Application of a rice field eel bone antioxidant peptide in preparation of antioxidant products

Abstract

The application discloses application of an orange bony fish bone antioxidant peptide in preparation of antioxidant products and belongs to the technical field of antioxidant active peptides.The amino acid sequence of the orange bony fish bone antioxidant peptide is shown as SEQ ID NO.3-14, the 12 polypeptides are proposed to have antioxidant activity for the first time, the ABTS activity and ORAC activity of the polypeptides are 1.70-4.29 times and 2.79-5.90 times of Trolox respectively.The polypeptides are proposed to activate the enzyme and non-enzyme antioxidant system in zebra fish by regulating the Keap1-Nrf2 pathway, so that the resistance of the body to oxidative stress is enhanced.The potential application of the orange bony fish bone antioxidant peptide in functional products has important significance.

Description

Technical Field

[0001] This invention belongs to the field of antioxidant active peptide technology, and particularly relates to the application of an antioxidant peptide from eel bone in the preparation of antioxidant products. Background Technology

[0002] In living organisms, excessive production of free radicals can lead to oxidative stress, thereby damaging DNA, proteins, lipids, and other cellular components. This complex process is closely related to the occurrence and development of various diseases, such as cardiovascular disease, neurodegenerative diseases, diabetes, and cancer. Therefore, maintaining a balance of free radicals is crucial for overall health and physiological homeostasis. Simultaneously, oxidation reactions caused by free radicals directly affect food quality, altering its nutritional composition, flavor, and texture. Antioxidants have the ability to reduce free radicals, thereby alleviating oxidative stress in organisms and food systems. Compared to chemically synthesized drugs, natural antioxidants exhibit minimal or almost no adverse side effects. Therefore, natural antioxidants have significant prospects and potential in the health and food sectors.

[0003] The utilization of protein resources from by-products in the production of antioxidant peptides has received widespread attention in recent years. Fish bones are one of the main by-products generated during aquatic product processing, rich in high-quality structural proteins such as collagen and osteocalcin, possessing high reuse value and potential for high-value development. The swamp eel (Monopterus albus) exhibits strong antioxidant potential due to its rich protein diversity, balanced amino acid composition, and excellent nutritional characteristics. However, systematic research reports on the antioxidant activity of swamp eel bone protein peptides are currently lacking. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of eel bone antioxidant peptides in the preparation of antioxidant products, such as protein peptides with amino acid sequences shown in SEQ ID NO. 3-14 that exhibit significant antioxidant activity. Another purpose of this invention is to provide a method for accurately screening and preparing eel bone antioxidant peptides.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of eel bone antioxidant peptides in the preparation of antioxidant products, wherein the amino acid sequence of the eel bone antioxidant peptides is at least one of those shown in SEQ ID NO.3-14.

[0006] Preferably, the effective concentration of the antioxidant peptides from the eel bone is 80-120 μM.

[0007] Preferably, the product includes pharmaceuticals, food, or cosmetics.

[0008] This invention provides a method for preparing antioxidant peptides from eel bone, comprising the following steps: enzymatically hydrolyzing eel bone with Alcalase to obtain an eel bone hydrolysate; separating and purifying the eel bone hydrolysate using gel column chromatography, collecting the eluent according to peak shape, and selecting the eluent with good antioxidant activity for UPLC-Q-TOF-MS / MS identification; predicting the bioactivity score of the identified peptide using the PeptideRanker database, and predicting the sensitization, potential toxicity, and physicochemical properties of the obtained active peptide using the AllerTOP and ToxinPred databases to obtain eel bone antioxidant peptides with high antioxidant activity; wherein the amino acid sequence of the eel bone antioxidant peptide is at least one of SEQ ID NO. 3-14.

[0009] Preferably, Alcalase alkaline protease solution is added according to 1-7 w / w% of the protein content of the eel bone.

[0010] Preferably, the enzymatic hydrolysis conditions include: pH 7.8-8.2, temperature 53-57℃, and time 4-8h.

[0011] Preferably, the conditions for the gel column chromatography include: a Sephadex G-25 gel chromatography column, water as the eluent, and wavelengths of 210-218 nm and 250-258 nm.

[0012] Preferably, the chromatographic column used for UPLC-Q-TOF-MS / MS identification is ACQUITYUPLC HSS T3, and the column temperature is 40-50℃.

[0013] Preferably, the mobile phase A identified by UPLC-Q-TOF-MS / MS is an aqueous solution containing 0.1% formic acid, and the mobile phase B is an 80% acetonitrile solution. The flow rate is set to 0.4 mL / min, and the gradient elution program is as follows: 0-2.0 min, 92.0% A; 2.0-45.0 min, 92.0-72.0% A; 45.0-55.0 min, 72.0-60.0% A; 55.0-56.0 min, 60.0-5.0% A; 56.0-66.0 min, 5.0% A.

[0014] The present invention provides an antioxidant product, wherein the active ingredient of the antioxidant product includes at least one of the amino acid sequences shown in SEQ ID NO.3-14.

[0015] The beneficial effects of this invention are: The amino acid sequences of the eel bone antioxidant peptides described in this invention are shown in SEQ ID NO. 3-14. This invention is the first to propose that the above 12 polypeptides possess antioxidant activity, with their ABTS and ORAC activities being 1.70-4.29 times and 2.79-5.90 times that of Trolox, respectively. Furthermore, it is proposed that these polypeptides activate the enzymatic and non-enzymatic in vivo antioxidant systems of zebrafish by regulating the Keap1-Nrf2 pathway, thereby enhancing the body's resistance to oxidative stress. The potential applications of the eel bone antioxidant peptides provided by this invention in functional products are of great significance.

[0016] This invention also proposes for the first time that the nitrogen-hydrogen bond and tyrosine phenolic hydroxyl group in the tryptophan indole ring are key sites for the antioxidant activity of the aforementioned 12 eel peptides. An LPS-induced oxidative damage model in zebrafish embryos was established. The results showed that the aforementioned 12 peptides, by activating the Keap1-Nrf2 pathway, promoted the expression of HO-1 and enzymes involved in glutathione synthesis (such as GCL and GSS), significantly increased the GSH / GSSH ratio, and enhanced the enzyme activities of SOD, CAT, and GSH-PX, thereby improving the total antioxidant capacity of zebrafish and inhibiting LPS-induced oxidative damage. This indicates that eel bone antioxidant peptides have the potential to improve oxidative damage in zebrafish. Attached Figure Description

[0017] Figure 1 The protein recovery rate, degree of protein hydrolysis (A), and antioxidant activity (B) of the enzymatic hydrolysis products are evaluated.

