Preparation,Purification And Structure Identification Of Antioxidant Peptides From Gracilaria Lemaneiformis

Gracilaria lemaneiformis is widely distributed in the coastal areas of Guangdong,Hainan,Shandong and Fujian provinces in China.It is a kind of economic Macroalgae and ranks the second in the cultivation yield of seaweed in China.Modern research shows that G.lemaneiformis has the advantages of high protein,high dietary fiber,low calorific value,and rich in vitamins and minerals.G.lemaneiformis contains a wide range of amino acids,including Leu,Ser,Arg and other amino acids with important physiological activities.It has high nutritional value of protein and can be used as a natural protein source of high quality bioactive peptides.Firstly,optimization of ultrasonic assisted alkaline extraction and acid precipitation for protein extraction parameter from G.lemaneiformis by RSM.Secondly,the hydrolysis conditions of G.lemaneiformis protein was obtained by the single factor experiment and orthogonal experiment with alkaline protease,and the antioxidant activity,amino acid composition and FT-IR spectra characteristics of the hydrolysate products from G.lemaneiformis protein were determined.Thirdly,separation and purification of antioxidant peptides from G.lemaneiformis by GFC and RP-HPLC.The amino acid sequence and molecular weight of the antioxidant peptides from G.lemaneiformis were identified by LC-MS/MS.Finally,the antioxidant peptides of G.lemaneiformis were synthesized,and the antioxidant activity and stability of the synthesized peptides were determined.The physical and chemical properties of peptides were evaluated by online prediction platform,and the mechanism of antioxidant effect of peptides was explored by molecular docking.The results are as follows:1.The method for extracting protein from dried G.lemaneiformis powder using ultrasonic assisted alkali dissolution and acid precipitation was optimized by single-factor experiment and Box-Behnken central composite design.The results indicated that the optimal extraction conditions were as follows:alkali concentration 0.2 mol·L^-1,liquid-solid ratio 24:1 m L·g^-1,ultrasonic time 70 min and ultrasonic power 482 W.The extraction rate of protein was 73.78%.At the same time,the antioxidant activity of G.lemaneiformis protein was studied.The results showed that protein from G.lemaneiformis had a certain antioxidant capacity.The half FRAP inhibitory concentration,half DPPH radical scavenging concentration and half ABTS radical scavenging concentration of its reducing power were 1.83 mg·m L^-1,2.92 mg·m L^-1 and 1.67 mg·m L^-1,respectively.The amino acid analysis showed that protein from G.lemaneiformis was an ideal plant protein source with high quality.2.The extracted method of enzymolysis products of protein from the G.lemaneiformis and its basic characteristics were studied in the paper.The degree of hydrolysis and antioxidant activity as evaluation indexes.Papain,alkaline protease,plant protein complex enzyme,trypsin and pepsin were used to hydrolyze protein from G.lemaneiformis.The enzymatic hydrolysis conditions were further optimized by single factor experiment and orthogonal experiment to obtain G.lemaneiformis protein hydrolysate(GLPH).The optimal hydrolysis conditions of alkaline protease were as follows:enzyme dosage 2%,hydrolysis time 2 h,substrate concentration 1 g·(100m L)^-1,p H 8.0.The half ABTS radical scavenging concentration,half DPPH radical scavenging concentration and FRAP inhibitory concentration of GLPH were 0.35 mg·m L^-1,1.44mg·m L^-1 and 1.46 mg·m L^-1,respectively.The amino acid content of GLPH was higher than that of G.lemaneiformis protein.The hydrolysate of G.lemaneiformis protein had good biological value.FT-IR spectra showed that G.lemaneiformis protein and its hydrolysates had different structural characteristics.3.The protein antioxidant peptides from G.lemaneiformis were separated by Sephadex G-25 gel column and reversed-phase high performance liquid chromatography(RP-HPLC).The main antioxidant component C2 was obtained.The component C2 was analyzed by LC-MS/MS,combined with PEAKS Studio version X+software,the mass spectrum data,the protein source of G.lemaneiformis in Uni Prot database,BIOPEP-UWM database and average local confidence(ALC,%),three peptides with amino acid sequences of LSPGEL,VYFDR and PGPTY were identified as antioxidant peptides of G.lemaneiformis.The half ABTS radical scavenging molar concentration was determined,and PGPTY had the best antioxidant activity(IC₅₀=0.45 m M).The p H and thermal stability of peptides LSPGEL,VYFDR and PGPTY were investigated respectively,and the simulated gastrointestinal digestion experiment was carried out.The results showed that peptides LSPGEL,VYFDR and PGPTY had good thermal stability,but the effect of acid-base change on antioxidant activity would have great difference.Finally,the docking energies of LSPGEL,VYFDR and PGPTY with Keap1 were-3.88kcal·mol^-1(LSPGEL),-3.49 kcal·mol^-1(VYFDR),-4.49 kcal·mol^-1(PGPTY),and the inhibition constants were 1.43 m M(LSPGEL),2.79 m M(VYFDR)and 0.51 m M(PGPTY),respectively.The docking energy value and inhibition constant of PGPTY were smaller than those of the other two peptides.The results showed that it had better antioxidant activity,which was consistent with the results of free radical scavenging activity.