Properties Of Peptides From Chlorella Vulgaris Protein Hydrolysates During Gastrointestinal Digestion And Purification Of Antioxidant Peptides

Chlorella is a kind of green unicellular algae with abundant protein and can be used as a raw material for preparation of bioactive peptides.Excessive reactive oxygen species lead to oxidative stress which can drive cell or tissue damage.Under normal circumstances,the endogenous antioxidant defense system plays a leading role,but exogenous antioxidants are equally crucial for maintaining free radical homeostasis between oxidative damage and antioxidant defense.In this paper,the antioxidant peptides were prepared from Chlorella vulgaris protein by enzymatic hydrolysis,the structural characteristics（microstructure,secondary structure and amino acid content）of Chlorella vulgaris protein antioxidant peptides during digestion and its potential effect on antioxidant activity were studied,and the antioxidant peptides were purified and identified.The research contents and results are as follows:（1）The chlorella defatted crude protein was extracted by water extraction and alcohol precipitation method.Then,a variety of proteases were applied for enzymatic hydrolysis of chlorella defatted crude protein,and the alkaline protease was determined as the best enzyme for hydrolysis.The optimal enzymatic hydrolysis process of the Chlorella vulgaris defatted crude protein was optimized by single factor and response surface experiments.The optimal enzymatic conditions were as follows:enzymatic hydrolysis temperature 55℃,p H 8.75,enzyme substrate ratio2.15%.At this time,the DPPH,ABTS,hydroxyl radical scavenging rate,and Fe²⁺chelating rate of polypeptides were 53.19±0.99%,76.86±0.28%,53.39±1.74%,and 22.56±2.97%,respectively.（2）The structural characteristics of Chlorella polypeptides M₃（>3 k Da）and L₃（<3 k Da）during gastrointestinal digestion were investigated using the INFOGEST model.M₃and L₃polypeptides exhibited reticular formation during digestion,and are structurally dominated by beta-sheet structure,in which lysine showed better digestive resistance.L₃exhibited stronger antioxidant activity in the stomach with DPPH free radical scavenging ability and Fe²⁺chelating ability of 64.29±2.97%and71.68±2.97%,respectively.After gastrointestinal digestion,the content of hydrophobic amino acids was reduced which resulted in the decrease in the antioxidant activity of L₃,but L₃possessed a higher antioxidant activity than M₃.Therefore,L₃with smaller molecular weight exhibited better gastrointestinal digestive stability and antioxidant activity.（3）The antioxidant peptides of Chlorella vulgaris were separated and purified by ultrafiltration and Sephadex G-15 gel chromatography,the scavenging capacity of the C1 component（4 mg/m L）for DPPH free radicals and Fe²⁺chelating ability were59.62±1.29%and 49.78±1.55%,respectively.152 peptide sequences were identified from C1 by LC-MS.Through peptide activity score,prediction of free radical scavenging activity and further screening of molecular docking,six peptide sequences were finally determined.Then the physical and chemical properties,toxicity and allergenicity of peptides were predicted.Among them,SGHHKPL has good digestive stability,non-toxicity and good water solubility,SLPGGYGL and WHMHKTF has no-toxicity and no sensitization,so they may be promising safe and effective antioxidants.