[0018] Figure 2 Chromatogram (A) of the Alcalase hydrolysate of eel bone after separation by Sephadex G-25 and antioxidant activity of each component (B).

[0019] Figure 3 Statistical analysis of peptides from the bones of the yellow eel. (A) Distribution of peptide number (outer ring) and distribution of amino acid composition (inner ring); (B) Composition and distribution of amino acids; (C) Amino acid distribution of peptides of different lengths.

[0020] Figure 4 To identify the antioxidant peptides NVGW (A) and WALN (B) from the F4 fraction using LC-MS / MS.

[0021] Figure 5 Active site analysis of NVGW, DFLRY, WGKLD, RWPVDL, WLDQPH, RDWPDAR, and WGDLSRP protein peptides based on HOMO and Mulliken charge distribution and bond length.

[0022] Figure 6Active site analysis of WALN, GYLPR, YVDRF, WDPDAR, GPYLDLK, WGDLSPK, and WDAAPGVPR protein peptides based on HOMO and Mulliken charge distribution and bond length.

[0023] Figure 7 The binding conformations of NVGW, DFLRY, WGKLD, and RWPVDL with myeloperoxidase (MPO) are shown in three-dimensional and two-dimensional representations.

[0024] Figure 8 The binding conformations of WALN, GYLPR, YVDRF, and WDPDAR with myeloperoxidase (MPO) are shown in three-dimensional and two-dimensional representations.

[0025] Figure 9 The binding conformations of WLDQPH, RDWPDAR, and WGDLSRP with myeloperoxidase (MPO) are shown in three-dimensional and two-dimensional representations.

[0026] Figure 10 The binding conformations of GPYLDLK, WGDLSPK, and WDAAPGVPR with myeloperoxidase (MPO) are shown in three-dimensional and two-dimensional representations.

[0027] Figure 11 To investigate the effect of antioxidant peptides on ROS levels in zebrafish embryos: (A) Fluorescence images, (B) Quantitative analysis of fluorescence intensity using ImageJ software.

[0028] Figure 12 The effects of antioxidant peptides on various indicators in zebrafish embryos: (A) MDA, (B) GSH / GSSG, (C) SOD, (D) CAT, (E) GSH-pX, (F) T-AOC, (G) Keap1, (H) Nrf2, (I) HO-1, (J) GCL and (K) GSS.

[0029] Figure 13 To explore the potential mechanism by which antioxidant peptides alleviate LPS-induced oxidative damage in zebrafish embryos. Detailed Implementation

[0030] This invention provides the application of eel bone antioxidant peptides in the preparation of antioxidant products, wherein the amino acid sequences of the eel bone antioxidant peptides are at least one of those shown in SEQ ID NO. 3-14. The amino acid sequences of the eel bone antioxidant peptides shown in SEQ ID NO. 3-14 of this invention are, in order: DFLRY, GYLPR, WGKLD, YVDRF, RWPVDL, WDPDAR, WLDQPH, GPYLDLK, RDWPDAR, WGDLSPK, WGDLSRP, and WDAPGVPR. This invention preferably uses WGKLD and RDWPDAR, which have higher antioxidant activity.

[0031] In this invention, the effective concentration of the antioxidant peptides from the eel bone is 80-120 μM, preferably 85-115 μM, and more preferably 100 μM. The effective concentration described in this invention is the concentration set during zebrafish antioxidant studies.

[0032] In this invention, the anti-product includes pharmaceuticals, food, or cosmetics.

[0033] This invention provides a method for preparing antioxidant peptides from eel bone, comprising the following steps: enzymatically hydrolyzing eel bone with Alcalase to obtain an eel bone hydrolysate; separating and purifying the eel bone hydrolysate by gel column chromatography, collecting the eluent according to peak shape, and selecting the eluent with the best antioxidant activity for UPLC-Q-TOF-MS / MS identification; predicting the bioactivity score of the identified peptide using the PeptideRanker database, and predicting the sensitization, potential toxicity, and physicochemical properties of the obtained active peptide using the AllerTOP and ToxinPred databases to obtain eel bone antioxidant peptides with high antioxidant activity; wherein the amino acid sequence of the eel bone antioxidant peptide is at least one of SEQ ID NO. 3-14.

[0034] This invention identified 12 novel antioxidant peptides from enzymatic hydrolysates of eel bones using computer simulation screening. These peptides exhibited ABTS and ORAC activities 1.70-4.29 times and 2.79-5.90 times higher than those of Trolox, respectively. Quantum chemical calculations and subsequent active site methylation experiments elucidated the crucial roles of hydrogen atoms on the indole nitrogen of tryptophan, the phenolic hydroxyl group of tyrosine, and the guanidino nitrogen atom of arginine in ABTS radical scavenging and ORAC activity. Furthermore, molecular docking analysis revealed that the ASE antioxidant peptides form stable binding bonds with myeloperoxidase (MPO) primarily through hydrogen bonding with Arg239, Arg333, Arg424, Asp98, His336, and Thr100, and electrostatic interactions with Arg239, Arg333, Glu102, and His336. The above research further demonstrates that zebrafish bone peptides activate the enzymatic and non-enzymatic in vivo antioxidant systems in zebrafish by regulating the Keap1-Nrf2 pathway, thereby enhancing the body's resistance to oxidative stress. This discovery provides a theoretical basis for the development of antioxidants based on zebrafish bone peptides and opens up new research directions for the prevention and treatment of antioxidant-related diseases.

[0035] In this invention, Alcalase alkaline protease solution is added at a protein content of 1-7 w / w% of the eel bone. Preferably, Alcalase alkaline protease solution is added at a protein content of 2-6 w / w% of the eel bone. More preferably, Alcalase alkaline protease solution is added at a protein content of 4 w / w% of the eel bone. The enzymatic hydrolysis conditions of this invention include: pH 7.8-8.2, temperature 53-57℃, and time 4-8 h; preferred enzymatic hydrolysis conditions include: pH 7.9-8.1, temperature 54-56℃, and time 5-7 h; further preferred enzymatic hydrolysis conditions include: pH 8.0, temperature 55℃, and time 6 h. The structural relaxation of the eel bone protein in the alkaline environment, coupled with the numerous cleavage sites of the Alcalase enzyme, allows it to effectively decompose peptide bonds in the eel protein, which is beneficial for generating peptides with stronger antioxidant properties.

