The Preparation Of ACE Inhibitory Peptides And Antioxidative Peptides From Oyster By Immobilized Enzyme

It has become one of the most effective means to study enzymatic preparation ofmarine bioactive peptides, in order to prevent and treat diseases such as hypertensionand free radicals oxidative damage by the method of dietotherapy, so as to meet therequirements of the human healthy diet and medical safety. Oyster is an importantsource to develop marine bioactive peptides as it is rich in nutrients andphamaceutical value. Furthermore, it has been widely applied and potentiallydeveloped in the industrialized production of marine bioactive peptides, because ofthe advantages of combination with immobilized enzyme and tangential flowultrafiltration membrane system in terms of operating structure and separation effect.The technology conditions of the preparation of oysters ACE inhibitory peptidesand antioxidant peptides have been studied by immobilized enzyme and tangentialflow ultrafiltration membrane system, then the active peptides were further separatedand purified by gel filtration chromatography and high performance liquidchromatography.The structure characteristics of polypeptide and molecular weightdistribution were analysed. The theoretical support was provided for the preparationtechnology and structure-activity relationship of marine active peptides, while,reference basis was provided not only for industrial application of immobilizedenzymes and ultrafiltration membrane system, but also for industrialized productionof marine bioactive peptide products. The main results were shown as follows:(1) The optimal conditions of oyster ACE inhibitory peptides digested withtrypsin are the protein concentration50mg/mL, temperature40℃, pH value8.0,enzyme concentration5000U/g, enzymolysis time8h, by detecting peptide yield anddegree of hydrolysis, ACE inhibitory rate was36%-37%, The optimal conditions ofoyster antioxidant peptides digested with trypsin are the protein concentration50mg/mL, temperature45℃, pH7.5, enzyme concentration6000U/g, enzymolysistime7.5h, DPPH free radical clearance rate was up to83.22%, the hydroxyl freeradical clearance rate up to95.69%. Variance analysis results indicate thatsignificant impact has been shown by the temperature for free enzyme preparationoyster ACE inhibitory peptides and antioxidant peptides, followed by the pH value,enzyme dosage and time.(2) The immobilized carrier material and the preparation process ofimmobilized enzyme have been studied, sodium alginate and chitosan gel werechoosen to produce the carrier for immobilized enzyme, the immobilized trypsin wasproduced by blending gel with the method of embedding and adsorption, theoptimum conditions are the concentration of glutaraldehyde0.4%, CaCl24.5%, theamount of enzyme1:15, crosslinking14h, ratio vitality of enzyme up to128.39U/g. Moreover, the stability, mechanical strength and efficiency have been improved.(3) The optimum conditions of ACE inhibitory peptides and antioxidantpeptides digested by immobilized trypsin have been studied. For the preparation ofoyster ACE inhibitory peptides, the optimum conditions are the protein concentration50mg/mL, temperature50℃, pH value9.0, enzyme concentration5g/g, enzymolysistime3.5h, the rate of ACE inhibitory is38.82%, immobilized enzyme can be usedfive times. For antioxidant peptides, the optimum conditions are pH value9.0,45℃,enzyme concentration3g/g, enzymolysis time1.5h, the rate of DPPH free radicalclearance reached93.68%, immobilized enzyme can be reused over seven times. itcan obtain higher activity of ACE inhibitory peptides and antioxidant peptidescompared with the free enzyme enzymolysis effect.(4) The molecular weight distribution and hydrophobicity of ACE inhibitorypeptides and antioxidant peptides were analysed by gel chromatography andRP-HPLC after separated by tangential flow ultrafiltration membrane system. Therelationship among the different molecular weight percolate, antioxidant activity,ACE inhibitory activity and polypeptide production rate was studied, it has beenshown the activity of ACE inhibitory peptides and antioxidant peptides are theoutcome of combined action of different molecular weight peptides. The activity ofantioxidant peptides are concentrated in the more than10kDa and less than3kDa, thepeptides which molecular weight are more than10kDa show the highest productionrate of peptides and hydroxy clearance rate, the peptides with molecular weight lessthan3kDa show secondly production rate of peptides and the highest DPPH freeradical clearance.The activity of ACE inhibitory peptides are mainly concentrated inless than1kDa, and show the highest production rate.Immobilized enzyme and tangential flow ultrafiltration membrane system couldbe used to prepare for oyster ACE inhibitory peptides and antioxidant peptidesthrough the verification and enlargementl test on the optimum conditions, thesepeptides of certain molecular weight have higher activity and more stable properties,all these can be applied for deep processing of seafood and industrial production ofmarine active peptide products.