Preparation, Separation And Purification Of Dual Renin And ACE Inhibitory Peptides From The Peach Kernel Protein Isolates

Hypertension, a major human chronic disease, has an important impact on public health. It is also a risk factor for heart diseases, cardiovascular diseases and kidney diseases. ACE inhibitors and renin inhibitors are believed to reduce blood pressure, but they have certain side effects. Combined use of ACE inhibitors and renin inhibitors can reduce not only blood pressure, but also the damage to other organs. The antihypertensive peptides derived from food proteins are considered to be milder and safer, as these peptides can reduce the damage to the target cells, generally have multifunctional properties and are easily absorbed compared with synthetic antihypertensive drugs. Therefore, we are interested in antihypertensive peptides derived from food protein.In this work the research focuses on preparation and purification of antihypertensive peptides from peach kernel protein isolates (PKPI). Firstly, PKPI was hydrolyzed by different proteinase, then, the hydrolysis condition was studied and optimized by the degree of hydrolysis, ACE inhibitory rate and renin inhibitory rate. The hydrolysate of PKPI was separated and purified by ultrafiltration, macroporous resin, gel filtration chromatography, high performance liquid chromatography, etc. These provide further evidence for PKPI resources development. The main results are as follows:1) The experiment of selecting protease. This experiment choosed protamex, neutral, Alcalase 2.4L, flavourzyme, papain, pepsin, trypsin, combined use pepsin and trypsin to test enzyme solution effect with the indicators of DH, ACE inhibitory rate and renin inhibitory rate. It is found that Alcalase 2.4L has the best ACE inhibitory rate and rennin inhibitory rate. Therefore, Alcalase 2.4L was selected for the following experiment.2) The experiment of optimizing enzymation proteolysis of PKPI. The preparation of dual rennin and ACE inhibitory peptides from peach kernel protein isolated with Alcalase 2.4L was investigated. ACE inhibitory rate and renin inhibitory rate in vitro were investigated with respect to hydrolysis parameters including temperature, pH, substrate concentration and enzyme dosage. As a result, the optimum conditions were found to be hydrolysis at pH 8.02, temperature of 50.6 ℃ and enzyme dosage of 3.06%. Under these conditions, the prepared peptides showed an ACE inhibitory rate of 69.60% and renin inhibitory rate of 56.29%.3) Purification of PKPI antihypertensive peptides by ultrafiltration and macroporous resin. The peach kernel protein isolates hydrolysates was divided into different molecule weight range by ultrafiltration with 50 kD、10 kD、5 kD、 1 kD Polyethersulfone (PES) membranes respectively, the results revealed that each fraction had varied ACE inhibitory activity and renin inhibitory activity, and the ACE inhibitory activity and renin inhibitory activity of the PKPI hydrolysates with molecule weight less than 1 kD was highest. The adsorption rate and desorption rate of the LSA-21, D101, XDA-6, DA201-C, LX-1180 and AB-8 macroporousresins were examined. It is found that DA201-C has the best highest purification effect, and the adsorption rate and desorption rate were 68.84% and 80.75% respectively. As a result, DA201-C was selected for the following experiment. The influences of sample concentration, pH, salt concentration, sample flow velocity, ethanol concentration and eluant flow rate on the purification of PKPI antihypertensive peptides were examined. As a result, the optimum conditions were found to be DA201-C purification at sample concentration of 20 mg/mL, pH 7.0, sample flow velocity of 0.5 mL/min, ethanol concentration of 75% and desorption flow rate of 1.0mL/min. When the ethanol elution concentration is 75%, the hydrolysate has the highest ACE inhibitory rate and renin inhibitory rate.4) Separation and purification of PKPI antihypertensive peptides by sephadex G-15 and half preparation RP-HPLC. The enzymatic hydrolysates were purified through Sephadex G-15 after macroporous resin, and the results showed that the optimum conditions were found to be Sephadex G-15 purification at the flow rate of 0.8 mL/min, the sampling volume of 1 mL and the sampling concentration of 150 mg/mL. Under these conditions, the purification effect was the best. It was separated into three fractions by sephadex G-15. The fraction G2 has the highest ACE inhibitory rate and renin inhibitory rate. Fraction G2 was further purified by analytical RP-HPLC and it was separated into five fractions, of which fraction c has the highest ACE inhibitory activity and renin inhibitory activity.