Preparation Of ACE Inhibitory Peptides From Mussel Protein And Research On Its Quantitative Structure-Activity Relationship

Mytilus edulis, a species of shellfish, is good resource of protein and favorite to Chinese. It belongs to phylum Mollusca, class Lanellibranchia, order Anisomyaria, tribe Mytilacea, family Mytidea. China has become the biggest producer of mussel in the world and its total annual yield is over 600000 tons. However, due to limitations in further processing technology,90% of them are traded in live, fresh, frozen, and dried forms. Therefore, mussel has a low commercial value and the waste of mussel protein is rather serious in China. In this thesis, protein composition, nutritional value and hydrolysis characteristic of mussel protein were evaluated. In addition, the preparation of mussel protein isolate (MEPI) and the production of angiotensinⅠ-converting enzyme (ACE) inhibitory peptides from MEPI by enzymatic hydrolysis were also studied. The purpose of this work was to provide some useful academic information and novel effective approach for deep processing of mussel protein, and to develop some high value-added products for human consumption.The chemical composition of the mussel powder was analyzed as follows:5.02% moisture, 13.09% ash,7.04% fat,48.44% protein and 23.75% polysaccharide. The results of fatty acids determination with GC and minerals analysis with ICP-MS showed that PUFA accounted for 31.09% of total fatty acids amounts and the contents of EPA and DHA were highest (12.21% and 7.18%, respectively). Palmitic acid was the major saturated fatty, and palmitoleic acid was the major monounsaturated fatty acid. Their contents were determined as 29.95% and 10.07%, respectively. Also, the mussel was an excellent source of minerals, such as Ca, Mg, Al, Fe, Zn and Se.The effect of four solvents on the defatting of mussel powder was studied. The results showed that 95% ethanol was the most suitable solvent. Four protein fractions (albumin, globulin, prolamine and glutelin) from defatted mussel powder (mussel protein) were fractionated by using the method of Osborne and then characterized by amino acid analysis and SDS-PAGE. Glutelin was the major fraction (40.78%) extracted, followed by albumin (24.59%), prolamine(6.88%), and globulin(1.62%). These protein fractions showed an excellent balance of amino acid, with a relatively high level of Glu, Asp, Lys and Ser. All the estimated mutritional quality parameters based on amino acid composition showed that mussel protein had good nutritional quality (E/T> 36%). Glutelin possessed the highest AAS and PER value. However, prolamine possessed the highest BV, followed by glutelin. Thus, considering both the yield and the quality parameters, glutelin was thought to possess the highest nutritional values in mussel protein.Hydrolysis of the mussel protein was investigated in the presence of various proteolysis enzymes. Alcalase was found to be the optimal protease with the highest hydrolysis degree (DH) (20.68% for 4h). The protein recovery (PR) and the ACE inhibitory activity of various hydrolysates of mussel protein were also compared, the hydrolysates obtained by the treatment with alcalse possessed the highest PR (85.77%) and ACE inhibitory activity, having a IC50 value of 66.34μg/mL. Moreover, the relative molecular weights of these hydrolysates were rather low, especially the hydrolysates obtained by alcalse were mainly distributed in the range of 200-1000 Da (54.36%).The relative molecular weight distribution and ACE inhibitory activity of four protein fractions hydrolysates with alcalse were studied. The result showed that glutelin was major fraction, which contributed more on ACE inhibitory activity of mussel protein. According to the composition feature of protein fractions and their contribution to the ACE inhibitory activity, the alkaline extraction-isoelectric precipitation technology was employed to prepare MEPI, and the parameters were optimized. Under the optimum conditions, i.e. solid-to-liquid ratio,1:30; pH,11.5; temperature,35℃; time,2.5h; precipation pH,3.5, the protein extraction ratio was 81.74%, and the protein content of the product was 74.32%.Taking the DH and the inverse of IC50 as responses, the hydrolysis parameters using alcalse were optimized