Atlantic salmon peptide with antioxidant activity, screening method and application thereof

Abstract

The present application relates to the field of biotechnology, specifically to Atlantic salmon peptide with antioxidant activity and screening method and application thereof; the amino acid sequence of the Atlantic salmon peptide is selected from one or more combinations of polypeptides shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8; the Atlantic salmon peptide has excellent free radical scavenging capacity and cell mitochondrial protection function, good water solubility, high safety and strong stability; wherein the three kinds of Atlantic salmon peptides shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.8 can be combined with docosahexaenoic acid (DHA) through intermolecular interaction, thereby protecting mitochondrial function and playing a more significant role in oxidative damage protection and cell viability maintenance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to Atlantic salmon peptides with antioxidant activity, their screening methods, and applications. Background Technology

[0002] Oxidative stress is a state of damage to the body caused by an imbalance between the production and clearance of free radicals, and it is closely related to aging, inflammation, apoptosis, and various chronic diseases. Developing safe, efficient, and natural antioxidants is a research hotspot in the fields of biomedicine and functional foods. DHA (docosahexaenoic acid), as an important Omega-3 polyunsaturated fatty acid, plays a crucial role in physiological activities such as brain function, visual development, and cardiovascular regulation. However, its molecule contains multiple unsaturated double bonds, making it chemically extremely unstable and susceptible to oxidative rancidity induced by factors such as oxygen, light, and temperature, which limits its application scenarios.

[0003] Currently, synthetic antioxidants pose potential safety risks; natural extracts suffer from poor compatibility, batch instability, and limited functionality. Bioactive peptides, on the other hand, offer advantages such as well-defined structures, controllable activity, high safety, and ease of modification. Some peptides exert antioxidant effects by scavenging free radicals and protecting cell structures, while others can form stable complexes with lipids, achieving lipid stabilization. Developing peptides that possess both high antioxidant activity and mitochondrial protective function, and can specifically bind to lipids, is an important direction for development in the fields of biomedicine and functional formulations.

[0004] Current methods for screening antioxidant peptides mostly employ random enzymatic digestion combined with activity tracking, lacking targeting specificity and failing to enrich peptides that bind specifically to lipids. Furthermore, they exhibit low capture efficiency for low-abundance, high-activity peptides. Simultaneously, there are no reports of peptide sequences derived from Atlantic salmon that possess both high-efficiency antioxidant and mitochondrial protective functions, and specifically bind to DHA, nor are there standardized preparation techniques. Systematic studies of related molecular interaction mechanisms are also lacking. This invention establishes a dedicated screening method combining affinity purification and unbiased proteomics to target and screen for highly active antioxidant peptides derived from Atlantic salmon. A solid-phase synthesis process is then used to achieve standardized preparation of these peptides, filling a technological gap in this field. Summary of the Invention

[0005] The first objective of this invention is to provide Atlantic salmon peptides with antioxidant activity; the second objective is to provide a screening method for Atlantic salmon peptides with antioxidant activity; and the third objective is to provide applications for Atlantic salmon peptides with antioxidant activity, solving problems such as low screening efficiency and poor targeting of existing antioxidant peptides, as well as safety, stability, and limited functionality of antioxidant technologies. Simultaneously, through molecular simulation technology, the interaction mode and stabilization mechanism between the antioxidant peptide and DHA are analyzed, providing a theoretical basis for its expanded applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides Atlantic salmon peptides with antioxidant activity, wherein the amino acid sequence of the Atlantic salmon peptides is selected from one or more combinations of the polypeptides shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8; wherein the polypeptides with sequences such as SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.8 specifically bind to DHA.

[0008] The present invention also provides a nucleic acid encoding the Atlantic salmon peptide as described above.

[0009] The present invention also provides an expression vector containing the nucleic acid described above.

[0010] The present invention also provides recombinant cells containing the expression vectors described above, wherein the recombinant cells include Escherichia coli, yeast, and mammalian cell lines.

[0011] The present invention also provides a method for screening Atlantic salmon peptides with antioxidant activity, comprising the following steps:

[0012] (1) Construction of molecular tags: Based on the specific interaction between biotin and streptavidin, biotin is covalently linked to the head group of phospholipids through chemical synthesis to construct a molecular hook system; the molecular hook system includes three biotin-tagged probes with different fatty acid chain combinations, namely biotinylated phospholipids containing double saturated fatty acid chains, saturated / polyunsaturated mixed fatty acid chains and double polyunsaturated fatty acid chains.

