Screening And Characterization Of Bovine Casein Peptides With Anticoagulant And Antihypertensive Activity

Casein?CN?accounts for about 80%of the total protein in milk.It mainly contains four types(?_s1-,?_s2-,?-and?-CN).In recent years,casein has been considered to be one of the excellent sources of bioactive peptides,such as anticoagulant peptides and antihypertensive peptides,and the related research is one of the hot projects in the research field.This study is based on the understanding of the structures of 4 types of casein molecules and the hydrolysis displine of casein.Using casein as the raw material,bioinformatics methodology was used to screen potential anticoagulant peptides and antihypertensive peptides in casein.Bioinformatics,circular dichroism,coagulation analysis?APTT,TT,PT?,thrombin chromogenic substrate method and molecular docking technology were combined to analyze the basic molecular characteristics and the mechanism of anticoagulant peptide.The primary molecular characteristics and activity mechanism of antihypertensive peptides were analyzed by bioinformatics,circular dichroism,high performance liquid phase ACE inhibition activity analysis method,Lineweaver–Burk plot analysis and molecular docking.At the same time,the digestive displine of casein in the gastrointestinal tract of mice,and the in vivo digestion and release of the target bioactive peptides were analyzed by mice intragastric experiments.In addition,the Caco-2 cell monolayer model was used to simulate the absorption of casein bioactive peptides in the small intestine.The molecular structures of casein were predicted,and hydrolysis displine of casein was analyzed.The spatial structure of?_s1-CN,?_s2-CN,?-CN and?-CN was predicted using RaptorX online website.The results showed that the spatial structure of?_s1-CN contains 32%?-helix,4%?-sheet and 63%of the loop;the spatial structure of?_s2-CN contains 49%?-helix and 50%loop;the spatial structure of?-CN contains 21%?-helix,1%?-sheet and 77%of loop;?-CN contains 3%?-helix,8%?-sheet and 87%loop in the spatial structure.Trypsin was used as a mode enzyme to analyze the enzymatic hydrolysis of casein.The results show that controlling the hydrolysis process can effectively control the composition of the product.With the increase of enzyme amount and hydrolysis time,the degree of hydrolysis was gradually increased,and the types of peptides identified in hydrolysate showed a decreasing trend.Anticoagulant peptides and antihypertensive peptides in casein molecules were predicted.The commonality of anticoagulant peptides was summarized by analyzing the molecular weight distribution,peptide chain length,N-terminal amino acid,C-terminal amino acid,isoelectric point,net charge,hydrophilicity and hydrophilic amino acid ratio of the reported anticoagulant peptides.The results showed that the molecular weight of anticoagulant peptides was mainly concentrated in 500-1000 Da,the number of amino acids was mainly concentrated in 2-10,and more than 85%of anticoagulant peptides contain-2-2 net charges,The value of pI in most anticoagulant peptides is between 4and 10,the ratio of hydrophilic amino acids is between 20%and 60%,and the N-terminal and C-terminal have their own common amino acids.Based on the above results,further use of molecular exclusion chromatography and TT,APTT,PT to analyze the molecular weight distribution and anticoagulant activity of the hydrolysates produced by trypsin,neutral protease,alkaline protease and trypsin digestion,and finally trypsin was selected for subsequent study.The bio-layer interferometry?BLI?,UPLC-Q-TOF-MS/MS and bioinformatics technology were used to screen and analyze the casein anticoagulant peptide in trypsin hydrolysates.The results indicate that the peptide AVPYPQR?Peptide-A?from?-casein has potential anticoagulant activity.Similarly,UPLC-Q-TOF-MS/MS and Innovagen,ProtParam,and molecular docking were used to screen and analyze the casein antihypertensive peptides.The results indicate that the peptides NMAINPSKENLCSTFCK?Peptide-B?and EKVNELSK?Peptide-C?may have the function of inhibiting angiotensin converting enzyme?ACE?.Peptide-A,Peptide-B and Peptide-C were identified as potential casein anticoagulant peptides and antihypertensive peptides.This study verified the activity of the potential casein anticoagulant peptide AVPYPQR?Peptide-A?,and analyzed its activity mechanism.The predicted anticoagulant activity of Peptide-A was analyzed using TT,APTT,and PT indicators.The results showed that Peptide-A??-CN,177-183?can inhibit coagulation from endogenous,exogenous and common coagulation pathways;the results of thrombin chromogenic substrate assay showed that this peptide can inhibit thrombin activity.The mechanism of action of peptides and thrombin was analyzed by molecular docking.The results showed that Peptide-A formed six hydrogen bonds with five amino acid residues in thrombin.Peptide-A also formed an electrostatic interaction with the amino acid Arg67 of thrombin,two hydrophobic interactions with the amino acid residue Tyr76 of thrombin.These interactions act to stabilize the enzyme-peptide complex and inhibit the activity of thrombin.This study also investigated the activity of two potential casein ACE inhibitory peptides?Peptide-B and Peptide-C?,and analyzed their activity mechanisms.The results showed that Peptide-B(?_s2-CN,25-41)and Peptide-C(?_s1-CN,35⁴2)can inhibit ACE,and their IC₅₀ values were 129.07?M and 5.998 mM,respectively.The inhibition mode of two ACE inhibitory peptides was analyzed by the Lineweaver-Burk double reciprocal equation.The results showed that two casein antihypertensive peptides exhibit mixed inhibition mode to ACE.The interaction sites between the peptide and ACE were evaluated using molecular docking techniques.The results showed that when Peptide-B coexisted with ACE,it could be hydrolyzed into smaller fragment NMAINPSKE by ACE,which could inhibit ACE.NMAINPSKE interacted with 10 amino acid residues on ACE to form 15 hydrogen bonds.Thus,the mixed inhibition of Peptide-B to ACE may be due in part to its product peptide NMAINPSKE,which exerts inhibitory activity by binding to the active center of ACE.Similarly,molecular docking analysis results of Peptide-C with ACE showed that Peptide-C can interact with 9 amino acid residues of ACE to form a total of 15 hydrogen bonds.This study investigated the in vivo digestion and absorption mechanism of casein bioactive peptides.The casein peptides produced in the gastrointestinal tract of mice were identified by UPLC-Q-TOF-MS/MS.The results showed that casein can be naturally digested into peptides with 7-27 amino acids in mice.The peptide-A,partial fragment of the Peptide-B?NMAINKSE?and Peptide-C,were all identified in the stomach of mice.These three peptides can be produced by in vivo digestion of casein and are resistant to be digested into smaller fragments by the stomach.The absorption of peptide-A,peptide-B,and peptide-C in Caco-2 cell monolayer model showed that Peptide-A,Peptide-B and Peptide-C are able to cross the monolayer reaching the basolateral compartment.