Abstract:Oxidative stress has arisen from an imbalance between reactive oxygen species (ROS) production and the body's antioxidant defense mechanisms, leading to ROS accumulation and subsequent cellular damage. As a significant contributor to the development of chronic diseases, including cardiovascular disorders, diabetes, and cancer. Oxidative stress presented a major global health challenge. Consequently, the development of effective and safe antioxidant interventions constituted a critical research priority. 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. As a crucial marine biological resource, sea cucumber have garnered considerable attention for their nutritional and medicinal value. The body wall of sea cucumber is rich in significant source of bioactive compounds, including polysaccharides, saponins, and lipids. These constituents exhibited notable biological activities, such as antioxidant, anti-inflammatory, and immunomodulatory effects, demonstrating substantial application potential in functional foods and biomedicine. Apostichopus japonicus gonads, commonly known as "sea cucumber flower", constituted an essential component of sea cucumber. Studies indicated that protein accounted for approximately 50% of the main components in sea cucumber gonads, while the protein content can reach as high as 80.9% in defatted gonads. Previous research on sea cucumber gonads has largely focused on assessing functional properties like antioxidant, immune modulation and so on, with few studies dedicated to the isolation, purification, and structural elucidation of the underlying antioxidant peptides. This study first determined the basic nutritional components of Apostichopus japonicus and employed a dual-enzyme approach (papain and lipase) using Apostichopus japonicus gonads as the raw material to prepare antioxidant peptides. The enzymatic hydrolysis parameters were optimized using response surface methodology (RSM). The resulting hydrolysate was then subjected to a multi-step purification process, including gel filtration chromatography and anion exchange chromatography, which yielded the D-1 fraction with the highest antioxidant activity. Amino acid sequences of the peptides in 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 utilisation of functional products derived from marine resources. Experimental results indicated that protein was the primary component of the gonads, while the lipid content was notably high at 15.40 g/100g.To meet the physiological demands of reproduction during sexual maturation, the Apostichopus japonicus gonads typically accumulated substantial lipid, suggesting that the tissue essentially constituted a lipoprotein complex. Consequently, when using Apostichopus japonicus gonads as a raw material for preparing antioxidant peptides, it is essential to remove lipid to ensure the feasibility of 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 efficient hydrolysis of triglycerides and phospholipids. By removing the lipid barrier, lipase alleviated the steric hindrance for protease action, thereby creating more favorable conditions for synergistic hydrolysis with papain and significantly 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 as follows: material-liquid ratio of 1:8 g/mL, enzymatic time of 5.5 h, enzymatic temperature of 53℃, and papain:lipase ratio of 1:2.4. Under these conditions, the degree of protein hydrolysis reached 56.47%±0.59%. The hydrolysate was initially separated using Sephadex G-25 gel chromatography, yielding three fractions (F-1, F-2, F-3), with F-2 exhibiting the strongest antioxidant capacity. F-2 fraction was further purified by DEAE-Sepharose Fast Flow anion exchange chromatography to obtain 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 enzymatic hydrolysate, the antioxidant activity of D-1 fraction significantly enhanced, although it remained lower than the positive control GSH. A total of 2806 polypeptide sequences from Apostichopus japonicus gonads were detected using LC-MS/MS. Through de novo sequencing analysis, 68 peptides with higher significance scores were screened. Based on bioactivity scoring, 13 potentially active peptides were further selected, from which two peptides, SGPW and TWP, were finally identified as possessing antioxidant potential. The results from synthetic determination confirmed that all of them exhibited high antioxidant activity,and SGPW was the most potent. Regarding the activity mechanism, the antioxidant activity of peptide SGPW was primarily attributed to the strong free radical scavenging ability of its C terminal tryptophan (W) residue. In contrast, the activity of peptide TWP was related to hydrophobic interactions within its structure, in which the hydrophobic aromatic ring of tryptophan (W). The rigid pyrrolidine ring of proline (P) may form a favorable hydrophobic stacking effect, thereby enhancing its overall antioxidant efficacy. These findings not only demonstrated a viable strategy for the valorization of sea cucumber processing by-products but also provided a theoretical basis for the development and utilization of marine resources functional products. Future work will focus on the chemical synthesis of these identified peptides for direct validation of their antioxidant activities in vitro and in vivo models.
