Separation And Purification Of ACE Inhibitory Peptides From Casein Hydrolysates

Angiotensin convert enzyme(ACE)plays an important physiologic role in the blood pressure prevention and regulation.At present,many kinds of ACE-inhibitory peptides have been derived from milk proteins.In this paper,ACE inhibitory peptides were prepared by casein hydrolysate by two enzymes.The hydrolysate was separated and purified by ultrafiltration,Sephadex G-15,capillary electrophoresis(CE)and reversed phase high performance liquid chromatography(RP-HPLC).And then molecular weight range was detected by Q-TOF LC/MS,peptides and amino acid was analyzed by LTQ XL.At last,amino acid sequences with high activated can be obtained.In the processing of hydrolysis,ACE inhibitory peptides were prepared by casein hydrolysate first with pepsin and then trypsin.Hydrolysate was separated by ultrafiltration membrane(MWCO=6Ku),and filtrate was purified by Sephadex G-15.It was determined that the half inhibition concentration(IC50)of ACE activity of separation products in vitro.Products by separation were purified and analyzed as follows.Capillary electrophoresis was used to analyze peptide spectrum.Reversed phase high performance liquid chromatography was used to analyze elements.Q-TOF LC/MS was used to detect molecular weight range.LTQ XL was used to detect and analyze peptide spectrum and amino acid sequences.The results showed that IC50 of casein hydrolysate was 560 ? g/mL,IC50 of filtrate was250?g/mL.The filtrate was purified with Sephadex G-15,and three components was obtained,that Component 1 contain 19 peptides and its IC50 was 123.41?g/mL,Component 2 contain 14 peptides and its IC50 was 66.67 ? g/m L,Component 3 contain 5 peptides and its IC50was64.29 ? g/mL.The molecular weight range of purification components was among 400~800u.Component 3 has the highest activation and analyzed by reversed phase high performance liquid chromatography.Its results revealed that it has four elements,and there are two main elements.The results of acid degradation of Component 2 indicated that it has eight amino acids such as Val,Ser,Pro,Leu,Phe,Glu,Asp,Tyr.The results of amino acid sequences of Component 2 are different between the protein ingredients.For the protein-?1,the amino acid sequence is LGYLEQLLR,FVAPFPEVFGK,and YVPLGTQYTDAPSF.For the protein-?2,the amino acid sequence is YQKFPQYLQY.For the protein-?,the amino acid sequence is PVVVPPFLQPEVM.For the protein-?,the amino acid sequence is SRYPSYGLNYYQQKPV and INNQFLPYPY.The results of acid degradation of Component 3 showed that it has two amino acids,they are Ser and Tyr.And Component 3 only has three possible amino acid sequences,they are Ser-Tyr(SY),Tyr-Ser(YS),or both of them.