[0036] In this invention, the conditions for gel column chromatography include: the chromatographic column is a Sephadex G-25 gel chromatography column, the eluent is water, and the wavelength is 210-218 nm and 250-258 nm; preferably, the wavelengths are 214 nm and 254 nm.

[0037] In this invention, the chromatographic column used for UPLC-Q-TOF-MS / MS identification was an ACQUITYUPLC HSS T3, with a column temperature of 40-50℃. The mobile phase A for UPLC-Q-TOF-MS / MS identification was an aqueous solution containing 0.1% formic acid, and the mobile phase B was an 80% acetonitrile solution. The flow rate was set to 0.4 mL / min, and the gradient elution program was: 0-2.0 min, 92.0% A; 2-45 min, 92.0-72.0% A; 45.0-55.0 min, 72.0-60.0% A; 55.0-56.0 min, 60.0-5.0% A; 56.0-66.0 min, 5.0% A. UPLC-Q-TOF-MS / MS identification was performed in electrospray ionization (ESI) mode, with a scan range of 50-2000 m / z. Data were analyzed using the Mascot database.

[0038] This invention provides an antioxidant product, wherein the active ingredient of the antioxidant product comprises at least one amino acid sequence as shown in SEQ ID NO. 3-14. In this invention, the product is preferably a pharmaceutical, food, or cosmetic.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Unless otherwise specified, the following embodiments are all conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] The materials used in the following examples were sourced as follows: Eel bones (protein content approximately 25%) were from the Zhuangxing Base of the Shanghai Academy of Agricultural Sciences (Shanghai, China) and pulverized through a 40-mesh sieve. Compound protease solution, papain solution, neutral protease solution, Alcalase alkaline protease solution, and flavor protease solution were all purchased from Beijing Dahonglihui Biotechnology Center. 6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid (Trolox) and 2,2'-azo[2-methylpropionamide]dihydrochloric acid (AAPH) were purchased from Beijing Yishan Huitong Technology Co., Ltd. 2,2-adiazon-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) and sodium fluorescein were purchased from Beijing Zhongcheng Jinnian Technology Co., Ltd. Sephadex G-25 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The synthesized peptides and peptides with methylated active sites were purchased from Nanjing Jietai Biotechnology Co., Ltd., including NVGW (SEQ ID NO.1), WALN (SEQ ID NO.2), DFLRY (SEQ ID NO.3), GYLPR (SEQ ID NO.4), WGKLD (SEQ ID NO.5), YVDRF (SEQ ID NO.6), RWPVDL (SEQ ID NO.7), WDPDAR (SEQ ID NO.8), WLDQPH (SEQ ID NO.9), GPYLDLK (SEQ ID NO.10), RDWPDAR (SEQ ID NO.11), WGDLSPK (SEQ ID NO.12), WGDLSRP (SEQ ID NO.13), and WDAAPGVPR (SEQ ID NO.14), all with a purity higher than 95%. All other reagents used in this experiment were analytical grade.

[0043] Example 1 5g of eel bone powder was dispersed in 200mL of water, and the pH was adjusted to 8.0. Then, Alcalase alkaline protease solution was added at 4% (w / w) of the eel bone protein content, and the mixture was enzymatically hydrolyzed at 55℃ for 6h. The enzyme was then inactivated at 100℃ for 10min. After cooling to room temperature, the supernatant was collected by centrifugation and lyophilized to obtain the polypeptide lyophilized powder.

[0044] Comparative Example 1 The difference from Example 1 is that the enzymes used are replaced with neutral protease solution (pH 7.0 and 50°C, Neutrase), flavor protease solution (pH 7.0 and 55°C, Flavorzyme), papain solution (pH 7.0 and 55°C, Papain), and complex protease solution (pH 7.0 and 55°C, Protease M), respectively. The enzymatic hydrolysis conditions of the corresponding enzymes are replaced with the conditions shown in parentheses. All other aspects are the same as in Example 1.

[0045] Example 2 The degree of hydrolysis and antioxidant activity of different enzymatic hydrolysates from Example 1 and Comparative Example 1 were compared using the following specific experimental methods: 1. Determination of degree of hydrolysis (DH) The OPA reagent (200 mL) was prepared as follows: 160.0 mg OPA was dissolved in 4.0 mL of ethanol, and 7.62 g borax, 200.0 mg SDS, and 176.0 mg DTT were dissolved in 150 mL of deionized water. The two solutions were mixed and the volume was increased to 200 mL. For the assay, 20 μL of the sample solution (the solution prepared from the lyophilized polypeptide powder) was mixed with 150 μL of OPA reagent. After reacting for 2 min, the absorbance (A) was measured at 340 nm. sample Deionized water served as a blank control (A). blank ), 0.1 mg / mL serine was the standard group (A) standard The serine-NH2 equivalent (Ser-NH2) and DH in the sample are calculated using the following formula: Where C sample (g / L) represents the protein concentration of the sample, α, β, h tot The values ​​were 1.0, 0.4, and 8.6 mmol / g, respectively. Furthermore, the molar concentration of serine at 0.1 mg / mL was 0.9516 mM.

[0046] 2. Determination of protein recovery rate The protein content of the enzyme hydrolysate was determined according to the Kjeldahl method in GB 5009.5-2016, and the recovery volume of the enzyme hydrolysate was also determined. The protein recovery rate was calculated according to the following formula.

[0047] Protein recovery rate (%) = ×100% 3. Antioxidant activity assay (1) Determination of ABTS free radical scavenging ability The specific steps are as follows: Mix an equal volume of 7 mM ABTS solution with 2.45 mM potassium persulfate, and incubate at room temperature in the dark for 12-16 hours to obtain ABTS· + Stock solution. Before use, dilute with 50 mM phosphate-buffered saline (PBS) at pH 7.4 to an absorbance of 0.700 ± 0.020 at 734 nm. Add 150 μL of the diluted stock solution to 50 μL of sample, incubate at 30 °C for 30 min, and measure the absorbance at 734 nm. Use PBS as a blank control and Trolox as a positive control. ABTS free radical scavenging rate is calculated using the following formula: Among them, A sample and A control The absorbance values ​​are for the sample group and the control group, respectively. Results are expressed as Trolox equivalents (μmol TE / μmol sample).