[0013] (2) Acquisition of polypeptides: Atlantic salmon muscle tissue was used as raw material. After grinding, tissue lysis, and centrifugation, the supernatant was collected. Trypsin was used to enzymatically hydrolyze the proteins in the supernatant to obtain a polypeptide mixture.

[0014] (3) Incubation and affinity capture: An experimental group and a control group were set up. The experimental group consisted of three biotin-labeled probes with different fatty acid chain combinations as described in step (1). The control group consisted of a solvent blank control group and a free biotin control group to exclude proteins that bind nonspecifically. Each group was mixed with the polypeptide mixture obtained in step (2) and incubated. Then streptavidin agar beads were added to capture the polypeptides. The polypeptides were washed 5 times with phosphate buffered saline solution containing 0.3% Tween. The captured polypeptides were obtained after washing.

[0015] (4) Mass spectrometry analysis: The captured peptides were analyzed by liquid chromatography-tandem mass spectrometry, and the sequence was identified by peptide mass fingerprint matching based on the Uniprot database;

[0016] (5) Peptide function prediction and screening of antioxidant active peptides: Based on the mass spectrometry analysis results of step (4), the peptide function is predicted by bioinformatics and Atlantic salmon peptides with antioxidant activity are screened.

[0017] The present invention also provides the application of the screening method described above in screening proteins or peptides that specifically bind to various phospholipids or fatty acids.

[0018] The present invention also provides the use of Atlantic salmon peptides with antioxidant activity as described above in the preparation of antioxidant preparations or cell protection preparations, the preparations including health foods, cosmetics, and functional food additives.

[0019] Preferably, the Atlantic salmon peptide with antioxidant activity is used alone as an active ingredient or in combination with other antioxidant active substances.

[0020] The present invention also provides the use of nucleic acids as described above in the preparation of antioxidant or cell protection agents, the agents including health foods, cosmetics, and functional food additives.

[0021] Preferably, the nucleic acid is used alone as an active ingredient or in combination with other antioxidant active substances.

[0022] The design principle of this invention is as follows: Addressing the shortcomings of existing antioxidant technologies and building upon previous research on polyunsaturated fatty acids, this invention targets the muscle of Atlantic salmon, which is rich in Omega-3, to discover natural antioxidant active substances. Through a targeted screening strategy of "affinity purification + unbiased proteomics," eight structurally well-defined Atlantic salmon-derived peptide sequences were ultimately identified. Their antioxidant properties were specifically verified, and the molecular interaction mechanism between the peptides and DHA was simultaneously elucidated, providing theoretical support for their application in various scenarios such as antioxidant preparations and cell protection products.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention provides eight specific polypeptide sequences derived from Atlantic salmon that have both high antioxidant and mitochondrial protection effects. The sequences are well-defined and their activities can be quantified. Among them, the polypeptide sequences shown in SEQ ID NO.2, 4 and 8 can be specifically bound to DHA by molecular docking verification. The binding free energy is ≤-6.7 kcal / mol, which can enhance cell protection and has the potential value of lipid stabilization, filling the gap in peptide sequences in this field.

[0025] (2) The antioxidant active peptides of the present invention have high safety, good water solubility and strong stability, and can achieve dual functions of free radical scavenging and cell protection. At the same time, based on a clear molecular interaction mechanism, they can be extended to DHA stabilization, breaking through the limitation of the single function of traditional antioxidants, and have a wide range of applications.

[0026] (3) This invention combines the targeting of affinity capture with the high-throughput advantage of proteomics, which can efficiently enrich peptides bound to unsaturated fatty acid chains and establish a dedicated screening method for targeted Atlantic salmon antioxidant peptides. It can accurately screen peptide sequences that are non-toxic, highly bioactive, and have excellent antioxidant properties, providing a new technical approach for screening similar active peptides.

[0027] (4) The antioxidant active peptides of the present invention are applicable to multiple fields such as biomedicine, functional preparations, cell culture, and lipid stabilization, providing new active ingredients for the development of related products.

