Preparation,Isolation,Structural Analysis And Activity Study Of Collagen Antioxidant Peptide From Rhopilema Esculentum

Rhopilema esculenta is a kind of food-borne organisms,which is rich in collagen.The collagen can be hydrolyzed to form peptides that can be directly absorbed by the human intestines,and has a strong antioxidant effect.In this study,firstly,collagen was extracted from Rhopilema esculenta,and its detailed structure were characterized.Secondly,antioxidant peptides were prepared with the extracted collagen as raw materials,and their structural properties,separation and identification,and antioxidant activity were studied to provide experimental data support for the search for natural antioxidants.The specific research content and experimental results were as follows:(1)The high-purity type ? collagen was successfully extracted from Rhopilema esculenta using hydrochloric acid-pepsin methodology,and its subunit structure was[?1(I)]3.Rhopilema esculenta collagen contained 15.94%imino acids,and the most abundant amino acid was glycine(25.99%),without cysteine and tryptophan.The thermal shrinkage temperature was 43.79?,which had good thermal stability.The collagen presented a compact triple helix structure,and the microstructure was mainly irregular networks consist mainly of multilayered fibers.(2)The results of the optimization test of enzymatic hydrolysis of Rhopilema esculenta collagen antioxidant peptides indicated that trypsin was the most suitable enzyme,and the enzymatic hydrolysis conditions were temperature 41.0?,time 5.9 h,pH 7.9,and enzyme dosage 8.5 u/mg.Under this condition,the activity of Rhopilema esculenta collagen antioxidant peptide was positively correlated with its mass concentration.The maximum absorbance of the Fe3+reducing power of Rhopilema esculenta collagen antioxidant peptide was 0.4125,and the IC50 values of DPPH·,·OH,ABTS+·radicals were 32.28,3.12 and 0.74 mg/mL,respectively.The structural characteristics of the study showed that the antioxidant peptides were mainly peptides below 1000 Da,and the contents of aromatic amino acids,hydrophobic amino acids and essential amino acids were significantly higher than before enzymatic hydrolysis.Moreover,Enzymatic hydrolysis destroyed the original triple helical structure of Rhopilema esculenta collagen,reduced the crystallinity of the molecule,weakened the degree of order,and the fiber structure was destroyed,showing an irregular fragmented structure at the microscopic level.(3)Ultrafiltration,Sephadex G-15 chromatography,Nano-LC-ESI-Q-Orbitrap-MS/MS and Denovo technology were used to separate,identify and screen the highly active components of Rhopilema esculenta collagen antioxidant peptides.And then ten peptides with amino acid sequence in line with the characteristics of antioxidant peptides were selected.The peptides were YLGPK,YGLPNLK,YAPFD,AYPNM,FPLP,PNLGAPGS,VGDLGPR,VPLP,LVVHYP,LVGDLGPR.(4)The antioxidant activity of 10 peptides was tested and compared by chemical and cellular methods.Chemical antioxidant tests showed that YAPFD had the largest DPPH·and·OH radical scavenging rates,LVVHYP had the largest ABTS+·radical scavenging rate and Fe3+reducing power.There were both synergistic and antagonistic effects between peptides of different amino acid sequences.Cellular antioxidant test of HepG2 cells showed that AYPNM had the strongest scavenging effect on lipid free radicals induced by ABAP,and LVGDLGPR had the strongest scavenging effect on ROS induced by H2O2.