with RSM as follows:enzyme to substrate ratio 1.73%, pH 9.2 and temperature 56.8℃. Under these conditions, MEPI hydrolysate was prepared with a DH of 25.4% and an IC50 value of 35.21μg/mL. Also, the stability of MEPI hydrolysate was examined. The results showed that the influence of food processing, such as heat and adjustment of pH on its ACE inhibitory activity is not significant, and MEPI hydrolysate showed resistance to in vitro digestion by gastrointestinal proteases. Moreover, MEPI hydrolysate was found to be competitive ACE inhibitors.Effects of DH on chemical composition, relative molecular weight distribution, amino acids composition and ACE inhibitory activity of MEPI hydrolysate were also studied. With the increase of DH, protein content increased slowly, the contents of hydrophobic amino acids, aromatic amino acids and branched-chain amino acids increased significantly, while the content of acidic amino acids decreased markedly. Also, the contents of fraction in the range of 200-1000 Da increased obviously at first, and then become a stable level. All above results can explain the effects of DH on ACE inhibitory activity of MEPI hydrolysate to some extent. The ACE inhibitory activity of MEPI hydrolysate significantly increased with the increase of DH, then increased slowly, and finally decreased slowly. Dynamic characteristics results of hydrolysis process showed that the kinetics fitting equations for DH and ACE inhibitory rate were as follows:DH=-4.24195+5.43497 In (T-2.73073) and I(%)-47.68838+0.91943T-0.00559T2 +1.30899×10-5 T3-1.06897X 10-8T4.Using ACE inhibitory activity as an index, MEPI hydrolysate was purified firstly by ultrafiltration (UF). The influence of UF conditions on flux and the molecular weight cutoff (MWCO) of membrane on ACE inhibitory activity were studied. The results showed that the ACE inhibitory peptides in MEPI hydrolysates could be effectively separated and enriched by membrane (MWCO:10 KDa) under suitable conditions (sample concentration of 15 g/L, operation pressure of 20 psi and temperature of 25℃). Three kinds of ACE inhibitory peptides with strong activities were isolated from MEPI hydrolysates by sequential Sephadex G-10, LH-20 and two-step RP-HPLC, and their amino acid sequences were identified by UPLC-MS as VW, LGW and MVWT with IC50 values of 1.7μM 30.0μM and 143.3μM respectively. Stability tests showed that these peptides could not be hydrolyzed in the presence of ACE, indicating that three ACE inhibitory peptides could be regarded as the competitive inhibitors, which means that three peptides could have antihypertensive effect in vivo.In order to study the quantitative structure-activity relationships of angiotensin I-converting enzyme (ACE) inhibitory peptides, a database including 182 dipeptides,237 tripeptides,94 tetrapeptides,94 pentapeptides and 88 hexapeptides was constructed on the basis of published literatures.18 models were computed using partial least squares regression (PLS), support vector machine (SVM) and principal component analysis (PCA)-SVM based on the VHSE descriptors of 20 coded amino acids and further validated by cross-validation. The results showed that SVM and PCA-SVM achieved better performance in QSAR than PLS for ACE inhibitory tetrapeptides, pentapeptides and hexapeptides. While SVM and PCA-SVM achieved equivalent predication results with PLS and better fitness than PLS for tripeptides. Also, equivalent results were obtained in comparision with three QSAR models for ACE inhibitory dipeptides. In addition, variable importance in the projection (VIP) analysis of individual amino acid residues at each position revealed that the C-terminal amino acid residues of dipeptides, the middle amino acid residues of tripeptides, the N-terminal tripeptide residues in tetrapeptides, the C-terminal tripeptide residues in pentapeptides and the C-terminal tetrapeptide residues in hexapeptides were more important to their ACE-inhibitory activity than the other residues in the peptide sequences.The models were validated with ACE inhibitory peptides from MEPI hydrolysates. The results showed that the QSAR models constructed in this paper could predicate activity of ACE inhibitory peptides exactly. Especially, the predicated results of SVM and PCA-SVM models were very well.