[0028] (5) This invention elucidates the interaction mechanism between peptide and DHA through molecular docking, providing possible theoretical support for the subsequent sequence modification, activity optimization and application expansion of the peptide. Attached Figure Description

[0029] Figure 1 This is a thin-layer chromatography (TLC) chromatogram of the phospholipid biotin-labeled reaction system.

[0030] Figure 2 This is a thin-layer chromatography (TLC) image of the organic phase after extraction of the phospholipid biotin-labeled product.

[0031] Figure 3 Mass spectrum of the product synthesized from Biotin-PE (16:0-16:0);

[0032] Figure 4 Mass spectrum of the product synthesized from Biotin-PE (16:0-22:6);

[0033] Figure 5 Mass spectrum of the product synthesized from Biotin-PE (22:6-22:6);

[0034] Figure 6 The mass spectrometry image shows the solid-phase synthesized antioxidant peptide with the sequence shown in SEQ ID NO.1.

[0035] Figure 7 The mass spectrometry image shows the sequence of the solid-phase synthesized antioxidant peptide as shown in SEQ ID NO.2.

[0036] Figure 8 The mass spectrometry image shows the sequence of the solid-phase synthesized antioxidant peptide as shown in SEQ ID NO.3.

[0037] Figure 9 The mass spectrometry image shows the sequence of the solid-phase synthesized antioxidant peptide as shown in SEQ ID NO.4.

[0038] Figure 10 The mass spectrometry image shows the sequence of the solid-phase synthesized antioxidant peptide as shown in SEQ ID NO.5.

[0039] Figure 11 The mass spectrometry image shows the sequence of the solid-phase synthesized antioxidant peptide as shown in SEQ ID NO.6.

[0040] Figure 12 The mass spectrometry image shows the sequence of the solid-phase synthesized antioxidant peptide as shown in SEQ ID NO.7.

[0041] Figure 13 The mass spectrometry image shows the sequence of the solid-phase synthesized antioxidant peptide as shown in SEQ ID NO.8.

[0042] Figure 14 The in vitro reactive oxygen species scavenging capacity of eight antioxidant peptides is shown in the figure.

[0043] Figure 15 This is a cell viability protection diagram of antioxidant peptides with sequences as shown in SEQ ID NO.1-4;

[0044] Figure 16 This is a cell viability protection diagram of antioxidant peptides with sequences as shown in SEQ ID NO.5-8;

[0045] Figure 17 A graph showing the cell viability protection effect of eight antioxidant peptides combined with DHA;

[0046] Figure 18 Flow cytometry data showing the protective effect of three antioxidant peptides on mitochondrial membrane potential.

[0047] Figure 19 A statistical graph showing the protective effects of three antioxidant peptides on mitochondrial membrane potential.

[0048] Figure 20 This diagram illustrates the rescue effect of three antioxidant peptides on ATP production in cells under oxidative stress.

[0049] Figure 21 A diagram illustrating how three antioxidant peptides combined with DHA enhance cellular superoxide dismutase activity.

[0050] Figure 22 A diagram illustrating how the combination of three antioxidant peptides with DHA enhances the total antioxidant capacity of cells.

[0051] Figure 23 This is a schematic diagram showing the docking and binding patterns of three antioxidant peptides with DHA molecules. Detailed Implementation

[0052] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0053] Example 1

[0054] The screening of short peptides that may bind to polyunsaturated fatty acids involves the following steps:

[0055] (1) Molecular tag construction: Biotin was covalently linked to the head group of phospholipids through chemical synthesis to construct a molecular fishing hook system. The molecular fishing hook system includes three biotin-tagged probes with different fatty acid chain combinations, namely biotinylated phospholipids containing double saturated fatty acid chains, saturated / polyunsaturated mixed fatty acid chains, and double polyunsaturated fatty acid chains. The specific steps are as follows:

[0056] This step is based on the extremely strong and highly specific non-covalent interaction between biotin and streptavidin to construct a molecular fishing hook system.

[0057] The key experimental reagents used in this embodiment are shown in Table 1.

