Study On The Purification Of Antioxidant Peptide From Loach Protein And Its Antifatigue And Anticancer Activities

In our study, antioxidant peptides were prepared from loach 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, and cellular models were used to evaluate their anti-cancer activities. Several isolation and separation technologies were done on loach 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 and the enzymes in a simulated gastrointestinal digestion on the antioxidant peptides were also studied.Loach is a kind of food material whose protein content is high (17.4%) and fat content is low, which makes it very appropriate for enzymatic hydrolysis to obtain bioactive peptides. The antioxidant activities of loach protein hydrolysates are highly related to the enzymes and the degree of hydrolysis (DH). When the DH is increased to 23%, the hydrolysates prepared with papain showed high antioxidant activities, with the scavenging activities of hydroxyl, DPPH?, ABTS?+ radicals and the reducing powers were 56.08%, 95.48%, 2.8033 mM Trolox and 1.458, respectively.Five different enzymes (including Alcalase2.4L, Protamex, Papain, PTN6.0S, Flavorzyme 500MG) were used to hydrolyze loach portein. Results showed that appropriate hydrolysis on the loach protein could improve its nutritional values. Considering the protein recovering ratios, peptide yield, DH, the scavenging activities for DPPH?,?OH and O2-?, papain was the best enzyme for the hydrolysis of loach protein. Response Surface Methodology (RSM) was used to optimize the processing conditions of hydrolysis, the results were: E/S = 3‰(the activity of papain is 600,000 U/g), temperature = 55℃, time = 4.5h, the actual EC50 value of the hydroxyl radical scavenging activity of the hydrolysate was 17.00±0.54 mg/mL, without significant difference with the predicted value 16.76mg/mL (P>0.05).Six different chemical models in vitro were used to evaluate the antioxidant activities of loach peptide. Results showed that its scavenging activities for hydroxyl, DPPH? radicals and metal chelating activities were 0.4, 0.46, 0.78 and 3.11 fold stronger than those of GSH's at the same molecular concentration (the average molecular weight of loach peptide is about five fold of that of GSH). When the mass concentration of loach peptide was 3.5 fold of that of GSH, there was no significant difference in their reducing power. Loach peptide could improve the activities of the endogenous cellular antioxidant enzymes in mice. It could increase the activities of SOD, CAT and GSH-Px by 1.7% - 8.5%, 36% - 49% and 13% - 27%, respectively. These indicate that loach peptides have potential antioxidant activities.Animal models were used to evaluate the antifatigue effects of loach peptide. Results showed that, compared with the control group, loach peptide could significantly increase the swimming time of NIH mice by 20-28%, could delay or decrease the consumption of glucose and liver glycogen in mice, could improve the elimination of lactic acid and blood urea nitrogen and improve the activities of the endogenous cellular antioxidant enzymes in mice. Therefore, loach peptide could improve the mice's endurance to exercise and has antifatigue effects. Results also showed that the antioxidant activities of loach peptide had good relationship with its antifatigue effect.Cellular models were used to evaluate the anti-cancer activities of loach peptide. Results showed that loach peptide had significant inhibition effects on the growth of HepG2, Caco2 and MCF-7 cells, with the EC50 values of 16.69 mg/mL, 9.92 mg/mL and 15.91mg/mL, respectively. The inhibition effect was increased when the concention of peptide was increased.Loach peptide was isolated and purified by ultrafiltration and consecutive chromatographic methods including ion-exchange chromatography, gel filtration chromatography and a two-step reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptide was identified as Pro-Ser-Tyr-Val (464.2 Da) using RP-HPLC connected on-line to electrospray ionization (ESI) mass spectrometer. The purified peptide showed a 9.14-fold higher scavenging activity for hydroxyl radical compared with the crude loach peptide, and 11.6 fold stronger than that of GSH's. The purified peptide is the first tetrapeptide to be found which has significant antioxidant activities (the EC50 value of scavenging activity for hydroxyl radical is 1.86±0.19mg/mL).The effects of temperature, pH, light, drying, metal ions and food ingredients on the DPPH? and ABTS?+ radical scavenging activities of loach peptide were investigated to determine the stability of the antioxidant peptides. Results showed that loach peptide had good stabilities of temperature but bad stabilities of light. The peptide was stable in acid and neutral conditions, but lost its antioxidant activities quickly in alkaline condition. The Cu2+ and Zn2+ at an increasing concentration significantly decreased the antioxidant activity of loach peptide, while Mg2+, Ca2+ and K+ ions (P>0.05) had no effect. Sugar improved the antioxidant activity of loach peptide in the order of glucose >sucrose >trehalose. Lyophilization and spray drying had no significant difference on its antioxidant activity.A two-stage in vitro digestion model system (a pepsin treatment for 2 h followed by a pancreatin treatment for 2 h, both at 37°C) was used to simulate the process of human gastrointestinal (GI) digestion to determine the changes in antioxidant activities of loach peptide. Results showed that pepsin hydrolyzed the peptide into smaller fractions and pancreatin hydrolyzed the peptide completely and released more free amino acids. The final GI digests contained 38.1% free amino acids, with short chain peptides (<500 Da) making up the rest of the biomass, which confirmed that peptide was absorbed in the forms of dipeptide, tripeptide and tetrapeptide, not totally amino acid. Enzymatic breakdown of the GI digests increased their hydroxyl (12% increase), ABTS?+ (5% increase) radical scavenging activity, the reducing power (77% increase) and the chelating ability of Cu2+ (12% increase), compared to the blank. The results showed that the digestion by gastrointestinal proteases can be used to produce antioxidant peptides, with the advantage that the peptides formed will resist physiological digestion in GI tract.