[0048] (2) Determination of oxygen free radical absorption capacity (ORAC) The specific method included: adding 120 μL of 117 nM sodium fluorescein to 20 μL of sample and reacting at 37°C in the dark for 15 min; then adding 60 μL of 40 mM AAPH, and detecting fluorescence every minute at an excitation wavelength of 485 nm and an emission wavelength of 520 nm for a total of 100 min. PBS and Trolox were used as blank and positive controls, respectively. Fluorescence intensity f i The AUC was calculated with the initial f0, and the net fluorescence decay area was obtained by linear regression. The results are expressed as Trolox equivalents (μmol TE / μmol sample).

[0049] Where AUC represents the area under the fluorescence decay curve; f i f is the fluorescence intensity measured at minute i; f0 is the initial fluorescence intensity; f i / f0 is the normalized relative fluorescence intensity. AUC sample and AUC blank represents the area under the curve for the sample group and the blank group, respectively, and Net AUC is the difference between the two.

[0050] The results are as follows Figure 1 As shown, compared with the other four enzyme hydrolysates, Alcalase enzyme hydrolysate exhibited significantly higher protein recovery and antioxidant activity. p <0.05).

[0051] Example 3 Purification, identification and screening of antioxidant peptides (1) Purification of antioxidant peptides: The lyophilized peptide powder solution (obtained by mixing the lyophilized peptide powder prepared in Example 1 with water) was separated and purified using Sephadex G-25 gel chromatography (3.7×27.8cm). Deionized water was used for elution, and the absorbance was monitored at 214nm and 254nm. The eluted fractions were collected according to the peak shape. The results are as follows: Figure 2 As shown in (A), the collected eluent was subjected to antioxidant activity determination according to the method for antioxidant activity determination in Example 2, and the results are as follows. Figure 2As shown in (B), the results indicate that F4 exhibits higher activity. The component with higher activity was selected for further identification.

[0052] (2) Identification of antioxidant peptides The sequences of the bioactive peptides were identified using UPLC-Q-TOF-MS / MS (Triple TOP 5600+ LC / MS system, AB SCIEX, USA). An ACQUITY UPLC HSS T3 column was used at 45℃. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was 80% acetonitrile. The flow rate was set to 0.4 mL / min, with a gradient elution program: 0–2.0 min, 92.0% A; 2–45 min, 92.0–72.0% A; 45.0–55.0 min, 72.0–60.0% A; 55.0–56.0 min, 60.0–5.0% A; 56.0–66.0 min, 5.0% A. Electrospray ionization (ESI) was used in the scan range of 50–2000 m / z, and the data were analyzed using the Mascot database. Using a filter setting with a threshold of ≥85%, 526 peptides were identified from F4 by UPLC-Q-TOF-MS / MS. The lengths of these peptides, as well as the properties and distribution of their constituent amino acids, are shown in [reference needed]. Figure 3 .

[0053] The results showed that these peptides were mainly composed of pentapeptides (17.5%), hexapeptides (26.4%), heptapeptides (15.4%), octapeptides (11.2%), and nonapeptides (11.4%). Figure 3 (A) - Outer ring). Hydrophobic amino acids accounted for the highest proportion, at 56.2%, followed by polar, uncharged amino acids at 16.8%. Basic amino acids accounted for 15%, and acidic amino acids accounted for 12%. Figure 3 (A) - Inner Ring). From Figure 3 (B) It can be seen that the amino acids composing these peptides include 20 different amino acids. Among them, G, P, L, A, R, and D are the six amino acids with relatively high frequency, accounting for 16.74%, 12.76%, 7.42%, 7.34%, 6.96%, and 6.80%, respectively. The proportion of hydrophobic amino acids at the N-terminus of these peptides is 43.73%, and at the C-terminus it is 41.83%. The results indicate that the eel bone peptides have significant antioxidant potential. The distribution of amino acids in peptides of different lengths was analyzed using Seqlogo analysis; the larger the letter of the amino acid, the higher its frequency. Figure 3(C) It can be seen that hydrophobic amino acids remain the most abundant amino acids in peptides of different lengths, followed by polar, uncharged amino acids and basic amino acids. Meanwhile, as the peptide length increases from pentapeptide to undecapeptide, the proportion of hydrophobic amino acids shows an increasing trend (from approximately 52.04% to 65.45%), while the proportions of acidic and basic amino acids gradually decrease, from 17.39% and 18.91% to 5.71% and 9.09%, respectively. The proportion of basic amino acids at the C-terminus of pentapeptides to nonapeptides is as high as 38.27% or more.

[0054] (3) Screening of antioxidant peptides and verification of their chemical antioxidant activity PeptideRanker was used to score the potential biological activity of peptides, and AllerTOP and ToxinPred were used to assess sensitization, potential toxicity, and physicochemical properties. Finally, eel bone peptides that were non-toxic, non-sensitizing, rich in antioxidant and hydrophobic amino acids, and had high biological activity scores were selected. The results are shown in Tables 1 and 2.

[0055] PeptideRanker is a predictive bioactivity server used to predict and screen antioxidant peptides. A peptide is considered active if the predicted value is above a threshold of 0.5. From 539 peptides, 45 peptides with scores greater than 0.5, and which were non-allergenic and non-toxic, were screened. Aromatic amino acids (especially Y and W) and hydrophobic amino acids can enhance the efficacy of antioxidant peptides (Table 1).

[0056] Table 1. Computer simulation screening of peptides (including predicted physicochemical properties and activity scores).

[0057] Based on the high ratio of aromatic and hydrophobic amino acids, 14 peptides (NVGW, WALN, DFLRY, GYLPR, WGKLD, YVDRF, RWPVDL, WDPDAR, WLDQPH, GPYLDLK, RDWPDAR, WGDLSPK, WGDLSRP, and WDAAPGVPR) were selected from 45 peptides for synthesis. As shown in Table 2, the synthesized peptides had high scores, typically consisting of 4-9 amino acids, with hydrophobicity ranging from -0.63 to 0.13, steric hindrance values ​​between 0.47 and 0.71, and amphiphilicity of 0 or 0.73. In the ABTS radical scavenging assay and ORAC assay, the TE values ​​of all 14 peptides were greater than 1.0 μmol TE / μmol, indicating that their activity exceeded that of the positive control Trolox (Table 2). A search in NCBI revealed NVGW and WALN as newly discovered peptide sequences; the secondary mass spectra of these two new peptides are shown below. Figure 4Furthermore, through a search in BIOPEP, these 14 peptides were identified as novel antioxidant peptides. Five peptides—WALN, GPYLDLK, PDWPDAR, WWGLSRP, and WDAPGVPR—showed significantly higher antioxidant capacity than the other peptides (Table 2). The ABTS free radical scavenging capacity of these five peptides ranged from 3.02 ± 0.03 μmol TE / μmol to 3.65 ± 0.02 μmol TE / μmol, with ORAC values ​​of 4.11 ± 0.11 μmol TE / μmol and 5.90 ± 0.28 μmol TE / μmol, respectively. The strong antioxidant capacity of these peptides is likely closely related to their amino acid composition and sequence structure.

