltration separation from four components,respectively TPep??<1 kDa?,TPep??1¹0 kDa?,TPep??10¹00 kDa?,TPep??>100 kDa?,including component TPep??<1 kDa?ACE inhibitory activity of the highest,93.29%.Se-TPep by ultrafiltration separation from four components,respectively,Se-TPep??<1 kDa?,Se-TPep??1¹0 kDa?,Se-TPep??10¹00 kDa?,Se-TPep??>100 kDa?,part of the Se-TPep??1¹0 kDa?the activity of the highest,94.78%.Ultrafiltration income Se-TPep?and TPep?via gel Sephadex G-25,averaging two component elution,and the content and?ACE inhibitory activity is higher,the rich selenium content is 2.25 PPM,selenium is ACE inhibitory activity of IC₅₀0 was 0.93 mg/ml,and not the rich selenium components IC₅₀0 ACE inhibitory activity is 1.45 mg/ml.?3?By electron microscopy figure can clear to see the appearance of the sample appearance,natural proteins and peptides ordinary flake form more thick,more loose thin compared with protein and polypeptide forms.Proteins and peptides were detected in the sample,N-H,O-H,NH₂ groups,C=O,C-N,aromatic ring C=C,C-O,C-S keys,etc.And in C-S position,natural selenium-rich tea protein and peptide absorption intensity increasing,may be replaced by the Se,S under the influence of C-Se keys.Comprehensive analysis,could be a part of the aromatic amino acid proteins and peptides.Through the Se-TPep?and TPep?of amino acid composition,Se-TPep?content of each amino acid in the than TPep?is higher,and in the N side contains Gly,Ala,Val,Arg activity such as amino acids,of which the highest content of Ala,Val.C contains Leu,Tyr,Phe,such as hydrophobic amino acids,of which the highest content of Leu.C contains Leu,Tyr,Phe,such as hydrophobic amino acids,of which the highest content of Leu.Se-TPep components?<1 k?after NanoLC-Q-TOF-MS analysis,get a peptide sequence AETGEIKGHY,molecular weight of 1103.53.Components of the Se-TPep??1¹0 kDa?are two peptide sequence,one of the components and Se-TPep?<1k?peptides are exactly the same,another LGVPIVMHDY peptide sequence,its molecular weight is 1158.57,and amino acids containing selenium is often combined with Met.Component TPep??<1kDa?is a peptide sequence LQPSLGFP,the molecular weight of 857.46.