| Depending on high content of protein, with all the hydrophobic amino acids, aromatic amino acids and basic amino acids included, flesh of silver carp fish has been considered a suitable resource for the preparation of angiotensin-converting enzyme (ACE) inhibitory peptide. Currently, synthetic drug of anti-hypertension inevitably causes some side effects to a certain degree. However, no report about side effects against human body has ever been found among ACE inhibitory peptide (or antihypertensive peptides) extracted from foodborn protein. It is incomparably beyond, and has been one of the topic of general interests for antihypertensive drugs. Based on silver carp fish as raw material, ACE inhibitory constituent was obtained according to the hydrolysis of protease and separation and purification of ultrafiltration, gel chromatography and RP-HPLC. The amino acid of the constituent has been sequenced, and the main results were as follows:1For the hydrolysis of silver carp fish, alkaline protease, neutral protease, flavor protease, pepsin and trypsin were considered as the candidates. Flavor protease has been concluded the optimal protease for hydrolysis in the study, hydrolysate processed by flavor protease showed a high ACE inhibitory rate which was up to63.24%, and the degree of hydrolysis was27.88%. Single factor experiments have validated that factor of pH value, temperature, time and water content showed a significant effect on the hydrolysis of flavor protease against silver carp protein. Based on Box-Beknken response surface, the models for each factor level have been fitted. The regression equation was as follow:Y=85.1500+0.3058A+0.6267B+0.1150C+0.1208D-2.1583A2-2.0321B2-1.1258D2+0.5700A*B-0.5250A*C-1.2575A*D-0.7000B*D.According to the software analysis of MINITABLE16, optimum conditions of ACE inhibitory peptide hydrolyzed by flavor protease against silver carp protein have been concluded:original pH value7.54, temperature50.56℃, time4.5h and water content46.67ml/10g flesh. Optimum relation of Y for ACE inhibitory rate was81.21%, ACE inhibitory rate was81.35%among validation tests.2Barrier separation for the hydrolysate of silver carp protein has been studied with a ultrafiltration membrane of lOKDa intercepting, tests showed that ACE inhibitory rate of constituent less than10KDa was83.27%, that above lOKDa was67.78%. Maximum absorption peak was scaned at220nm towards constituent less than lOKDa with UV spectrum. Three constitutes named F1, F2, F3have been separated from constituent less than lOKDa by SephadexG-25gel chromatography. Of the three constituents, F2showed a high ACE inhibitory rate about88.47%. By RP-HPLC,4main constituents of F2have been separated, the highest rate up to94.64%has been detected in F2d. It has been confirmed that peak of F2d was relatively single, with a peak area of84.00%.3The molecule weight and amino acid sequence have been identified by LC-ESI-MicroTOFQ-Ⅱ after purified. Polypeptide A and B have been obtained with molecule weight916.8672Da and1180.4606Da respectively. Part of the amino acid sequence has been identified as Pro-Val-Ile/Leu-Ser andlys-Ala-Ala-Asp-Glu-Pro respectively which started from N terminal. |
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