[0058] Table 2 Physicochemical properties, antioxidant activity, and quantum computing energy of 14 peptides

[0059] Note: Data with different letters in the same test indicate significant differences. p <0.05).

[0060] Example 4 1. Density Functional Theory (DFT) Calculation The initial conformations of the 14 antioxidant peptides (NVGW, WALN, DFLRY, GYLPR, WGKLD, YVDRF, RWPVDL, WDPDAR, WLDQPH, GPYLDLK, RDWPDAR, WGDLSPK, WGDLSRP, and WDAAPGVPR) obtained in Example 3 were constructed using Chem 3D software. Density functional theory (DFT) optimization was then performed at the M062X / 6-31G(d) level using the Gaussian09 software package, and frequency calculations confirmed the conformations were stable and free of imaginary frequencies. Subsequently, the HOMO, EHOMO, Mulliken charge distribution, and bond length properties of the molecules were calculated based on the optimized conformations. The results are as follows: Figures 5-6 Tables 3 and 4.

[0061] Table 3 shows the predicted atomic charge distribution of 14 polypeptide active sites. Table 4. Mullicken charge distribution and bond length of predicted active sites in 14 peptides

[0062] To further investigate the relationship between the structure and properties of antioxidant peptides, the peptide structures were calculated using quantum chemical methods. The ability of a molecule to donate electrons is closely related to the effectiveness of its antioxidant activity. According to the frontier molecular orbital theory, a higher EHOMO indicates a stronger ability to donate electrons. Furthermore, the HOMO, as a frontier orbital, plays a crucial role in reactions with nucleophiles, especially hydrogen-withdrawing and electron-withdrawing radicals. Figures 5-6 The HOMO distributions of 14 synthetic peptides are shown, focusing on the indole molecule on the W ring, the phenolic hydroxyl group on tyrosine, and the guanidinium group on arginine. These findings suggest that these specific sites are most likely to lose electrons when peptides interact with free radicals. Furthermore, previous studies have shown that antioxidants primarily scavenge free radicals at specific sites within the HOMO orbital regions, which are typically associated with relatively negatively charged atoms and longer bond lengths. These characteristics enhance the ability of antioxidants to donate hydrogen atoms during scavenging. Table 3 details the Mulliken atom charge distribution and bond lengths within the HOMO orbital regions of these 14 peptides. These peptides exhibit similar charge distribution patterns, with negative charges primarily located on the nitrogen atom of the tryptophan indole ring, the oxygen atom of the tyrosine phenolic hydroxyl group, and the nitrogen atom of the arginine guanidinium group. It is speculated that the active sites of NVGW, WALN, DFLRY, GYLPR, WGKLD, YVDRF, RWPVDL, WDPDAR, WLDQPH, GPYLDLK, RDWPDAR, WGDLSPK, WGDLSRP, and WDAAPGVPR may be located at positions 32N-61H, 10N-44H, 45O-92H, 40N-83H, 10N-52H, 14O-61H, 24N-77H, 10N-62H, 49N-104H, 18O-72H, 63N-121H, 10N-65H, 10N-67H, and 10N-77H, respectively (Table 4). These sites are considered to be hydrogen donors for scavenging free radicals, which helps to enhance their antioxidant activity. These findings indicate that, regardless of whether tryptophan and tyrosine are located at the N-terminus or C-terminus, the nitrogen-hydrogen bond on the tryptophan indole ring and the phenolic hydroxyl group of tyrosine play crucial roles in antioxidant activity. Quantum chemical calculations further confirm the key roles of tryptophan and tyrosine.

[0063] 2. Validation of antioxidant active sites To verify the active sites predicted by the HOMO distribution, these sites were methylated to block their activity. The hydrogen atoms 32N-61H, 10N-44H, 45O-92H, 40N-83H, 10N-52H, 14O-61H, 24N-77H, 10N-62H, 49N-104H, 18O-72H, 63N-121H, 10N-65H, 10N-67H, and 10N-77H at the active sites in the peptide sequences sequentially represented by SEQ ID NO.1-SEQ ID NO.14 were methylated, yielding 14 new antioxidant peptides. The modified peptides were identified as NVGW(me), W(me)ALN, DFLRY(me), GYLPR(me), W(me)GKLD, Y(me)VDRF, RW(me)PVDL, W(me)DPDAR, W(me)LDQPH, GPY(me)LDLK, RDWPDAR(me), W(me)GDLSPK, W(me)GDLSRP, and W(me)DAAPGVPR (Table 5). The in vitro antioxidant activity of the modified peptides was evaluated. The results showed that the ABTS radical scavenging ability of the methylated peptides ranged from 0.0016 ± 0.0001 μmol TE / μmol to 0.3914 ± 0.0023 μmol TE / μmol, with 14 peptides exhibiting only 0.04%–5.57% and 23.02% of their original values ​​(Table 5), respectively. GYLPR(me) and W(me)DPDAR were 18.02% and 23.02% of their original values, respectively. The ORAC values ​​of the 14 modified peptides were only 2.67%–14.36% of their original values. These results indicate that the modified active peptides almost completely lost their ABTS radical scavenging and ORAC abilities. This demonstrates that methylation of the HOMO-predicted active sites weakens their ability to scavenge free radicals in vitro. The hydrogen atoms 32N-61H, 10N-44H, 45O-92H, 40N-83H, 10N-52H, 14O-61H, 24N-77H, 10N-62H, 49N-104H, 18O-72H, 63N-121H, 10N-65H, 10N-67H, and 10N-77H at the active sites of the 14 peptide sequences make important contributions to the antioxidant capacity of the antioxidant peptides.

[0064] Table 5. Antioxidant activity of 14 methyl-modified peptides

[0065] Note: Data with different letters in the same test indicate significant differences. p <0.05).

