| In thie paper, study porcine bone was treated. Porcine bone Collagen albumen was getted out by hydrogen dioxide(H2O2)soaking method. Used the trypsinase to hydrolyze it and gained collogen polypeptide of enzyme hydrolysis with ACE inhibitory activity. For further exploration of the feasibility of extracting ACE inhibitory activity peptide from procine bone, the ACE inhibitory activity of the collogen polypeptide of enzyme hydrolysis were tested in some measurements of ACE inhibitory activity in vitro.1. Fist of all, preprocessing porcine bone by smashing, defatting, pickling, liming, balancing and so on, to made protein content account above 85% of bone dry weight. One-Way and orthogonal experiments on bone with H2O2 soaking preprocessed were designed to sure the best condition of extraction technology and Collagen protein extraction rate was the experiment index. The results showed that the best condition of extraction technology is H2O2 concentration 0.5%, extraction temperature 100℃, pH8.0, extraction time 7h, from the four factors three levels orthogonal experiments with H2O2 concentration, extraction temperature, pH and extraction time, and collagen protein extraction rate was 86.69% in this condition.2. One-Way and orthogonal experiments were desigend to study the enzyme hydrolysis function of trypsinase on Porcine bone collagen protein. The best trypsinase hydrolysis process was determined by the relationship between the degree of hydrolysis (DH) and ACE inhibitory ratio. The results showed that correlation of degree of DH and ACE inhibitory ratio of Collagen polypeptide was the positive. From the five factors four levels orthogonal experiments with temperature, pH, the concentration of substrate, the ratio of enzyme and substrate and time, the best condition of enzymolysis was temperature 50℃, pH7.5, the 6% of concentration of substrate, the 8000u/g of the ratio of enzyme and substrate,6h. Under this condition, the degree of hydrolysis (DH) was 10.34% and ACE inhibitory ratio was 65.92%.3. The Porcine bone Collagen polypeptide was filtrated respectively by ultra filtration tube with molecular weight lOKDa,3KDa. Then studied the protein concentration and the ACE inhibitory ratio of the separated components of different molecular weigh. The results showed that the ability of ACE inhibitory activity of each constituent:the constituent with molecular weight below 3KDa> collogen polypeptide stock solution> the constituent with molecular weight 10KDa～3KDa> the constituent with molecular weight above 10KDa.The each group has significant difference (P<0.01). The constituent of molecular weight of Collagen polypeptide with below 3KDa was the lowest protein concentration, but it was the most ability of ACE inhibitory activity. The protein concentration was 30.11μg/mL and ACE inhibitory activity was 74.79%.4. Collagen polypeptide with below 3KDa molecular weight was prepared into the solution with the seven concentration by 4,8,10,12,16,20,25μg/mL, and the positive control drug (Captopril) was prepared into the solution with the seven concentration by 0.4,0.8,1.2,1.6,2.0,2.4,2.8ng/mL also, and then mensurating the ACE inhibitory activity of the two solution. The results showed that the IC50 of ACE inhibitory activity of Collagen polypeptide with below 3KDa molecular weight was 11.73μg/mL. These shows Collagen polypeptide from trypsinase enzymolysised Porcine bone collagen protein had the fairly low IC50 of ACE inhibitory activity, so it had the fairly powerful ACE inhibitory activity in vitro. The IC50 of ACE inhibitory activity of control drug was 1.35×10-3μg/mL. This result accords with IC50 of ACE inhibitory activity of Captopril (7.5×10-10～2.2×10-8 mol/L or 2×10-4～6×10-3μg/mL)that had been reported. This showed the assay method of ACE inhibitory activity of our laboratory is feasibility and reliability.5. Collagen polypeptide with below 3KDa molecular weight was prepared into the 0,10,20μg/mL solution by concentration, and studying ACE activity in 5.00μ4.50,4.00,3.50mmol/L of concentration of HHL. For founding ACE inhibitory kinetic model, reacting production quantity of Hip, and suring the relationship of the seep (V) and concentration of substrate (S) by studying OD of ACE activity. The results showed that Collagen polypeptide with below 3KDa molecular weight competitively inhibits combination of ACE and the substrate. In the parameter of dynamics, Michaelis constant (Km) increase and the maximum velocity (Vm) did not alter basicly.6. The digestive enzyme digested the Collagen polypeptide stock solution and Collagen polypeptide with below 3KDa molecular weight respectively, under the condition of imitating physiological gastrointestinal digestion. The each deal group:①Porcine bone Collagen polypeptide+pepsin②Porcine bone Collagen polypeptide+pepsin+chymotrypsin③Porcine bone Collagen polypeptide+ pepsin+chymotrypsin+trypsinase. Studying the change of ACE inhibitory ratio after using digestive enzyme to digest Porcine bone Collagen polypeptide and evaluate the digestive stability of Collagen polypeptide. The results showed that after ACE inhibitory ratio, ACE inhibitory ratio of Collagen polypeptide with below 3KDa molecular weight downed from 72.83% to 68.21%, but it also had high ACE inhibitory ratio. Thses explained it resists hydrolysis of digestive enzyme and it was digestive enzyme stability. The each deal group deferented from un-deal group significantly (P<0.05), and among the each deal group had no significantly deference (P>0.05). ACE inhibitory ratio of the un-deal group downed from 58.96% to 44.81%. The each deal group deferent from un-deal group significantly (P<0.05), and among the each deal group had significantly deference (P<0.05)... |
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