The Preparation And Identification Of Duck Egg White Derived ACE Inhibitory Peptide

In order to use salted duck egg white resources effectively and exploit peptides with angiotensin I-converting enzyme(ACE)inhibitory activity,the desalted duck egg white was used as raw material and the hydrolysis enzyme was selected from seven different enzymes.The optimal hydrolysis parameters were determined by the single factor test and response surface analysis test,when the ACE inhibition rate was taken as the main index.The hydrolyzed product was preliminary separated by ultrafiltration and dextran gel chromatographic column.By means of HPLC-ESI/MS/MS,the primary structures of peptides were identified.Then the ACE inhibitory peptides were synthesized and the Moe software was used to simulate the molecular docking process between ACE and the synthesized ACE inhibitory peptides,to determine the active sites and the way of interaction.The result provides more useful information on revealing the structural characteristics of ACE inhibitory peptides.1.Preparation of desalted duck egg white peptides with high ACE inhibitory activityProtamex was chosen as the optimal enzyme among seven different enzymes.The result of response surface analysis test showed that the relationship between the in vitro ACE inhibitory activity of peptides and degree of hydrolysis was not a linear relationship,and the interaction between various factors was significant.The optimal hydrolysis parameters were:temperature 50?,p H 6.5,hydrolysis time 4 h,substrate concentration33.04 g/L,enzyme dosage 11876.99 U/g.The ACE inhibitory rate was 84.94%.Under the optimal conditions,the ACE inhibitory rate was close to the predicted result through response surface analysis.2.The structure identification of duck egg white derived ACE inhibitory peptidesThe duck egg white peptides were fractionated by ultrafiltration,the result showed the molecular weight<1 k Da fraction has the highest ACE inhibitory rate.Then the fraction was separated by Sephadex G-15 with three peaks.However,each had the lower ACE inhibitory rate than that of the fractions(Mw<1 k Da).Compared to protein database,83 duck egg white derived peptides were identified by HPLC-ESI/MS/MS technique.Among which,18 peptides had been estimated with ACE inhibitory activity according to structure-activity relationship rule,and the polymerization degree was between 2-5.Four kinds of peptides including IR,HPA,ILKP,INSW were synthesized.The peptide ILKP has tde highest ACE inhibitory activity with IC₅₀ of 166.45?g/m L.3.The molecular docking of duck egg white derived ACE inhibitory peptidesThrough the molecular docking of synthetic peptides ILKP,INSW,IR,HPA and ACE respectively,it wasfound that the interaction including hydrogenbond,electrostatic interaction,metal coordination bond,hydrophilic,hydrophobicand conjugated interations,together maintained stable dockingstructure.ACE amino acid residues Glu362 and Ala332 were taken as the main active site.In the combination of peptide Ile(I)and ACE,it was easier for peptide with N-terminal amino,C-terminal carboxyl or branched chain amino to interact with ACE.