Preparation,Purification,Structure Identification Of Antioxidant Peptide From Grateloupia Livida

Recently,with the continuous progress of culture technology and the gradual expansion of culture scale,the processing and utilization of Grateloupia livida has gradually attracted people's attention.At present,the research on the active substances from Grateloupia livida is focused on the extraction and activity of terpenes and polysaccharides,while there are few studies on proteins and poly-peptides.Therefore,to promote the development and utilization of Grateloupia livida,it was used as raw material in this paper,the protein extraction process was studied,and the properties of the G.livida protein were determined;the antioxidant activity of the hydrolysates was studied,and the effect of decolorization on the antioxidant activity of the hydrolysates was investigated;the antioxidant peptide from G.livida was purificated and identified,and bioinformatics analysis was conducted through databases and online tools.The main research contents and results are as follows:Part 1:Extraction and antioxidant activity of protein from G.lividaWith G.livida as the raw material and the protein extraction rate as the index,the effect of different wall-breaking techniques on their protein extraction was explored.The results showed that the highest protein extraction rate of the swelling method,freeze-thawing method,and bead milling method was?29.66±0.86?%,?24.52±0.04?%,and?26.52±0.79?%,respectively,all lower than those of ultrasonic-assisted water extraction method.The ultrasonic-assisted water extraction by single-factor analysis and orthogonal test was optimized.The results showed that the best optimal conditions of extracting protein from G.livida by ultrasonic-assisted water extraction?the protein extraction rate can reach 58.16%?are as follows:liquid-to-solid ratio of 160 m L·g^-1,total ultrasonic time of 60 min,ultrasonic power of 1440 W and p H of 6.0.The crude protein was purified by ammonium sulfate salting-out.The optimum saturation of ammonium sulfate for protein precipitation is 65%,and the highest protein recovery is?84.19±0.71?%.The SDS-PAGE analysis of the G.livida protein was found.The results showed that the main molecular weight range of the G.livida protein was 16?66 k Da.The antioxidant activity of the G.livida protein was detected.The results showed that the G.livida protein had strong antioxidant activity at the concentrations of 1?8 mg·m L^-1(its reducing power at 8 mg·m L^-1 was?0.43±0.01?,and the IC₅₀ of DPPH free radicals and ABTS free radicals of the crude protein were?4.00±0.97?and?3.96±0.99?mg·m L^-1,respectively.Part 2:Preparation and antioxidant activity of protein hydrolysateThe protein of G.livida was hydrolyzed by alkaline protease,protamex1.5L,and neutral protease.The DH and antioxidant activities of all kinds of enzymatic hydrolysates was compared.The results showed that the neutral protease hydrolysate?it was enzymatic for an hour?have the highest antioxidant activity,the IC₅₀ of DPPH radical scavenging rate and IC₅₀ of ABTS radical scavenging rate are?3.96±0.41?mg·m L^-1,?0.88±0.13?mg·m L^-1,respectively.And its reducing power at 8 mg·m L^-1is?0.487±0.007?.To investigate the effect of chromophore on the antioxidant activity of G.livida hydrolysate,the protein enzymolysis product of G.livida was decolorized.The results showed that after decolorization of activated carbon,the peptide in the enzymatic hydrolysis product was lost,and the antioxidant activity was decreased.The DPPH radical scavenging rate and ABTS radical scavenging rate of the neutral protease digested for 1 h decrease by5.62%and 7.86%,respectively.Part 3:Purification and structure analysis of protein hydrolysateThe antioxidant peptides of G.livida were purified by Reverse-phase High Performance Liquid Chromatography?RP-HPLC?,and three components were obtained.The antioxidant activities of each component was compared.The results showed that F₃have the highest antioxidant activity,and the IC₅₀ of DPPH radical scavenging rate is?2.43±0.59?mg·m L^-1,and its reducing power at 4 mg·m L^-1 is?0.263±0.005?.Liquid Chromatography-tandem Mass Spectrometry?LC-MS/MS?was used to identify the amino acid sequence of F₃,and 10 kinds of polypeptides were obtained.Through bioinformatics prediction of the physical and chemical properties,toxicity,sensitization and ADMET properties of 10 kinds of peptides,one of the peptides?LYEEMKESKVINADK?may be a good novel antioxidant peptide.