仿刺参性腺抗氧化肽的制备、分离纯化及结构鉴定OA

Oxidative stress is caused by an imbalance between reactive oxygen species(ROS)production and antioxidant defense mechanisms,leading to ROS accumulation and subsequent cellular damage.Oxidative stress is a major contributor to the development of chronic diseases,including cardiovascular disorders,diabetes,and cancer.Oxidative stress presents a global health challenge.Therefore,the development of effective and safe antioxidant interventions is a research imperative.Antioxidant peptides have emerged as promising therapeutic candidates due to their ability to mitigate oxidative damage through mechanisms such as free radical scavenging and metal ion chelation. Apostichopus japonicus(sea cucumber),a crucial marine biological resource,has attracted attention due to its nutritional and medicinal value.The body walls of sea cucumbers are a major source of bioactive compounds,including polysaccharides,saponins,and lipids,with antioxidant,anti-inflammatory,and immunomodulatory effects,demonstrating their substantial potential for application in functional foods and biomedicine.Approximately 50％of A.japonicus gonads,commonly known as"sea cucumber flowers",are protein,which can be as high as 80.9％in defatted gonads.Research on sea cucumber gonads has focused on assessing functional properties,such as antioxidant activity and immune modulation,with few studies dedicated to the isolation,purification,and structural elucidation of the underlying antioxidant peptides. In this study,the basic nutritional components of A.japonicus were determined and a dual-enzyme approach(papain and lipase)was used on A.japonicus gonads to prepare antioxidant peptides.Enzymatic hydrolysis parameters were optimized using response surface methodology(RSM).The resulting hydrolysate was subjected to a multistep purification process,including gel filtration and anion exchange chromatography,which yielded the D-1 fraction with the highest antioxidant activity.The amino acid sequences of the peptides in the D-1 fraction were identified using liquid chromatography-tandem mass spectrometry(LC-MS/MS).Subsequently,the bioactivity and physicochemical properties of identified peptides were predicted using online bioinformatics tools,including PeptideRanker for bioactivity scores,ToxinPred and AllerTOP for safety assessment,and AnOxPePred-1.0 for specific antioxidant potential evaluation to screen for the optimal sequence. This study aimed to provide a theoretical basis for the subsequent development and utilization of functional products derived from marine resources.Protein was the primary component of the gonads,with high lipid(15.40 g/100 g).To meet the physiological demands of reproduction during sexual maturation,the A.japonicus gonads typically accumulate substantial amounts of lipid,suggesting that the tissue essentially constitutes a lipoprotein complex.Consequently,when using A.japonicus gonads as a raw material for preparing antioxidant peptides,it is essential to remove lipids to ensure the feasibility of the subsequent extraction processes and the purity of the resulting products.During the screening of optimal enzymes,lipase demonstrated unique advantages due to its specific catalytic activity towards ester bonds,enabling the efficient hydrolysis of triglycerides and phospholipids.By removing the lipid barrier,lipase ameliorated the steric hindrance to protease action,thereby creating more favorable conditions for synergistic hydrolysis with papain,substantially enhancing the overall enzymatic efficiency. Therefore,a combination of lipase and papain was selected for the subsequent dual-enzyme hydrolysis experiments.The RSM optimal enzymatic hydrolysis process parameters were:material-liquid ratio of 1∶8.5 g/mL,enzymatic time of 5.1 h,enzymatic temperature of 53.0 ℃,and papain:lipase ratio of 1∶1.9.Under these conditions,the degree of protein hydrolysis was 61.41％±0.54％.The hydrolysate was initially separated by Sephadex G-25 gel chromatography,yielding three fractions(F-1,F-2,F-3),with F-2 exhibiting the strongest antioxidant capacity.F-2 was purified using Diethylaminoethyl-Sepharose Fast Flow anion exchange chromatography to obtain the D-1 fraction,which had DPPH,ABTS,and OH scavenging rates of 81.13％±0.86％,78.55％±1.28％,and 70.82％±1.46％,respectively.Compared with the enzymatic hydrolysate,the antioxidant activity of the D-1 fraction was substantially enhanced,although it was lower than that of the positive control,GSH. A total of 2806 polypeptide sequences from A.japonicus gonads were detected using LC-MS/MS.Using de novo sequencing analysis,68 peptides with high significance scores were identified.Based on bioactivity scoring,13 potentially-active peptides were selected,of which two peptides,SGPW and TWP,were identified to possess antioxidant potential.The results of the synthetic determination confirmed that all exhibited high antioxidant activity,with SGPW being the most potent.The antioxidant activity of peptide SGPW was primarily attributed to the strong free radical scavenging ability of its C-terminal tryptophan(W)residue.The activity of TWP was related to hydrophobic interactions within its structure with the hydrophobic aromatic ring of tryptophan(W).The rigid pyrrolidine ring of proline(P)is conducive to hydrophobic stacking,thereby enhancing its antioxidant efficacy.These findings not only demonstrate a viable strategy for the valorization of sea cucumber processing by-products,but also provide a theoretical basis for the development and utilization of marine resource functional products.Future work will focus on the chemical synthesis of these identified peptides for direct validation of their antioxidant activities in in vitro and in vivo models.