[0066] Example 5: Docking antioxidant peptides with MPO protein molecules Molecular docking analysis was used to study the interaction between the antioxidant peptide and the MPO protein. The crystal structure of the MPO protein (PDB ID: 3F9P) was downloaded from the protein database (https: / / www.rcsb.org / structure / 3F9P). Water molecules were removed using PyMol software, and hydrogen atoms and charges were added using MGLTools. The 3D structure of the antioxidant peptide was plotted using ChemDraw 20.0 software. Subsequently, molecular docking of the ligand with the MPO protein was performed using Autodock Tools 1.5.6, and the complex with the highest docking score was considered the best docking result. Finally, the complex structure was visualized using PyMol and Discovery Studio 4.5 software. The results are as follows: Figures 7-10 As shown in Table 6.

[0067] Based on the molecular docking results, the molecular mechanism of the antioxidant activity of the synthesized peptides was further analyzed. Figures 7-10 Table 6 shows the optimal conformations and main interactions between 14 peptides and the MPO receptor, primarily including hydrogen bonds (conventional hydrogen bonds, C-H bonds), electrostatic interactions (salt bridges, attracting charges, Pi-cations, Pi-anions), and hydrophobic interactions (alkyl groups, Pi-alkyl groups, Pi-Sigma, Pi-Pi T-shapes, amide-Pi superposition, Pi-Pi superposition, and Pi-sulfur bonds). Generally, a binding energy less than -5 kcal / mol indicates good binding strength, and a binding energy less than -7 kcal / mol indicates very strong binding. The binding energies of the synthetic peptides to MPO protein were -8.4 kcal / mol (NVGW), -9.3 kcal / mol (WALN), -9.2 kcal / mol (DFLRY), -9.2 kcal / mol (GYLPR), -8.6 kcal / mol (WGKLD), -9.2 kcal / mol (YVDRF), -9.7 kcal / mol (RWPVDL), -10.6 kcal / mol (WDPDAR), -10.4 kcal / mol (WLDQPH), -8.3 kcal / mol (GPYLDLK), -9.4 kcal / mol (RDWPDAR), -9.1 kcal / mol (WGDLSPK), -9.8 kcal / mol (WGDLSRP), and -9.2 kcal / mol (WDAAPGVPR), indicating that these eel protein peptides can bind tightly to MPO protein. Figures 7-10The 3D diagram shows the docking of the eel peptide molecule with the active cavity of MPO. When the peptide binds to MPO, it interacts with 35 amino acid residues, which are detailed in Table 6. Among them, key residues, including Arg239, Arg333, Arg424, Asp 94, Asp98, Gln91, Glu102, His95, His336, Pro145, Thr100, and Thr329, mainly interact with the peptide through hydrogen bonds and electrostatic interactions. Hydrogen bonding interactions mainly occur between Arg239, Arg333, Arg424, Asp 94, Asp98, Gln91, Glu102, His95, His336, Pro145, and Thr100. These electrostatic interactions mainly occur between basic amino acids (Arg239, Arg333, Arg424, His336, and His502) and acidic amino acids (Glu102 and Asp94) in MPO and tryptophan and C-terminal carboxyl groups in the 14 peptides.

[0068] Table 6. Docking sites and interactions between synthetic peptides and MPO receptors

[0069] Note: "+" indicates the number of interactions between the peptide and the receptor (green indicates hydrogen bonds; red indicates electrostatic interactions; purple indicates hydrophobic interactions).

[0070] Example 6: In vivo study of zebrafish 1. Zebrafish rearing and embryo collection: Wild-type AB series zebrafish were purchased from the National Zebrafish Resource Center (NZRC). The fish were maintained at 28±0.5℃ with a 14-hour light cycle followed by 10 hours of darkness. They were fed twice daily, at 09:00 and 17:00, with Artemia larvae until satiated. The amount of food was adjusted daily based on the previous day's food consumption, ensuring the fish finished all food within 3-5 minutes. After a 2-week acclimatization period, healthy and sexually mature zebrafish were selected and transferred to the breeding tank at a male-to-female ratio of 1:2 overnight. Fertilized embryos were collected at 10:00 the following day. After rinsing the embryos three times with ultrapure water, they were transferred to E3 embryo culture medium composed of 5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, and 0.33mM MgSO4. The experiment was conducted in accordance with the ethical guidelines approved by the Experimental Animal Welfare and Ethics Committee of the Guangdong Provincial Engineering Technology Research Center for Human Microecology (Approval No.: IACUC MC 0811-01-2025).

[0071] 2. Study on the mechanism of antioxidant peptides: Effects of antioxidant peptides on ROS content, MDA content, GSH / GSSG, SOD, CAT, GSH-PX activity and T-AOC in zebrafish embryos. (1) Detection of ROS levels in zebrafish embryos Normally developing 3-day post-fertilization (dpf) zebrafish embryos were randomly selected and placed in 48-well cell culture plates, with 10 zebrafish per well and one well per group. The experiment was divided into four groups: the normal group received E3 aqueous solution; the model group received 25 μg / mL LPS solution; the positive group received 250 μM NAC solution and 25 μg / mL LPS solution; and the test sample group received 100 μM eel bone peptide solution and 25 μg / mL LPS solution. 2 mL of solution was added to each well, and the culture plates were incubated at 28.5℃ for 72 hours, with the solution replaced every 24 hours. After incubation, the solution in the wells was discarded, and the zebrafish were washed three times with E3 water. Then, 20 μg / mL DCFH-DA solution was added, and the plates were incubated for 1 hour in the dark. After incubation, the zebrafish were washed three more times with E3 water and observed and photographed under an inverted fluorescence microscope. The fluorescence intensity in zebrafish was quantitatively analyzed using ImageJ software, and the ROS level (%) was calculated using the following formula: ROS level (%) = fluorescence intensity of the treatment group / fluorescence intensity of the blank group × 100. The treatment groups included the model group, the positive group, and the eel bone peptide treatment group. Results are as follows... Figure 11 As shown.

