Preparation Of Antioxidant Peptides From Chinese Soft-shelled Turtle And Its Anti-aging Activity

Chinese soft-shelled turtle is a traditional tonic in China.Chinese traditional medicine proposes that Chinese soft-shelled turtle has the function of prolonging life,previous study has shown that Chinese soft-shelled turtle was rich in nutritional value,but few reseacches have reported on its anti-aging effect.In this study,the antioxidant peptides were prepared by enzymatic hydrolysis using Chinese soft shelled turtle meat protein as raw material,and its anti-aging activity was evaluated by S.cerevisiae.The fraction was purified and its amino acids sequence was further identified,and the anti-aging mechanism was explored by molecular docking.The main research contents and results were as follows:The nutritional value and extraction of Chinese soft shelled turtle meat protein.The contents of protein in meat was 18.61%,and the proportion of essential amino acids in Chinese soft-shelled turtle meat in accordance with FAO/WHO model According to the solubility of protein at different pH,the protein were extracted by alkali solution acid precipitation at pH 5.0 and pH 11.0,respectively.SDS-PAGE showed that the relative molecular weight of the protein was mainly 29-200 ku,and myogenic fibronectin was the main protein.Preparation and physicochemical properties of enzymatic hydrolysates of Chinese soft-shelled turtle protein.The best protease,alcalase,was used to hydrolyze the protein of the Chinese soft-shelled turtle with antioxidant activity?DPPH radical scavenging activity,hydroxyl radical scavenging activity,ferrous ion chelating ability and inhibition of linoleic acid autoxidation?as the main index and DH as the assistant index.The optimal conditions were obtained by single factor and orthogonal experiments:solid-liquid ratio of 1:20,the enzyme addition of 8000 U/g and time of 5 h.The DH of the STPH was 19.90%,and the DPPH radical scavenging activity,hydroxyl radical scavenging activity,ferrous ion chelating ability and inhibition of linoleic acid autoxidation of STPH at 5 mg/mL were 87.89%,10.83%,65.56%,and 63.24%,respectively.The proportion of peptides with relative molecular weight less than 1000 u was 87.69%,and the contents of hydrophobic amino acid,aromatic amino acid,and aromatic amino acid were 36.94%,28.26%,and 8.55%,respectively.Effects of STPH on the chronological lifespan?CLS?of S.cerevisiae.At CLS 6,compared with the blank group,the survival rate of S.cerevisiae treated with low,medium and high dose STPH?1,2,and 4 mg/m L?increased by 1.85%,5.77%,and 10.99%,respectively.At CLS 6,compared with the blank group,the activities of SOD,CAT,and POD of cells in the high dose group increased by 47.4%,35.4%,and 27.2%,respectively,and the level of lipid peroxidation decreased by 44.7%.The STPH can inhibit the production of ROS and improve the morphology of senescent cells.The correlation between the ability of STPH to chelate Fe²⁺and the survival rate of S.cerevisiae at CLS 6 was 0.816.Purification and sequence identification of STPH and its molecular docking with Tor.The fraction F_3-1 with the strongest chelating ability of Fe²⁺was separated and purified using two-steps RP-HPLC,and compared with the original hydrolysate,its chelating rate and survival rate of S.cerevisiae at CLS 6 increased by 10.83%and 7.77%,respectively.The sequence of F_3-1 was identified by LC-MS/MS,and 6 peptides were obtained:TEAPLNPK,LVRGPR,KLLDPDGK,SKHPNLSAS,APLNPK,and KYPHQK.Among them,the TEAPLNPK,LVRGPR,and KLLDPDGK were combined by molecular docking with FKBP12-FRB.All three peptides can not only form a more stable complex through hydrogen bond and other interactions but also enhance the interaction between FKBP12 and FRB.