[0058] Table 1 Key Experimental Reagents

[0059]

[0060] Biotin labeling of phospholipids: 20 mg (0.024 mmol) of 22:6-22:6 PE, 16:0-22:6 PE, and 16:0-16:0 PE (dissolved in chloroform, 10 mg / mL) were transferred to round-bottom flasks. NHS-Biotin (8.98 mg, 0.026 mmol) was then added and dissolved in methanol (1 mL). Triethylamine (6.65 μL) was then added to the round-bottom flasks using a syringe. The flasks were sealed with rubber stoppers and allowed to react at room temperature for 5 h.

[0061] After the reaction was completed, thin-layer chromatography (TLC) was used to confirm the reaction status, such as... Figure 1As shown, the polarity difference between the product and the reactants / reagents, and the spot matching, confirm that the reaction occurred and the target product was generated. The developing solvent was dichloromethane:methanol (DCM:MeOH) = 3:1. Iodine vapor was used for color development. The Rf value of the target product was higher than that of the phospholipid reactants, confirming the complete reaction. After confirming the reaction status, the reaction solution was initially concentrated by rotary evaporation. The concentrated crude product was dissolved and diluted with DCM, transferred to a separatory funnel, and the organic phase was washed with small amounts of 10% ammonium chloride solution and saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate, and the extraction was confirmed by TLC. Figure 2 As shown, the distribution of spots containing the product in the organic phase and not in the aqueous phase confirms that the extraction operation successfully transferred the product from the reaction system to the organic phase. The developing solvent was DCM:MeOH (3:1). The product was concentrated using a rotary evaporator to obtain the biotin-labeled phospholipid product. The chemical reaction formula for this step is as follows:

[0062] Synthesis of Biotin-PE (16:0-16:0):

[0063] ;

[0064] Synthesis of Biotin-PE (16:0-22:6):

[0065] ;

[0066] Synthesis of Biotin-PE (22:6-22:6):

[0067] .

[0068] Mass spectrometry characterization: The obtained product was analyzed by liquid chromatography, and the characterization results are as follows. Figure 3 , 4 As shown in Figure 5: Figure 3 The target peak appears at 916.5866 (HRMS (ESI) m / z calculated for C). 47 H 88 N3O 10 PS [MH]+:916.5855; found: 916.5866), corresponding to a molecular weight of 916.5855 for Biotin-PE (16:0-16:0);

[0069] Figure 4 The target peak appears at 988.5869 (HRMS (ESI) m / z calculated for C). 53 H 88 N3O 10PS[MH]+: 988.5855; found: 988.5869), corresponding to a molecular weight of 988.5855 for Biotin-PE (16:0-22:6);

[0070] Figure 5 The target peak appears at 1060.5861 (HRMS (ESI) m / z calculated for C). 59 H 88 N3O 10 PS [MH]+: 1060.5855; found: 1060.5861), corresponding to a molecular weight of 1060.5855 for Biotin-PE (22:6-22:6).

[0071] Chromatographic conditions: reversed-phase C18 capillary column (75 μm × 25 cm, 2 μm packing), mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid acetonitrile solution, gradient elution, flow rate 300 nL / min;

[0072] Mass spectrometry conditions: Qexactive HFX mass spectrometer, positive ion mode, scan range m / z 350-1800, first-order resolution 60,000, second-order resolution 15,000.

[0073] (2) Acquisition of peptides: Fresh Atlantic salmon muscle tissue was ground with liquid nitrogen and then lysed with lysis buffer. The mixture was centrifuged at 4°C and 12,000 rpm for 20 min and the supernatant was collected. Trypsin (enzyme-to-substrate ratio 1:100) was added and the mixture was placed at 37°C for 4 h to obtain a mixed solution of Atlantic salmon peptides.

[0074] (3) Incubation and affinity capture: Experimental and control groups were set up. The experimental groups were biotinylated phospholipid 1 [Biotin-PE (16:0-16:0)], biotinylated phospholipid 2 [Biotin-PE (16:0-22:6)], and biotinylated phospholipid 3 [Biotin-PE (22:6-22:6)]. The control groups were the simple solvent (Vehicle) group and the biotin N-hydroxysuccinimide ester (NHS-Biotin) group. Each group was mixed with the polypeptide mixture and incubated overnight at 4°C. Then streptavidin agar beads were added and incubated at room temperature for 2 hours. The peptides were washed 5 times with phosphate buffered saline (PBST) containing 0.3% Tween to obtain the captured peptides.

