| Hypertension, is a major human chronic diseases, has an important impact on public health, Which is risk factor for heart diseases, cardiovascular diseases, kidney diseases. The number of hypertension patient is continually growing. And it is estimated that200million people now suffers from high blood pressure. Therefore, treatment and prevention of hypertension is a very important topic in the present society. Angiotensin-I-converting enzyme (ACE) play a key role in regulating blood pressure, and ACE inhibitor is the effective drug treatment for hypertension. Captopril, one of the ACE-inhibitory drugs, is first reported in1977. These synthetic inhibitors are believed to reduce blood pressure, However, as well as have certain side effects, such as cough, taste disturbances and skin rashes. The ACE inhibitory peptides derived from food proteins are considered to be milder and safer comparedwith synthetic drugs; furthermore, these peptides usually have multifunctional properties and are easily absorbed. Therefore, we are interested in researching for inhibitors derived from food protein. Since the first discovery of ACE inhibitors from snake venom in1965, many animal proteins can be used as materials for producing ACE inhibitors. For example, such as come from soybean, oats, rice bran, corn, milk, spirulina, bonito fish, sea bream.At present, more than500million pigs are slaughtered each year to provide meat in China, and approximately1.5million blood is collected in the slaughter operation, Which porcine blood proteins is rich. Porcine blood is a valuable protein source, which has high value. It is great significance to development and utilize porcine blood reasonably and to provide more high quality protein as well as functional food. In addition, it is known that bioactive peptides is isolated from the hydrolysate of porcine hemoglobin including highly ACE-inhibitory effect to other bioactive peptides derived from protein resources. In recent years, there are domestic and foreign scholars’ studies about isolation of ACE inhibitor from hydrolysate of porcine hemoglobin.This paper focuses on the research of preparation ACE inhibitory peptides from porcine hemoglobin and its purification. This paper begins with the enzymation of porcine hemoglobin, Firstly, selecting the appropriate protease, getting the result of single factor experiments and optimization experiment through the Central Composite Design response surface. Moreover, macroporous resin, glucan gel, half preparation RP-HPLC in turn purified porcine hemoglobin ACE inhibitory peptides. Finally, obtaining the amino acid sequence, which provides reference for porcine blood protein resources further development. The main results are as follows:(1) The experiment of selecting protease This experiment chooses pepsin, trypsin, neutral, papain, protamex and alcalase to test enzyme solution effect with the indicators of DH and ACE inhibitory rate. Finally, pepsin has the best ACE inhibitory rate,70.09%. Therefore, selecting pepsin does the next experiment.(2) The optimizing enzymation experiment of porcine hemoglobinThis paper has the single factor experiment of enzyme temperature, enzyme pH, substrate concentration, enzyme quantity and enzyme time, before the optimization and get the better values, on which the Central Composite Design response surface basing obtained the best technical parameters of temperature of37.60℃, enzyme pH1.98, substrate concentration4.98%, enzyme quantity3.04%and enzyme time of4h. In these conditions, the IC50of enzymic hydrolysates is1.53mg.ml-1,the curve equation:Y=68.28+1.14X1-0.12X2-0.063X3+0.012X4-0.069X1X2+0.044X1X3+0.16X1X4-0.44X2X3-0.13X2X4-0.094X3X4-1.90X12-1.70X22-2.07X32-2.09X42.(3) The experiment of macroporous resin purification porcine hemoglobin ACE inhibitory peptidesThe experiment examines the adsorptionrate and desorptionrate of the DA201-C, HPD-826, DM130, HPD-400, HPD-100, D101, HPD-800and AB-8macroporous resins. The result was the macroporous resin of DA201-C had highest purification effect, adsorptionrate and desorptionrate respectively59.83%and89.78%. As a result, selecting DA201-C does the next experiments.The experiment examine the influence of sample concentration, pH, sample flow velocity, ethanol concentration and desorption flow rate on the purification of porcine hemoglobin ACE inhibitory peptides. And its results were as follow:40mg/mL of sample concentration, pH5.0, sample flow velocity0.5mL/min, ethanol concentration75%, desorption flow rate1.0mL/min velocity. When the ethanol elution concentration is75%. The eluent has the highest value of hydrophobicity and aromatic amino acid content, respectively4.90kJ/mol and8.43%. In these conditions, the eluents IC50was0.87mg.mL-1.(4) Sephadex LH-20purification porcine hemoglobin ACE inhibitory peptidesIt had three peaks after Sephadex LH-20purifying eluent, and their IC50respectively were0.64of a-Ⅰ,0.21of a-Ⅱ,0.49of a-Ⅲ. Hence selecting a-Ⅱ does the next experiments.(5) The half preparation RP-HPLC purification and structure identification of porcine hemoglobin ACE inhibitory peptidesThrough half preparation RP-HPLC purification, it found6peaks, including2-IV having the highest ACE inhibitory activity, IC500.02mg.mL-1. After the amino acid sequence analysis of2-IV, it got its sequence:VGln-Glu-Leu-Pro-Gly.(6) The research on the physical and chemical properties of porcine hemoglobin ACE inhibitory peptidesThe lowest solubility of ACE inhibitory peptides of porcine hemoglobin (a-Ⅱ) is50.37%at pH7; and this fraction (a-II) demonstrated high stability against gastrointestinal proteases, temperature25-55℃and pH not7; but ACE inhibitory activity could reduce rapidly when the NaCl concentration exceeded0.6mol/L.This paper is part of the research of Southwest university basic scientific research business expenses special funding for the project (Number:XDJK2009C055) and National high technology research and development program (863program) project "protein biogenesis and refined key technology research and development (Number:2013AA102201)"... |
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