Preparation Of Water Soluble Protein From Pseudosciaena Crocea Eggs Oxidation Active Peptide And The Activity Research

Pseudosciaena crocea eggs are rich in protein,unsaturated fatty acid and phospholipid,which showmuch high nutrition.At present,only a few fish eggs are processed into food,feed,etc.Most of fish eggs have been treated as waste,which seriously restricts the utilization of Pseudosciaena crocea eggs.In this paper,water-soluble proteins were extracted from defattedPseudosciaena crocea eggs.Four antioxidant peptides were isolated,purified and identified from the hydrolysate of this water-soluble proteins.This study provided theoretical basis for the development of new products ofPseudosciaena crocea eggs.?1?The basic components of the defatted Pseudosciaena crocea eggs was analyzed.The contents of protein,water,fat and ash were 85.1%,8.3%,2.4%,and 4.0%,respectively.The results indicated that the defatted fish eggs had a low-fat,high protein characteristics.The conditionsfor extractingwater-soluble proteins from the defatted eggs wereoptimized.The yield of water-soluble proteins was up to 33.56g/100g when the optimized extraction conditions were exerted.The results of amino acids analysis showed water-soluble protein was rich in some kind of amino acids related to the bioactivity peptides,such as Glu,Ser,Gly et al.?2?Alcalase,neutral protease and papain were used for hydrolyzing the water-soluble proteins fromthedefattedP seudosciaena crocea eggs.By comparing the extraction rate of peptide and the scavenging rates on DPPH,the alcalase was used as the experimental enzyme.Through the response surface optimization,it was concluded that the concentration of alcalase 4000U/g,enzymolysis time 1.5h,enzymolysis temperature 45 ?,the yield of polypeptide was 430±15.6mg/g.?3?The PCPE was isolated by ultrafiltration membrane with molecular weight cut-off of 10KDa and 5KDa.Three components PCPE1?>10KDa?.PCPE2?5?10KDa??PCPE3?<5KDa?were obtained.PCPE3?<5KDa?showed the strongest antioxidant activity,the IC50 values of total antioxidant capacity,DPPH·,·OHand O2 ·-scavenging ability were 10.179±0.055,4.114±0.026,0.591±0.003,2.714±0.162mg/mL,respectively.?4?The PCPE3 was further fractionated by Sephadex G15 gel filtration and gained five components named PCPE3-l?PCPE3-5.PCPE3-1 showed the strongest antioxidant activity,the IC50 values of total antioxidant capacity,DPPH-,·OHand 02· scavenging ability were 4.214 ±0.067,1.813± 0.001,0.419 ±0.089,1.773±0.037mg/mL,respectively.?5?The PCPE3-1 was further fractionated by Semi preparation RP-HPLC and gained five components named PCPE3-1-1?PCPE3-1-5.The antioxidant activity of each component was determined.The results showed that PCPE3-1-1?PCPE3-1-4 had antioxidant activity.LC-MS/MS was used to identify the amino acid sequence,and four peptides were identified as Asn-Pro-Cys-Ala-Ser-Arg?NPCASR,645.99Da?,Gln-Glu-Ser-Glu-Glu-Tyr?QESEEY,7 82.99Da?,Asn-Glu-Val-Gly-Ala?NEVGA,487.99Da?,Ala-Met-Cys-Cys?AMCC,425.99Da?.?6?To evaluate the antioxidant capacity of the four antioxidant peptides,a H₂O₂ induced oxidative damage in HepG2 cells model was established.The four peptides were found to reduce H₂O₂ induced oxidative damage in HepG2 cells,increase the cell viability,reduce ROS content,enhance SOD,CAT,GSH-Px activity,and increase GSH level in the cell.The antioxidant capacity of the four peptides:NPCASR>QESEEY>NEVGA>AMCC.The peptide NPCASR,which had the highest antioxidant activity,could significantly increase the expression of HO-1 protein.In the last step,it was proved that the antioxidant peptide could maintain the normal level of intracellular redox and eliminate the damage caused by oxidative stress.