Preparation And Activity Study Of Fish Oil And Antihypertensive Peptides From Squid Liver

In this research, fish oil and protein were extracted from squid liver, and angiotensin I-converting enzyme (ACE) inhibitory peptides were obtained using enzymatic method. The optimum conditions of enzymatic hydrolysis were studied and ACE inhibitory peptides were preliminary separated. It provided basic theories for the use of squid liver in the food, drugs and animal feed. And it also realized the full use of the protein and the fat in the squid liver, and decreased the discharge of the wastes.First, the constitute of squid liver was identified. The protein content is 14.15% (w/w) by using kjeldahl determination and the fat content is 32.24% (w/w) by using soxhlet extraction.Second, the protein and the fat was separated from squid liver. The fish oil was extracted by high pressured steam boiling law and the residual crude protein was acted on by acetone to eliminate fat. Then we find that protein only takes up 20% of the wet weight in squid liver.The third step is the refinement of the fat. Make use of phosphoric acid to degum, sodium hydroyide to deoxidisate, acitive floridin to decolor and rotary evaporator to deodorizate. Then we get the refined squid oil which is light yellow, clarified, with a thin fish smell. The physical and chemical indexes both live up to level standard of SC/T3502-2000. We analyse the fatty acid composed of squid oil by gas chromatography-mass spectrometry. It has higher polyunsaturated fatty acid which contains 13.1% EPA and 20.3% DHA.Fourth, squid liver protein was hydrolyzed by seven kinds of protein enzymes. We choose pepsin as the tool enzyme finally. According to ACE inhibitory activity and degree of hydrolysis in the pepsin hydrolysates of squid liver protein, we finally defined 2.5% substrate, 2% proportion of enzyme to substrate, 20 hours reaction time, 37℃as the optimized conditions.Fifth, we prepared bioactive peptide through filtrating by using 10000Da filtration membrane, and carried through the preliminary separation by using Sephadex G-25 chromatography. After that, five eluting peaks was obtained. The componentⅡhave the best ACE inhibitory activity. It's IC50 is 1.63mg/mL. It's ACE inhibitory activity did not change between 4～100℃, pH2～12, and the ACE inhibitory activity altered unconspicuously after being acted by pepsin,chymotrypsin and trypsin one by one. The kinetics of the enzymatic action was established to analysis the inhibition type. Results showed that the inhibition type of ACE was competitive.Finally, we make componentⅡthrough further purity by using DEAE Anion exchange column and Sephadex LH-20. After that com- ponentⅡwas separated to two parts. we examine its purity by HPLC. The results of this research show it doesn't absolutely purification. But it provided the basic theories of further separation, purify and identify of ACE inhibitory peptide.