| Sardine(Sardina melamosticta) is one of the most important fishery resources in the southeast coast of China. Since its high productivity, rapid propagation and bargain price, sardine is a fish resource with great potential for development. In our study, sardine antihypertensive peptide was prepared by enzymolysis, and then its components and in vivo effect were studied. Firstly, different kinds of enzymes were utilized to prepare angiotensin-I-converting enzyme(ACE) inhibitory peptides from sardine. According to ACE-inhibitory rate, the optimal enzymes were screened and the conditions of enzymolysis were optimized. Then, the sardine protein hydrolysate was sequentially separated and purified by ultrafiltration and gel column chromatography. Fractions with strong ACE-inhibitory activity were further analyzed by RP-HPLC and amino acids analyzer. Finally, in vivo antihypertensive, physiological and biochemical effect of sardine protein hydrolysate was determined with spontaneously hypertensive rats(SHR) and wister kyoto rats(WKY). Results are presented as follows:1) The optimal combination of enzymes was: papain and alcalase. And the optimal condition of enzymolysis was: 30%(W/V) of sardine in water was hydrolyzed firstly by 4000 U papain per gram of sardine at 65 °C and pH 7.5 for 2.25 h at the first enzymatic step, and then by 5000 U alcalase per gram of sardine at 65 °C and pH 10.0 for 2.0 h at the second enzymatic step. After optimization of the enzymatic hydrolysis condition, the degree of hydrolysis(DH) was increased from 16.70% to 28.44%,and the ACE-inhibitory rate of hydrolysate(C=20.0 mg/m L) was also increased from 65.40% to 73.44%.2) When the ultrafiltration was completed, hydrolysates with molecular weight less than 5 kDa were obtained. After two-step gel column chromatography with G-25 and G-15, three fractions(P2-1, P2-2 and P3-2) with strong ACE-inhibitory activity were obtained and the ACE-inhibitory rates of P2-1, P2-2 and P3-2 were 69.48%, 70.42% and 75.43% at 1.0 mg/m L, respectively. According to RP-HPLC and amino acids analysis, the strong ACE-inhibitory activity might be attributed to suitable hydrophobicity and composition of amino acids.3) After one-time oral administration of the sardine ACE-inhibitory peptide, systolic blood pressure(SBP) of SHR were decreased in certain degree(P?0.01, P?0.05 or P?0.05), but the down-regulation of SBP could not be stable. Then, after chronic oral administration for 4 weeks, the SBPs of SHRs were significantly decreased(P?0.01 or P?0.05) and the down-regulation was stable. Furthermore, according to the detected organ index, serum and organ malondialdehyde(MDA) and ACE activity and concentration of angiotensin-II(ANG-II) in serum, sardine protein hydrolysate presented significantly positive effect on SHR(P?0.01 or P?0.05) without significantly physiological and biochemical effect on WKY(P?0.05). In addition, SBP and physiological and biochemical indexes were partially changed(P?0.01, P?0.05 or P?0.05) in the positive group of WKY.In conclusion, Sardine antihypertensive peptide, which was made from sardine, showed strong activity without any safety problems and side effects of chemical drugs. Hence, it possessed great development value and application prospect. |
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