| Spotted Spanish mackerel, one species of pelagic fish, is usually found in both temperate and tropical seas living along the coast or offshore. The mackerel is widely distributed in China, such as in and very famous for its deliciousness in Hainan. However, researches on the biomedical utilization of Spotted Spanish mackerel are limitied Therefore, the objective of this study was to investigate potential ACE inhibitory peptides derived from the hydrolysates of Spotted Spanish mackerel muscle proteins.The main work is listed as below:1. Enzymatic hydrolysis of the fish meat was performed using four commercial enzymes (pepsin, trypsin, protamex and alcalase) under their optimal conditions recommended by suppliers. The samples, which were adjusted to the appropriate pH and temperature for each enzyme, were digested with enzymes at the concentration of 1%(w/w) for 5h. Finally, the mixture was heated in a boiling water bath for lOmin to inactivate the enzyme. The hydrolysates obtained were tested for ACE inhibitory activity. The Alcalase hydrolysate exhibited the strongest ACE inhibitory activity (61.63%) at the concentration of 1mg protein/mL.2. The alcalase hydrolysate was initially fractionated into three different ranges of molecular weight (MH-Ⅰ>10KDa, MH-Ⅱ 5～10KDa and MH-Ⅲ<5KDa) using a LabscaleTM TFF system (Millipore, Bedford, MA) equipped with 10 KDa and 5 KDa molecular weight cutoff ultrafiltration membranes. Then the most active fraction MH-III was further fractioned into seven fractions by gel filtration chromatography on a Sephadex G-25 column. Subsequently, the most potent fraction E was purified by two-step RP-HPLC. As a result, a purified peptide was obtained with the purity of 97.9%.3. The molecular weight of fraction E3-Ⅰ was analyzed using MALDI-TOF MS and the result was 825.2Da. The N terminal triipeptide sequence of the purified peptide was determined as Ile-Tyr-Pro by using an auto amino acid sequencer.. |
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