Controllable Hydrolization Of Silkworm Pupae Protein For Preparation Of ACE Inhibitory Peptides And Research On Its QSAR

The hypertension is one of the most common cardiovascular diseases, it can cause the damage of cerebra, heart blood vessel and kidney, and it is also the most important reason of causing apoplexy, heart failure and coronary. Treatment and prevention hypertension was helpful to improve humanity's health and lengthen life. The functional factors of anti-hypertension in natural food materials were ideal choices for non-druggery treat hypertension, for the functional factors with no side effects. Peptides hydrolyzed from food protein by proteases were with different bioactivities, these peptides could be released from its former proteins during digesting and food processing.Silkworm pupae protein was with high nutritional value, and the output was abundant each year in China, but the thoroughly utilizing approach of silkworm protein was still faultiness, and its add-ons value were also not ideal. In this study, by the method of controllable enzymatic hydrolyzation, we pay our attention on the preparation of Angiotensin I-Converting enzyme inhibitory peptides from silkworm pupae protein and then study on the peptides Quantitative Structure-Activity Relationship (QSAR).Firstly, we set up a novel fast and convenient RP-HPLC method for Angiotensin I-Converting enzyme inhibitory peptides assay. And, we also optimized the super quantity of substrate HHL with the enzyme of ACE. The results was, 5mM HHL 6μL with 0.1U/mL ACE 10μL. Following assayed the hydrolysates inhibitory activity on Angiotensin I-Converting enzyme from silkworm protein, the results showed this RP-HPLC method was fast, convinent and precise.We utilized several kinds of protease to hydrolyze silkworm pupae protein, and got the hydrolysates by acidic protease (Aspergillus usamii NO. 537) with the highest inhibitory rate on ACE, and the hydrolysate was also with lower bitterness and super colour and stability. So, we took acidic protease as the preferred enzyme for hydrolyzing silkworm pupae protein.And after one factor experiment, fractional factorial design (FFD), and center composite (CCD) design methods, the optimal hydrolyzing conditions were as the follows: temperature was 35°C, the hydrolysis time was 4.92 hour, the E/S (w/w) was 1% (equal to 500U/g substrate), the substrate concentration (water: silkworm pupae protein) was 14.94 (w/v), and the suitable pH was 2.18. The ACE inhibition was improved from IC₅₀ 2.5 mg/mL of one-factor design to 1.38mg/mL.According to the different dissolve ability of protein components, we distilled four kinds of protein components by the method of Osborne, the results showed that the content characteristics of protein components was albumin > glutelin > prolamine > globulin. Albumin was the predominant component in silkworm pupae protein. And the composing analysis of amino acids showed, the necessary amino acids were 44.48% to total amino acids, and the necessary to un-necessary amino acids was 80.43%, these results was higher than the standard of WHO/FAO that necessary amino acid was account for 40% to total amino acids and the necessary to unnecessary amino acids was 60%.By the center composite design (CCD) methods, we got the optimal distilling conditions of albumin was, water/silkworm material was 6.8, distilling time was 5hour. The distilling rate of pure protein was increased from 17.39g/100g silkworm material to 26.41g/100g, the increase rate was 34%, and total protein distilling rate reached to 61.83%.Make clear the detail components of the protein which with potential anti-hypertension activity, and make sure which kind of component contribute more on the ACE inhibitory activity, were with great value for the protein further application. By studying on the molecular weight distribute characteristics of components in silkworm pupae protein and the relationships between the hydrolyzing characteristics of different protein components in silkworm pupae protein and Angiotensin I-Converting enzyme inhibitory activity (ACEIP), we got the hydrolysates of albumin with the highest ACE inhibitory rate and degree of hydrolysis (DH), it showed albumin was proned to be hydrolyzed by acidic protease ( Aspergillus usamii NO. 537), and the inhibitory activity of the hydrolysates peptides were very high.We separated the hydrolysates of albumin with ultrafiltration, Sephadex G-15 filtration, and the Ettan MDLC nanoflow/capillary LC system separation, and then by nanoscale reversed-phase LC-MS/MS analysis, we got a ACE inhibitory peptide, the sequence was "SEPTVF", and we synthesized this peptide by chemical method, assayed its inhibitory activity on ACE, the IC₅₀ was 324μM.Based on the published literature, we collected 20 compounds as training set to generate pharmacophore models for ACE inhibitory peptides (Hypo 1). The peptide which we got (ser-glu-pro-thr-val-phe) from silkworm pupae protein was docking onto the best hypothesis Hypo 1 to determine which amino acid compositions best adhere to the requirements of the pharmacophore model, the result showed the best pharmacophore model maps to later three amino acids " thr-val-phe".By study on the changing of artery shrink pression of spontaneously hypertensive rat (SHR) before and after they ate the hydrolysate of albumin, we got the hydrolysates with the effect of decreasing the artery shrink pression of SHR -22.7mmHg at the concentration of 80mg/kg for 4 weeks, and with no side effects.The results of this paper provided basic research data for thoroughly application silkworm pupae protein and utilizing it to produce high economical add-on antihypertensive peptides products.