[0075] (4) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS): After pretreatment of the captured peptides, liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used for detection. The obtained mass spectrometry data were compared with the Uniprot database, and a total of 1134 peptides were obtained. By comparing with the control group, non-specifically bound peptides were removed. Then, the peptides of the three groups of biotinylated phospholipids were compared to screen for peptides captured in the Biotin-PE (16:0-22:6) group and the Biotin-PE (16:0-22:6) group but not captured in the Biotin-PE (16:0-16:0) group. Peptides with DHA binding sites were screened according to the literature, and 31 candidate peptide sequences that may bind to the tail of phospholipid polyunsaturated fatty acids were screened. The results are shown in Table 2:

[0076] Table 2. Peptide sequences that may bind to DHA

[0077]

[0078] Example 2

[0079] Antioxidant peptides were screened based on the physicochemical properties, toxicity, and antioxidant capacity of the 31 peptides screened in Example 1.

[0080] (1) Screening short peptides based on the physicochemical property thresholds of food-derived short peptides:

[0081] Standards: molecular weight less than 1800 Da, net charge absolute value not greater than 2, hydrophobicity index not higher than 0.5, and proportion of hydrophobic residues ≤ 50%. Results indicated that 10 of the 31 peptides potentially binding to DHA met the criteria for food-derived short peptides, as shown in Table 3.

[0082] Table 3. Food-derived short peptides and their physicochemical properties

[0083]

[0084] (2) Potential toxicity prediction was performed using the ToxinPrep platform. The SVM algorithm was used to analyze the potential toxicity of the 10 peptide sequences that conformed to the physicochemical properties of food-derived peptides. The results indicated that of the 10 peptides, one was a potentially toxic peptide, while the remaining nine were non-toxic peptides, as shown in Table 4.

[0085] Table 4. Prediction of food-derived peptide toxicity

[0086]

[0087] (3) Antioxidant activity prediction: The antioxidant activity of the nine non-toxic peptides in Table 4 was predicted using the AnOxPP platform. The screening criterion was a re-Score value > 0.8. The results showed that eight peptides were antioxidant peptides and one peptide was a non-antioxidant peptide. The results are shown in Table 5.

[0088] Table 5 Food-derived antioxidant peptides

[0089]

[0090] The final screened antioxidant peptides are as follows:

[0091] SEQ ID NO.1: EIWGMEPSLEK;

[0092] SEQ ID NO.2: HGDSSVPVWSG;

[0093] SEQ ID NO.3: QFAAQANIIGPWIQTK;

[0094] SEQ ID NO.4: GYEEWLLTEIRR;

[0095] SEQ ID NO.5: DVGVHWVILGHSER;

[0096] SEQ ID NO.6: HGESEWNQYNK;

[0097] SEQ ID NO.7: TVDGPSHKDWR;

[0098] SEQ ID NO.8: PEITPPPSQSQPAR.

[0099] Example 3

[0100] Solid-phase synthesis of antioxidant peptides

[0101] In this embodiment, 2-chlorotriphenylmethyl chloride resin (2-CTC) was used as a solid-phase synthesis support. The eight Atlantic salmon-derived antioxidant peptides screened in Example 2 were prepared using the fluorene methoxycarbonyl (Fmoc) solid-phase synthesis method. The specific operation steps are as follows:

[0102] (1) Weigh 2-CTC resin, add dichloromethane (DCM) to swell and soak for 1 hour, pour out the DCM, and wash the resin 3 times with N,N-dimethylformamide (DMF) to complete the resin activation;

[0103] (2) According to the C-terminus to N-terminus sequence of the target peptide amino acid sequence, take 1 eq of the first amino acid protected by Fmoc and 1.5 eq of N,N-diisopropylethylamine (DIEA), prepare a reaction solution with DMF as solvent, mix with the activated 2-CTC resin, and react at room temperature for 2 hours;

[0104] (3) After the reaction is complete, the resin is dried and washed with DMF 3 times. A mixture of methanol and DIEA is added to the resin to seal the head. The reaction is carried out at room temperature for 1 hour to block the unbound active sites.

[0105] (4) After the head is finished, clean the resin, add 20% piperidine DMF solution to remove the Fmoc protecting group, react in two batches, 10 min each time, and wash the resin thoroughly after deprotection.