[0072] The DCFH-DA fluorescent probe can bind to reactive oxygen species (ROS) in cells, generating DCF with high fluorescence intensity. The green fluorescence intensity is positively correlated with the ROS content. ImageJ software was used to analyze the fluorescence microscopy images (…). Figure 11 (A)) Quantitative analysis was performed, and the results are as follows: Figure 11 As shown in (B). Figure 11 As can be clearly seen in (B), the fluorescence intensity of the zebrafish in the LPS-induced group was significantly higher than that in the control group. Except for the WGDLSPK treatment group, the fluorescence intensity of the 13 antioxidant peptide treatment groups was significantly lower than that in the model group. p <0.001). Data showed that the fluorescence intensity of the model group was 2.8329±0.0154 times that of the control group, indicating that LPS induction significantly increased the ROS content in zebrafish embryonic tissue ( p <0.001). Compared with the model group, the fluorescence intensity of the antioxidant peptide treatment group decreased by 27.56%±2.94% to 78.29%±0.99%. Among them, the fluorescence intensity of the NAC group decreased by about 74.29%±1.34%, and the ROS scavenging ability of the WDPDAR group was even stronger than that of the positive control group.

[0073] (2) Determination of MDA, GSH / GSSG, SOD, CAT, GSH-Px, and T-AOC in zebrafish embryos: Following the ROS level detection method described above, zebrafish embryos at 3 days post-fertilization (dpf) with normal development were randomly selected and placed in 6-well cell culture plates. 5 mL of solution was added to each well, and 40 zebrafish were placed in each well (3 wells per group). The culture plates were incubated at 28.5℃ for 72 hours, with fresh solution replaced every 24 hours. After incubation, 20 larvae were randomly selected from each group as a sample for subsequent testing. After euthanasia, the zebrafish larvae were homogenized with 600 μL of physiological saline. The homogenate was centrifuged at 4℃ and 2500 rpm for 10 minutes, and the supernatant was collected. Commercially available detection kits were used to detect MDA content and antioxidant enzyme activities, including SOD, CAT, and GSH-PX activities, as well as total antioxidant capacity (T-AOC). Results are as follows: Figure 12 As shown.

[0074] During oxidative stress, ROS react with unsaturated fatty acids in the cell membrane to generate MDA. MDA is often used as a marker of cell damage caused by oxidative stress in zebrafish models. Experimental results showed that the WGDLSPK treatment group, due to its higher ROS content, exhibited significantly increased MDA levels in zebrafish, reaching 3.0828 ± 0.0497 nmol / mg prot ( Figure 12 (A)); followed by the GPYLDLK treatment group, with an MDA content of 1.4458 ± 0.0847 nmol / mg prot. In contrast, the MDA content of other treatment groups was significantly lower than that of the LPS treatment group. These results indicate that LPS induces oxidative stress in zebrafish embryos, leading to a significant accumulation of ROS in vivo, while antioxidant peptides can effectively alleviate oxidative damage in zebrafish embryos, demonstrating a certain protective effect. GSH is often used as the first line of defense against oxidative stress. Figure 12 As shown in (B), the GSH / GSSG ratio in the control group was relatively high, at 1.0219 ± 0.0301, indicating that under normal physiological conditions, the GSH and GSSG contents in zebrafish are comparable. In contrast, the GSH / GSSG ratio in the LPS group decreased significantly to 0.7631 ± 0.0165. However, the GSH / GSSG ratios in 10 peptide treatment groups were significantly higher than those in the LPS group ( p<0.001), indicating that these peptides effectively mitigated oxidative damage by increasing the GSH / GSSG ratio. Among them, the WLDQPH and WDAPGVPR treatment groups had the highest GSH / GSSG ratios, at 0.97103±0.108 and 0.9787±0.0200 respectively, significantly higher than other peptide treatment groups; followed by the WGKLD and YVDRF treatment groups, with GSH / GSSG ratios exceeding 0.9247±0.0190. Furthermore, zebrafish contain abundant enzymatic antioxidant systems (such as SOD, CAT, GSH-Px, etc.), which help protect the body from oxidative damage. Figure 12 As shown in Figures (C)-(E), the activities of SOD, CAT, and GSH-Px in zebrafish treated with LPS were significantly reduced. p <0.001%, which were 0.8637, 0.5064, and 0.5503 times that of the control group, respectively, indicating that LPS stimulation activated the body's enzyme defense system, thereby accelerating the consumption of antioxidant enzymes. Compared with the LPS-treated group, the enzyme activities of the peptide-treated groups were restored to varying degrees. Specifically, the activities of SOD (73.8538±2.3500 U / mg protein), CAT (8.8815±0.3467 U / mg protein), and GSH-Px (224.9341±12.3437 U / mg protein) were all higher in the WGDLSRP-treated group. In addition, the RWPVDL, WDPDAR, WLDQPH, and GPYLDLK peptide-treated groups also showed higher antioxidant enzyme activities. In summary, compared with the LPS-treated group, antioxidant peptide treatment significantly improved the activity of non-enzymatic and enzymatic antioxidant systems in zebrafish, thereby significantly increasing the T-AOC value ( Figure 12 (F)), of which 13 groups showed highly significant differences ( p <0.001).

[0075] (3) Effects of antioxidant peptides on the expression of Keap1, Nrf2, HO-1, GCL and GSS genes Zebrafish were grouped using the same method as above. Total RNA was extracted from zebrafish homogenates using the G3640-50T kit (Seville Biotechnology Co., Ltd.), and then cDNA was synthesized using the KR128 FastKing one-step method (Beijing Tiangen Biotech Co., Ltd.), after which genomic DNA was removed. Real-time PCR reactions were performed on a Light Cycler 96 system (Roche), with the following parameters: pre-denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C and annealing at 60°C. Gene expression levels were normalized using Actin mRNA as an internal control and analyzed by... The method calculates the relative quantification of the target gene in each sample. The results are as follows: Figure 12 As shown.

[0076] The Keap1-Nrf2 pathway is a key signaling pathway for endogenous antioxidant stress. When zebrafish underwent LPS-induced oxidative stress, the transcriptional level of the Keap1 gene in the LPS group significantly increased. p <0.001, approximately 2.5402 times that of the control group; while the Nrf2 gene transcription level was significantly decreased ( p <0.001), which was 0.6470 times that of the control group. Compared with LPS, the antioxidant peptide treatment group significantly reduced the transcription level of the Keap1 gene, approximately 0.4253-0.8810 times that of the LPS group, while increasing the transcription level of the Nrf2 gene, reaching 1.1520-1.7083 times that of the LPS group (see <0.001). Figure 12 (G)- Figure 12 (H) Further molecular mechanisms revealed that antioxidant peptides activate antioxidant stress signaling pathways by upregulating the transcriptional levels of GCL, GSS, and HO-1 genes. Specifically, LPS treatment reduced the transcriptional levels of GCL, GSS, and HO-1 genes to 0.6060, 0.5604, and 0.5125, respectively, compared to the control group, while the protein peptide treatment group increased the transcriptional levels of all three genes to varying degrees (see [link to study]). Figure 12 (I)- Figure 12 (K)). This observation is consistent with the report by Ji and Wasik et al. Pearson correlation analysis showed that ROS levels in zebrafish were significantly correlated with the GSH / GSSG ratio, GSH-Px activity, and the expression of genes such as GCL, GSS, HO-1, Keap1, and Nrf2. p <0.01). This indicates that the main mechanism of action of the antioxidant peptides in eel bone is through regulating genes related to the Keap1-Nrf2 pathway, thereby upregulating the activities of GSH-Px and HO-1, improving the GSH / GSSG ratio, and ultimately effectively inhibiting oxidative stress and enhancing antioxidant capacity.

