Preparation Of ACE Inhibitory Pepdities From Hydrolysis Of Sesame Meal Protein By Immobilized Enzyme

To prepare ACE inhibitory peptides from sesame meal proteinby enzymatic hydrolysis in this research, the suitable tool enzyme was selected from aseries of commercial proteases, the process of preparation of magnetic chitosanmicrospheres immobilized Alkalase was researched, the optimum conditions ofpreparation of ACE inhibitory peptides by immobilized Alcalase had been exploredand testified by response surface methodology, the hydrolysate had been coarselypurified by ulterfiltration and gel filtration chromatography.The suitable protease which was used to hydrolyze the sesame mill proteinextracted from sesame cake had been selected from Papain, Trypsin, Bromelain,Alcalase, Nuetrase and Alkaline protease2709by means of the performances ofdegrees of hydrolysis, yield of peptide and ACE inhibitory activity under theirsuitable conditions. According to the evaluation parameters, Alcalase was the mostsuitable tool enzyme for preparing sesame peptide. Moreover, the hydrolysategenerated by Alcalase showed the highest ACE inhibitory activity, whose inhibitionrate was (67.42±1.75)%at the level of1mg/mL.Taking the magnetic chitosan microspheres (M-CS) as the carrier, the effects ofenzyme-to-carrier ratio, pH, glutaral concentration, reaction time on the activity andactivity recovery of immobilized Alcalase were investigated. The characterizationsand microstructure of immobilized enzyme were also studied. The results showed:Under the fellowing conditions, enzyme-to-carrier ratio of112000u/g, pH8.5,glutaral concentration of8%(V/V), reaction time of11h, The maximum activity ofimmobilized Alcalase reached86779u/g, and the activity recovery of immobilizedAlcalase was77.48%. Optimal reaction pHs of free and immobilized Alcalase were11and10.5respectively. both The optimal reaction temperatures for free andimmobilized Alcalase were60℃. The immobilized Alcalase showed higher thermaland pH stabilities than free Alcalase, The activity of immobilized Alcalase reused5times remained80.89%. According to Kmvalue, immobilized Alcalase showedstronger affinity than free Alcalase. Transmission electron microscope illuminated thatFe3O4and chitosan microspheres were all spherical nanopaticles with smooth surface;more enzyme could be bound due to high specific area. FT-IR revealed that Fe3O4was well wrapped up in M-CS, Vibrating sample magnetometer indicated thatimmobilized Alcalase had a good magnetic response.To obtain the optimum conditions of Hydrolyzing sesame meal protein usingmagnetic chitosan microsphere immobilized Alcalase, the single factor experimentand response surface were employed. Using Design-Expert8.05software analyzedthe experimental data to get the mathematical model, and the optimum conditionswere obtained: enzyme-to-protein ratio of7105.03u/g, temperature of59℃, pH10.96, hydrolysis time of4.3h, yield of peptide and ACE inhibition rate were86.35%and65.5%under these conditions.Finally, using ultrafiltration and Sephadex G-15purified the peptide. Afterultrafiltration peptide content was73.85%, slightly higher than that of originalhydrolysate. After desalination by Sephadex G-15gel chromatography, Comparingwith samples without gel filtration, the IC50value of recovery components wassignificantly decreased. The componentⅡ which owned the lowest IC50value of1.624mg/mL, showed the highest ACE inhibitory activity. After gel filtrationchromatography, peptide content increased significantly than those beforedesalination, whose desalination rate was all above80%and peptide recovery ratewas63.85%. Then, the purification effect was good, the method could be used topurify ACE inhibitory peptides prepared from sesame cake.