[0106] (5) Take 3 eq of the next Fmoc protected amino acid, 3 eq of 1-hydroxybenzotriazole (HOBT), and 3 eq of N,N'-diisopropylcarbodiimide (DIC), prepare a coupling solution with DMF as solvent, and react with the resin at room temperature for 1.5 hours to complete the coupling of the amino acid.

[0107] (6) Repeat the above steps (4)-(5) for deprotection and amino acid coupling operations to complete the linkage of amino acid residues of the target peptide sequence in sequence, and finally remove the Fmoc protecting group at the N end.

[0108] (7) After the full sequence assembly is completed, thoroughly clean the resin with DMF to remove residual reagents and dry the solvent on the resin surface.

[0109] (8) Prepare a cleavage reagent (trifluoroacetic acid: triisopropylsilane: ethylenedithiol: water in a volume ratio of 95:2:2:1), mix it with the dried resin, and react at room temperature for 2 hours to achieve peptide chain cleavage and removal of side chain protecting groups;

[0110] (9) After the reaction is complete, filter to remove the resin, collect the filtrate, wash the filtrate repeatedly with ice-cold ether, centrifuge and retain the precipitate to obtain crude polypeptide;

[0111] (10) The crude peptide was separated and purified by high performance liquid chromatography. After collecting the target peak, it was freeze-dried to obtain an antioxidant peptide product with a purity ≥95%. The mass spectrometry characterization results of the synthesized product are as follows: Figure 6-13As shown, the target peaks appear at their respective molecular weight positions. The molecular weight of the antioxidant peptide with the sequence shown in SEQ ID NO.1 is 1319.63 (purity 97.64%), the molecular weight of the antioxidant peptide with the sequence shown in SEQ ID NO.2 is 1128.13 (purity 95.06%), the molecular weight of the antioxidant peptide with the sequence shown in SEQ ID NO.3 is 1785.96 (purity 97.62%), the molecular weight of the antioxidant peptide with the sequence shown in SEQ ID NO.4 is 1565.71 (purity 96.57%), the molecular weight of the antioxidant peptide with the sequence shown in SEQ ID NO.5 is 1603.72 (purity 95.15%), the molecular weight of the antioxidant peptide with the sequence shown in SEQ ID NO.6 is 1392.36 (purity 95.24%), and the molecular weight of the antioxidant peptide with the sequence shown in SEQ ID NO.7 is 1298.53 (purity 95.22%). The antioxidant peptide shown in NO.8 has a molecular weight of 1505.65 (purity 96.06%).

[0112] Example 4

[0113] In vitro antioxidant activity assay

[0114] The in vitro antioxidant activity of eight high-purity antioxidant peptides synthesized in the solid phase in Example 3 was verified using a body fluid oxidation-activated oxygen luminol chemiluminescence assay. The peptide concentration was set at 1000 μg / mL, and each experiment was performed in triplicate. The scavenging rate was calculated by taking the average value. The results are as follows: Figure 14 As shown, at a concentration of 1000 μg / mL, all eight antioxidant peptides synthesized in Example 3 exhibited strong in vitro free radical scavenging capabilities. Among them, the relative reactive oxygen species residues of the antioxidant peptide groups shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.8 were all less than 45%, at 41.47%, 34.97%, 31.78%, and 34.97%, respectively, suggesting that these four antioxidant peptides have good antioxidant potential as food-derived peptides.

[0115] Example 5

[0116] Cell viability protection experiment

[0117] Using TM4 cells as a model, the protective effect of the eight antioxidant peptides of this invention on cell viability was evaluated using the H2O2 oxidative damage method. (1) Seed TM4 cells at an appropriate density in 96-well plates and cultured for 8 h until they adhered stably; (2) Grouping: set up a blank control group, a model group (H2O2 damage), and 8 antioxidant peptide treatment groups (final concentrations set at 20, 50, 100, and 200 nmol / L), and screened the optimal antioxidant peptide + DHA treatment group (i.e., DHA was added to the antioxidant peptide group to make the final concentration 50 nmol / L); (3) Except for the blank control group, each group was incubated with 500 μmol / L H2O2 for 2 h to establish an oxidative damage model; (4) Discard the culture medium, wash with PBS, add the corresponding concentration of antioxidant peptide culture medium to each group, and continue to culture for 24 h; (5) Use the CCK-8 method to detect cell viability, add 10 μL of CCK-8 reagent to each well, incubate at 37℃ in the dark for 1–2 h, measure the absorbance OD value at 450 nm, and calculate the relative cell viability.