[0077] (4) Mechanism study of antioxidant peptides inhibiting LPS-induced oxidative damage in zebrafish Reaction mechanism such as Figure 13As shown, LPS, acting as a free radical initiator, triggers the generation of a large amount of ROS when interacting with zebrafish. Excess ROS interacts with unsaturated fatty acids in the cell membrane, leading to a significant increase in MDA. Antioxidant peptides reduce the level of free radicals in zebrafish by directly reacting with the peroxide free radicals generated by LPS. GSH, as an important non-enzymatic antioxidant in zebrafish, plays a crucial role and is the main line of defense against oxidative stress. Under the action of GSH-Px, GSH is oxidized to GSSG. Excess GSSG can be converted back to GSH with the help of NADPH and glutathione reductase (GR), thereby effectively inhibiting lipid peroxidation. In addition, zebrafish also possess enzymatic antioxidant defense systems such as SOD, CAT, and GSH-Px. SOD converts O2... - Free radicals are converted into H2O2, which is then reduced to water by CAT, while GSH-Px converts H2O2 into water or reacts with GSH to produce water. Both non-enzymatic and enzymatic reactions maintain the dynamic balance between free radical generation and scavenging in vivo, directly or indirectly participating in redox reactions and maintaining normal cellular physiological functions. However, when zebrafish are exposed to free radicals for extended periods, their intracellular defense systems are unable to clear excess free radicals, leading to oxidative damage. Antioxidant peptides from zebrafish bone, by activating the Keap1-Nrf2 pathway, promote the expression of HO-1 and enzymes involved in glutathione synthesis (such as GCL and GSS), significantly increasing the GSH / GSSH ratio, as well as the enzyme activities of SOD, CAT, and GSH-PX, thereby enhancing the total antioxidant capacity of zebrafish and inhibiting LPS-induced oxidative damage. This indicates that antioxidant peptides from zebrafish bone have potential in improving oxidative damage in zebrafish.

[0078] In summary, the experiments revealed 14 novel antioxidant peptides (NVGW, WALN, DFLRY, GYLPR, WGKLD, YVDRF, RWPVDL, WDPDAR, WLDQPH, GPYLDLK, RDWPDAR, WGDLSPK, WGDLSRP, and WDAAPGVPR) through identification and computer simulation screening of antioxidant-rich components. These 14 peptides exhibited stronger activity than the positive control. Quantum chemical calculations indicated that the hydrogen atoms on the indole nitrogen of tryptophan, the phenolic hydroxyl group of tyrosine, and the guanidino nitrogen atom of arginine are key sites for antioxidant activity. Antioxidant studies in zebrafish embryos showed that eel bone peptides can activate the in vivo antioxidant system by regulating the Keap1-Nrf2 pathway, thereby exerting their antioxidant activity. The comprehensive screening strategy established in this study provides a possibility for high-throughput screening of antioxidant peptides. It also provides a theoretical basis for the functional activity research of eel bone antioxidant peptides and the development of high-value-added products.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of eel bone antioxidant peptides in the preparation of antioxidant products, characterized in that, The amino acid sequence of the antioxidant peptide from the eel bone is at least one of those shown in SEQ ID NO.3-14.

2. The application as described in claim 1, characterized in that, The effective concentration of the antioxidant peptides from the eel bone is 80-120 μM.

3. The application as described in claim 1, characterized in that, The products include pharmaceuticals, food, or cosmetics.

4. A method for preparing antioxidant peptides from eel bones, characterized in that, The procedure includes the following steps: enzymatic hydrolysis of eel bones with Alcalase to obtain an eel bone hydrolysate; separation and purification of the eel bone hydrolysate by gel column chromatography, collection of eluents according to peak shape, and identification of eluents with good antioxidant activity by UPLC-Q-TOF-MS / MS; prediction of the bioactivity score of the identified peptides using the PeptideRanker database, and prediction of the sensitization, potential toxicity, and physicochemical properties of the active peptides using the AllerTOP and ToxinPred databases to obtain eel bone antioxidant peptides with high antioxidant activity; the amino acid sequence of the eel bone antioxidant peptides is at least one of those in SEQ ID NO. 3-14.

5. The preparation method according to claim 4, characterized in that, Add Alcalase alkaline protease solution according to the protein content of the eel bone, which is 1-7 w / w%.

6. The preparation method according to claim 4, characterized in that, The enzymatic hydrolysis conditions include: pH 7.8-8.2, temperature 53-57℃, and time 4-8h.

7. The preparation method according to claim 4, characterized in that, The conditions for the gel column chromatography include: a Sephadex G-25 gel column, water as the eluent, and wavelengths of 210-218 nm and 250-258 nm.

8. The preparation method according to claim 4, characterized in that, The chromatographic column used for UPLC-Q-TOF-MS / MS identification is ACQUITYUPLC HSS T3, and the column temperature is 40-50℃.

9. The preparation method according to claim 4, characterized in that, The mobile phase A identified by UPLC-Q-TOF-MS / MS was an aqueous solution containing 0.1% formic acid, and the mobile phase B was an 80% acetonitrile solution. The flow rate was set to 0.4 mL / min, and the gradient elution program was: 0-2.0 min, 92.0% A; 2.0-45.0 min, 92.0-72.0% A; 45.0-55.0min, 72.0-60.0% A; 55.0-56.0min, 60.0-5.0% A; 56.0-66.0 min, 5.0% A.

10. An antioxidant product, characterized in that, The active ingredient of the antioxidant product includes at least one of the amino acid sequences shown in SEQ ID NO.3-14.

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