Preparation Of Angiotensin I-converting Enzyme Inhibitory Peptides From Enzymatic Hydrolysates Of Shrimp Byproduct

A large number of byproducts were produced during shrimp processing. In order toimprove the utilization of shrimp byproducts, in this thesis, enzyme hydrolysis methodwas chosen to prepare angiotensin converting enzyme (ACE) inhibitory peptides.First, sixty peptides derived from food protein resources, which were reported withACE inhibitory activities, were selected to analyze the main factors affecting ACEinhibitory activity. Amino acids’hydrophobic property was analyzed and the twentyamino acids’ frequency of occurrence at C-terminal and N-terminal was calculated. Itwas found that hydrophobic amino acid residues at C-terminal were positively correlatedwith ACE inhibitory activity. And amino acid residues at C-terminal were mainlyaromatic amino acid or proline. Then, according to the cleavage site of common protease,α-chymotrypsin and proline protease were chosen to produce ACE inhibitory peptides.Secondly, the protein content and amino acid composition were analyzed. Theprotein content of shrimp byproduct was59.94%. The proportion of aromatic amino acidand proline was14.24%. The hydrolysis time was optimized according to the hydrolysisdegree (HD) for α-chymotrypsin, and both HD and ACE-inhibitory activity for prolineprotease, the results showed that the optimal hydrolysis time for both enzymes were4hours. The half maximal inhibitory concentration (IC50) for ACE inhibition ofhydrolysate was1.645mg protein/mL.Then, the hydrolysate was separated into fraction1(0~500Da) and fraction2(500~1000Da) by molecular weight cut-off (MWCO) membrane. The IC50value offraction1and fraction2were0.333mg peptide/mL and1.320mg peptide/mL, respectively.Fraction1and fraction2were further separated by gel filtration chromatography.Fraction1was divided into five peaks and peak F1-G5showed49.24%ACE inhibitoryactivity, which was the highest among the5peaks; Fraction2was also divided into five peaks and peak F2-G2showed39.07%ACE inhibitory activity. Then peak F1-G5andpeak F2-G2were concentrated for further separation by RP-HPLC. Peak F1-G5wasdivided into six peaks. Peak F1-G5-H1and peak F1-G5-H4had higher ACE inhibitoryactivity than the other4peaks; Peak F2-G2was divided into four peaks and only peakF2-G2-H1and peak F2-G2-H2had ACE inhibitory activity. PeakF1-G5-H1, peakF1-G5-H4, peak F2-G2-H1, and peak F2-G2-H2were then concentrated fortime-of-flight mass spectrometry analysis.Twenty five peptides were found in peak F1-G5-H1, F1-G5-H4, F2-G2-H1andF2-G2-H2by time-of-flight mass spectrometry analysis. The C-terminal amino acids oftwenty two out of the twenty five peptides were proline or aromatic amino acid, whichwas corresponding to QSAR results.Finally, peak F1-G5-H1, F1-G5-H4, F2-G2-H1, and F2-G2-H2were hydrolyzed withgastrointestinal digestive enzymes. The ACE inhibitory activity of F1-G5-H1, F1-G5-H4,F2-G2-H1declined after hydrolysis by digestive enzymes. However, the ACE inhibitoryactivity of peak F2-G2-H1increased after hydrolysis.In conclusion, ACE inhibitory peptides with aromatic amino acid or proline atC-terminal could be produced byα-chymotrypsin and proline protease hydrolysis.Through this study, the source of ACE inhibitory peptides and the efficient utilization ofshrimp byproduct have been enriched.