| The present study deals with the preparation of antifatigue biopeptides from grass carp protein. The mechanisms of enzymatic hydrolysis for sarcoplasmic protein, myofibrillar protein and stroma protein were studied. The bioactivities of the peptides were evaluated. Ultrafiltration, ion-exchange chromatography, counter-current chromatography and gel filtration chromagraphy were applied to the separation of peptides. ESI-MS/MS was adopted for the identification of peptides. Moreover, the formula of beverage with antifatigue function was also studied.The protein content of sarcoplasmic protein, myofibrillar protein and stroma protein were 33.65±1.71%, 61.42±2.78% and 3.12±0.87%, respectively. The thermal denaturation temperatures of sarcoplamic protein, myosin, actin, and collagen, as determined by differential scanning calorimetry (DSC), were 59.83 oC, 51.49°C, 76.45°C , and 59.83 oC, respectively.Five proteases (papain, PTN6.0, bromelain, Neutrase1.5MG and Alcalase2.4L) were used to hydrolyze sarcoplasmic protein, myofibrillar protein and stroma protein. Papain, PTN6.0 and Alcalase2.4L were the most suitable enzymes for preparing peptides from the above proteins, respectively. Hydrolysates from stroma protein showed the highest hydroxyl radical scavenging ability, while hydrolysates from sarcoplasmic protein exhibited the lowest activity. The three proteases were applied to hydrolyze grass carp protein and hydrolytic conditions were optimized by response surface methodology (RSM). The minimum IC50 value (155.89±11.21μg/mL) which signified the maximum hydroxyl radical scavenging ability was obtained.The antioxidant activities of grass carp protein hdyrolysates (GCPHs) were evaluated by in vitro chemical reactions. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of GSPHs was as high as that of glutathione (GSH). The reductive potential and superoxide anion radical scavenging activity of GSPHs was a little bit less than that of GSH, while the ferrous ion chelating activity of GSPHs was much higher than that of GSH. GSPHs demonsatrated inhibition activities on lipid peroxidation in rat hepatocytes and also displayed activities on erythrocyte hemolysis induced by hydrogen peroxide solution.The evaluation of anti-fatigue function of GCPHs was studied by swimming endurance test and the analysis of biochemistry indexes. The result showed that GCPHs could significantly increase the swimming time of mice, blood testosterone and blood creatine kinase as well as decrease the content of blood urea nitrogen and blood lactic acid. The antifatigue activity of GCPHs was considered to be more related to its ability to balance the endocrine system of mice.Ultrafiltration and chromatography techniques were performed to purify GCPHs. It was found that basic peptides had greater capacity to the antioxidant activity of GCPHs than acidic peptides or neutral peptides. Hydrophobic peptides contributed more to the antioxidant activity of GCPHs than hydrophilic peptides. Also, the peptides with molecular weight between 1,000 Da and 3,000 Da was more important to the activity of GCPHs. The purified peptide was identified as Pro-Ser-Lys-Tyr-Glu-Pro-Phe-Val (966.3 Da) using RP-HPLC connected on-line to an electrospray ionization mass spectrometry.The antioxidant activity of GCPHs was more stable in neutral/acidic conditions than in basic conditions. Pasteurization has little effect on the activity of GCPHs, while sterilization under (121 oC, 0.1 MPa, 15min) caused greast loss of its activity. GCPHs could still keep 71.87±3.16% of its original activity even after the in vitro simulated gastrointestinal digestion. The formula for antifatigue beverage was as follows: 40% (v/v) of GCPHs,5% (v/v) of the extraction of ginger,20% (v/v) of the extraction of both chrysanthemum and medlar,3% (w/v) sucrose, 3% (w/v) glucose,18 mmol/L sodium chloride and the pH was adjusted to 4.4 with the mixture of sodium citrate, citric acid and lactic acid at a ratio of 1:2:1. Suitable amount of water was finally added。.
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