Separation And Purification Of Pig Femoral Collagen Antihypertensive Peptides

This paper focused on the femoral head in pigs, antihypertensive peptides were extracted by the enzymolysis of acid protease, neutral protease, and alkaline protease, two kinds of protease in each group. The ACE inhibition ratio and degree of hydrolysis (DH) are two indicators, as to screen out one kind of protease as the enzyme of the enzymolysis in each group. The enzymolysis condition was optimized, according to the single-factor experiment and response surface methodology of each protease. On this basis, a compound experiment was conducted under the optimum condition of each enzyme. Ultra-filtration, ion-exchange chromatography, and gel chromatography were applied to separate and purify the antihypertensive peptides.The acid, neutral, and alkaline protease which are screening out were papain, flavourzyme,and alkaline protease, separately.Using the gel chromatograpHy separation to separate the enzymolysis liquid whose molecular weight is less than 5kD(with an IC50 of 1.362mg/mL)after ultrafiltration separation. Study the effect of Separation and purification with the three factors,which is eluent, sample loaded and velocity.The optimal conditions for analysis were as follows:elution of ultrapure water,the flow rate is 0.6 mL/min, the sample amount is 1%,and the peak 2 had the highest ACE-inhibitory activity with an IC50 of 0.4016mg/mL.The solubility of Antihypertensive Peptide of Pig Femoral Collagen was better at pH 2-6,and this peak demonstrated high stability against gastrointestinal proteases temperature,and it has good acid and alkali resistance,salt resistance.In order to obtain high activity and purity of antihypertensive peptides,the method of ion exchange chromatography was used to separat the solution after the preliminary separation and charactenization by ultrafiltration and gel permea tion chromatography.The eluent pH, ionic strength, velocity of the three factors were studied.The optimal conditions for analysis were as follows:elution of pH =6.0,the flow rate is 0.8 ml/min, the ionic strength t is 1.0mol/L.The results sh owed:The fraction was then purified by ion exchange chromatography into 3 peaks,in which peak 1 had the strongest ACE inhibitory activity with an IC50 of 0.1012mg/mL;And the peak 1was further desalinationted by gel permeation c hromatograpHy to obtain 4 peaks,and the peak 2 had the highest ACE-inhibitor y activity with an IC50 of 0.0836 mg/mL, the desalination rate 86.60%±0.5%.In order to obtain high activity and purity of antihypertensive peptides,the method of RP-HPLC was used to separate the solution after the preliminary s eparation and charactenization by ultrafiltration,gel permeation chromatograpHy and ion exchange chromatograpHy. Studying the effect of Separation and purifi cation on the factors of elution conditions, The optimal conditions for analysis were as follows:0-5min 0%～80%A;5-8min 80%～100%A;8～15min 80%～60%A; 15～20min 60%～20%A. The results showed:The fraction was then purified by frist RP-HPLC into 3 peaks, in which peak Z2 had the strongest ACE inhibitor y activity with an IC50 of 0.0437mg/mL.The peak Z2 was desalinationted by th e second RP-HPLC, the peak Z21 (denoted by component H) has the highest ACE inhibitory activity which with an IC50 of 0.0352 mg/mL, it’s purity at 96. 7%. Through the analysis of the amino acid of component H and identification of LC-MS,it contains six kinds of amino acids (Glu,Ala,Val,Leu,Phe,Pro),the molecular weight is 764.8D.