The Preparation Of Hydrolysates, Extraction, Isolation And Structure Of Antihypertensive Peptide From Mytilus Coruscus

Mussel is an important aquaculture species worldwide, but people can't make more profits if only mussel is sold directly. Mussel not only is a traditional tonic food, but also has a high nutritional value and variety of pharmacological effects.The objectives of this study are to prepare hydrolysates from Mytilus coruscus by enzyme hydrolysis, separate and isolate the activity peptides from Mytilus coruscus hydrolysates (MCHs) by a series of separation and analysis technologies, including ultra-filtration, gel chromatography and reversed-phase high performance liquid chromatography, and identify the active peptides sequences by mass spectrum technology. The antihypertensive effect is evaluated in vitro. The work will be a base for development of antihypertensive functional food production and explore the potential of Mytilus coruscus as a functional Ingredient for value-added foods. The major results obtained in this study are as follows:Mytilus coruscus protein was hydrolyzed using five different proteases. The results showed that Alcalase 2.4L had the best hydrolyzing capacity, the higher protein recovery and the stronger ACE (AngiotensinⅠ-coverting Enzyme) inhibitory activity. Response surface methodology (RSM) was applied to optimize the hydrolysis conditions using Alcalase 2.4L. The optimum values for enzyme/substrate ratio, pH and temperature were found to be 2.18%,8.8 and 55℃respectively. Under those conditions, the ACE inhibitory rate was 83.12%(1.Omg/mL).The IC50 of the fraction MCP separated by ultra-filtration was 0.308 mg/mL. Then Sephadex G-10 gel filtration chromatography was used to separate MCP, eluted by H2O. Consequently, three fractions, MCP-1, MCP-2 and MCP-3 were obtained. In Anti-hypertension test, MCP-3 showed best ACE inhibition effects, with its IC50 value of 0.163 mg/mL, which retained activity under various temperature and pH treatments, and showed resistance to in vitro digestion by gastrointestinal proteases. Lineweaver-Burk plots suggested that the MCP-3 acted as competitive inhibitors against ACE.MCP-3 was further separated by semi-preparative RP-HPLC, and 5 fractions were obtained, among which fractions MCP-3-4 showed the highest ACE inhibition activity. RP-HPLC analysis of MCP-3-4 suggested it was pure. Amino acid sequence of the pure peptide was identified with LC-MS/MS, its structure was Glu-Phe.