Preparation,Isolation And Biological Activity Of Peptides From Porcine Cerebral Protein

Biological active peptides have the effects of adjusting human bodys’ physiological functions. As the basic nutrients, biological active peptides have good processing properties as well as high bioavailability. The porcine brain contains rich protein. In recent years, the hydrolysates by enzymolysis from porcine cerebral protein have often been used to treat functional disorder diseases in brain. Researches had found that the biological activities were closely related with its small molecular peptides from cerebral protein hydrolysates. However, based on few study of preparation, isolation, structure identification and bioactivity researches of porcine cerebral peptides, this study was to investigate the preparation and its properties of porcine cerebral hydrolysate peptides by using countercurrent and pulsed ultrasound (UPCHPs). Study the reactions kinetics and thermodynamics of enzymolysis from ultrasonic porcine cerebral proteins (PCP). Study the activities of sedation and hypnosis, improvement of lead-induced learning and memory and antioxidant activities of UPCHPs. Obtain a purified fraction after separation and purification (UPCHPs-F31), identify the structions and investigate the in vitro antioxidant activities of UPCHPs-F31. The research can provide the basis for the researched and applications in the field of food from porcine brains and its polypeptides, which has important theoretical and practical significance. The step experimental procedures and results of the study are given below:(1) To prepare porcine cerebral hydrolysate peptides by using countercurrent and pulsed ultrasound assisted enzymolysis and analyze its properties. Obtain UPCHPs from porcin brain by using countercurrent pulse ultrasound assisted digestion after centrifugation, collection and freeze-drying the supernatant. Porcine cerebral hydrolysate peptides (PCHPs) were obtained from the traditional digestion. The results showed that their peptides yields were 81.5% and 66.7%, peptide contents were 45.12% and 31.44%, molecular weight distributions were 3000-5000 Da and 6000～11000 Da of UPCHPs and PCHPs, respectively. These results showed that countercurrent and pulse ultrasound assisted enzymolysis was better than the traditional enzymolysis. Compared with the traditional enzymolysis, the ultrasound assisted enzymolysis can significantly increase the hydrophobic peptides and reduce the emulsification of peptides, foaming ability and foam stability (P<0.05). They could expand the application of UPCHPs in the field of food.(2) To study the reactions of kinetics, thermodynamics and laws of kinetics PCP enzymolysis by using countercurrent and pulsed ultrasound and Michaelis-Menten equation, first-order kinetic model, Arrhenius law, and transition state theory. Compared with traditional enzymolysis, countercurrent and pulsed ultrasound significantly increased the affinity between enzyme and PCP and the values of reaction rate contant k(P<0.05). The thermodynamics parameters Ea, △H and △S of enzymolysis were significantly reduced (P<0.05). The results indicated countercurrent and pulsed ultrasound could make the enzymolysis proceeding more easily. A reaction model of DH=5.78ln[1+(3.4946e0/s0+0.1219)t] was obtained from PCP by using ultrasound assisted enzymolysis (DH: degree of hydrolysis, e0:the initial concentration of enzyme, s0:the initial substrate concentration). The model can provide theoretical basis for the preparation of porcine cerebral peptides. The ultrasonic porcine cerebral hydrolysate peptides (UPCHPs) were investigated their biological activities.(3) Study the activities of sedation and hypnosis, improvement of lead-induced learning and memory and antioxidant activities of UPCHPs through the animal behavior and chemical activitant in vitro model. Sleeping induced by pentobarbital sodium in mice was used to study sedative-hypnotic activity of UPCHPs. The results indicated that UPCHPs showed inhibitory effect on locomotion activity of mice and synergistic effect on pentobarbital induced sleep in mice, by shortening the sleep latency and prolonging the sleeping time significantly (P<0.05). Besides, peptides promoted a significant protection on nikethamide (NKTM)-induced convulsion by prolonging the death latency and decreasing mortality (P<0.05). The sedative-hypnotic activity of UPCHPs may be related to the GABAergic system. The experimental results of improvement of learning and memory in lead-induced mice showed that 0.025% of lead water significantly reduce the learning memory function of mice. UPCHPs can significantly shorten the incubation period in directional navigation of Morris water maze (P< 0.05). The concentrations of lead were significantly reduced and the concentrations of zinc and iron were significantly increased in lead-induced blood and brain tissue (P< 0.05). The levels of ROS and MDA were significantly reduced (P< 0.05), the levels of GSH and the activities of SOD, GSH-Px, CAT and NOS were significantly increased in brain tissue of lead-induced mice (P<0.05). Different antioxidant chemical models in vitro were used to evaluate the antioxidant activities of UPCHPs. The results showed that the EC50 value to scaveng DPPH·,ABTS+ and hydroxyl radicals were 2.40,0.63 and 10.7 mg/mL, respectively. Metal Fe2+ chelating activity and reducing power were 17.6% and 0.723 of 5 mg/mL UPCHPs, respectively. Therefore, UPCHPs has good antioxidant activity in vitro.(4) To separate, purify and structural identification of UPCHPs. UPCHPs were isolated and purified by ultrafiltration, ion-exchange chromatography and Tricine-SDS-PAGE. The stronger antioxidant activity components (UPCHPs-F31) were obtained through the determination of ABTS radical scavenging ability. UPCHPs-F31 was identified as Ile-Tyr-Arg (IYR) and Glu-Ala-Ser-Ile-Glu-Trp-Ser-Arg-Lys (EASIEWSRK) using MALDI-TOF-TOF-MS combined with TOF-MS/MS. An in vitro digestion model system was used to simulate the process of human gastrointestinal (GI) digestion. The digestive product of UPCHPs-F31 (UPCHPs-F31-GI) was determined the antioxidant activity of ABTS free radical scavenging. The results showed that the EC50 in ABTS free radical scavenging ability reduced by 73.5% of UPCHPs-F31-GI. UPCHPs-F31 can be hydrolyzed in body to produce small peptides fragments which had the stronger activitity to scaveng ABTS free radical. To study the antioxidant activity of purified components from porcine cerebral hydrolysate peptides. UPCHPs-F31 exhibited strong antioxidant activities mainly through terminating free radical and the EC50 of scavenging activities for DPPH, ABTS and hydroxyl radicals were 0.80,0.18 and 3.36 mg/mL, respectively. Metal Fe chelating activities and reducing power were 52.1% and 1.504 of 5 mg/mL UPCHPs-F31, respectively.1 mg/mL UPCHPs-F31 has significant inhibitory effects of lipid peroxidation, P-carotene linoleic acid oxidation system, free radicals caused by BSA and plasmid DNA oxidative damage. Through the Cu2+/H2O2 induced rat tissue homogenate oxidative damage system, UPCHPs-F31 significantly inhibited Cu2+/H2O2 induced tissue protein, lipid oxidative damage and improved Cu2+/H2O2 induced antioxidant enzyme activity in rat brains (P<0.05). Therefore, UPCHPs-F31 has good antioxidant activity in vitro.