Study On The Purification Of Antioxidant Peptide From Pacific Saury Protein And Its Antifatigue Activities

In our study, antioxidant peptides were prepared from pacific saury protein through controlled enzymatic technology. Chemical models in vitro and animal models in vivo were used to evaluate their antioxidant activities. Animal models were also used to evaluate their antifatigue activities. Several isolation and separation technologies were done on pacific saury peptide and RP-HPLC-ESI-MS/MS was used to identify the amino acid sequence of the peptide. The effects of the processing and storage conditions on the antioxidant peptides were also studied. Moreover, the formula of beverage with antifatigue function was also studied.The composition of pacific saury shows it has high content of protein(18.18%), and the fish is rich in amino acids. The composition of amino acids is reasonable, which contains essential amino acids, flavor amino acids, the antioxidant amino acids and branched chain amino acid and so on. It makes pacific saury a good substrate for active peptide. When compared to flavourzyme, neutrase, Alcalase, and papain, it was proved that pancreatin produced antioxidant peptides most efficiently because the resulting hydrolysate showed the highest activity. Then the influences of enzyme dosage, hydrolysis time and water-material ratio for enzymatic hydrolysis by pancreatin on pacific saury protein were inspected. Response surface methodology was used to optimize the enzymolysis technology parameters for antioxidant peptides from pacific saury. And the results showed that that the optimal enzymolysis conditions were as follows: enzyme dosage 1085 U/g, liquid-to-solid ratio 3:1 and time 8 h, p H 8, temperature 50°C. Using these parameters, the experimental protein recovery ratio was 76.86%, and the oxygen radical absorbance capacity(ORAC) value was 883.40 μmol Trolox equivalent/g, both of which were not significantly different from their predicted values.Three different chemical models in vitro and animal models were used to evaluate the antioxidant activities of pacific saury peptide. Results showed that its scavenging activities for DPPH? radicals were 0.5 fold stronger than those of GSH’s at the same mass concentration. When the mass concentration of loach peptide was 15 and 6 fold of that of GSH, there was no significant difference in their scavenging activities for O2-? radicals and reducing power. pacific saury peptide could improve the activities of the endogenous cellular antioxidant enzymes in mice. It could increase the activities of SOD and CAT by 4.97%-15.11% and 20.89%-34.74%, but the activities of GSH-Px die not increased significantly when compared to control group. In a word, these indicate that pacific saury peptides have potential antioxidant activities in vitro and in vivo.Animal models were used to evaluate the antifatigue effects of pacific saury peptide. Resultsshowed that, compared with the control group, pacific saury peptide could significantly increase the swimming time of the mice by 8.67%-34.69%. It could delay or decrease the consumption of liver glycogen in mice and increase the content of liver glycogen by 1.09-1.17 fold. It could improve the elimination of lactic acid and blood urea nitrogen and decrease the content of lactic acid and blood urea nitrogen by 8.60%-14.33% and 6.79%-22.95%. It could improve the activities of the creatine kinase by 29.96%-36.24%. Therefore, pacific saury peptide could improve the mice’s endurance to exercise and has antifatigue effects.Pacific saury peptide was isolated and purified by macroporous resin adsorption, gel filtration chromatography and RP-HPLC. The purified peptide showed 9.9 fold and 4.18 fold higher ORAC value compared with the crude pacific saury peptide, and 4.47 fold and 1.17 stronger than that of GSH’s.The two purified antioxidant peptide was identified as Ser-Gly-Ala-Ala-Met(435Da) and His-Gly-Glu-Glu(470Da) using RP-HPLC connected on-line to ESI-MS/MS. The purified peptide is the first tetrapeptide to be found which has significant antioxidant activities.The effects of temperature, p H, drying, metal ions, storing means and sterilization means on the DPPH? radical scavenging activities and ORAC value of pacific saury peptide were investigated to determine the stability of the antioxidant peptides. Results showed that pacific saury peptide had good stabilities of temperature. The peptide was stable in acid and neutral conditions, but lost its antioxidant activities quickly in alkaline condition. The Zn2+ and Cu2+ at an increasing concentration significantly(P<0.05) decreased the antioxidant activity of pacific saury peptide, while Mg2+, Ca2+and K+ ions(P>0.05) had no effect. Lyophilization and spray drying had no significant difference on its antioxidant activity(P>0.05). The influence of pasteurizer sterile process on antioxidant ability is less than that of boiling water, and the influence of autoclaves sterile process is the greatest. Pacific saury peptide can keep more than 90% of antioxidant activity when peptide was packed in liquid refrigerant packing and solid vacuum packing. However, solid vacuum packing is more advantageous than the liquid refrigerant packing. Moreover, the formula for antifatigue beverage was studied and the formula as follows: 40%(v/v) of diluted pacific saury protein hydrolysates, 40%(v/v) of the extraction of both candied date and medlar, 0.7%(v/v) blueberry fragrant semen, 0.7%(v/v) the mixture of sucralose and honey, 0.7%(g/v) of sodium chloride and the p H was adjusted to 5.5 with the lactic acid. Suitable amount of water was finally added.