Optimization Of Enzymatic Conditions And Isolation Of Antihypertensive Peptides From Pig Femoral Head

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.The results showed that the optimum hydrolysis conditions of papain were as follows:the temperature was55℃, PH value was6.14, time was4.38h, and the enzyme-substance ratio was6000U/g, substance concentration was8%, by this time, the ACE inhibition ratio was71.50%; the optimum hydrolysis conditions of flavourzyme were as follows:the temperature was53.8℃, PH value was7.0, time was4.85h, and the enzyme-substance ratio was12000U/g, substance concentration was6%, by this time, the ACE inhibition ratio was85.87%; the optimum hydrolysis conditions of alkaline protease were as follows:the temperature was58.2℃, PH value was10.09, time was7h, and the enzyme-substance ratio was12000U/g, substance concentration was6%, by this time, the ACE inhibition ratio was80.09%.Under the optimum hydrolysis conditions of each protease, the enzyme was added in an order as follows:alkaline protease, papain, and flavorzyme. On this condition, the ACE inhibition ratio and DH of the enzymatic hydrolysate were the highest. The ACE inhibition ratio was93%, DH was23.42.Ultra-filtration centrifugal tubes with lOKDa and5KDa MWCO(molecular weight cutoff) were adopted to filtrate and isolate the enzymatic hydrolysate. Three molecular weight peptide fragments were isolated, which were M>10KDa, lOKDa>M>5KDa, and M<5KDa. The peptide fragment with a molecular weight<5KDa had the highest ACE inhibition ratio, and IC50value was1.4022mg/mLIEC(ion exchange chromatography) was adopted to do further purification on the peptide fragment with a molecular weight <5KDa. By analyzing the effects of elution flow rate, ionic strength and the pH value of the eluent on the isolation effect, we could obtain the optimum isolation and purification conditions. The results showed that A liquid was sodium acetate, pH=4.0,0.02mol/L;B liquid was sodium acetate, along with1mol/L NaCl in it, pH=4.0,0.02mol/L; flow rate was1mL/min. Using IEC to isolate and purify the ACE inhibitory peptides could acquire the best effect. Under this separating condition,4peaks was showed, and peak2had the best inhibitory activity, IC50value was0.4884mg/mL.Aiming at peak2, another isolation and purification was performed by gel chromatography, according to the influence of elution flow rate on the isolate effect, optimum isolate flow rate was established, which was0.5mL/min. Under this condition, four peaks were acquired, and peak2had the highest inhibitory activity, value was0.1953mg/mL.