[0118] The results are as follows Figure 15-16 As shown, compared with the H2O2 model group, all eight antioxidant peptides of this invention can significantly improve the survival rate of oxidatively damaged cells. The optimal concentration is determined by a significant increase in cell survival rate compared with the damage group, i.e., P<0.05. Specifically, the optimal concentration of the antioxidant peptide shown in SEQ ID NO.1 is 20 nM, the optimal concentration of the antioxidant peptide shown in SEQ ID NO.2 is 20 nM, the optimal concentration of the antioxidant peptide shown in SEQ ID NO.3 is 100 nM, the optimal concentration of the antioxidant peptide shown in SEQ ID NO.4 is 20 nM, the optimal concentration of the antioxidant peptide shown in SEQ ID NO.5 is 20 nM, the optimal concentration of the antioxidant peptide shown in SEQ ID NO.6 is 200 nM, the optimal concentration of the antioxidant peptide shown in SEQ ID NO.7 is 50 nM, and the optimal concentration of the antioxidant peptide shown in SEQ ID NO.8 is 20 nM.

[0119] The optimal concentrations of each antioxidant peptide were combined with docosahexaenoic acid (DHA) to act synergistically on cells, and the effects of these eight antioxidant peptides on DHA were observed. The results are as follows: Figure 17 As shown in the figure, the three antioxidant peptides shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.8, when combined with DHA, can significantly improve cell survival rate, which is significantly higher than that of the DHA-only group, and is statistically significant.

[0120] The above results demonstrate that all eight antioxidant peptides of this invention can effectively reduce free radical damage to cells and enhance the cells' ability to resist oxidative damage. Among them, the antioxidant peptides shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.8 may have a synergistic effect with DHA, exhibiting more significant protective effects against oxidative damage and maintenance of cell viability. The following examples focus on investigating the related mechanisms of the cell-protective effects of these three antioxidant peptides that may have a synergistic effect with DHA.

[0121] Example 6

[0122] Cellular mitochondrial protection experiment

[0123] Using TM4 cells as an experimental model, an H2O2 (500 μmol / L) oxidative damage model was established to verify the mitochondrial protective function of three antioxidant peptides that may synergize with DHA in the examples. TM4 cells were divided into a blank control group, an oxidative damage model group, a DHA treatment group (final concentration 50 nmol / L), and an antioxidant peptide + DHA treatment group (i.e., DHA was added to the antioxidant peptide group to a final concentration of 50 nmol / L, and each antioxidant peptide had a final concentration of 20 nmol / L). Each group had three replicates. After 24 h of culture, mitochondrial membrane potential and adenosine triphosphate (ATP) content were measured (mitochondrial membrane potential was detected using the JC-1 fluorescent probe, and ATP content was determined using a luciferase assay kit). The results are as follows: Figure 18 , 19 As shown, compared with the oxidative damage model group, the mitochondrial membrane potential of TM4 cells treated with antioxidant peptides combined with DHA, as shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.8, was significantly restored, and the content of adenosine triphosphate (ATP) was significantly increased. Figure 20 As shown, the three antioxidant peptides shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.8, combined with DHA, can significantly increase the mitochondrial membrane potential and adenosine triphosphate (ATP) content in oxidatively damaged cells, effectively improve cell viability, protect the structural and functional integrity of mitochondria at the cellular level, reduce cell apoptosis caused by oxidative damage, and have excellent cell protection effects.

[0124] Example 7

[0125] Cellular antioxidant capacity assay

[0126] The effects of three antioxidant peptides, which may synergize with DHA, on cellular antioxidant capacity were detected using a superoxide dismutase (SOD) assay kit (WST-8 method) and a total antioxidant capacity assay kit (ABTS rapid method). The results are as follows: Figure 21 and Figure 22As shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.8, the combined action of antioxidant peptides and DHA significantly enhances the activity of superoxide dismutase (SOD) and total antioxidant capacity of cells, effectively improving cellular antioxidant capacity and maintaining normal cellular function.

[0127] Example 8

[0128] Simulation verification of the interaction between antioxidant active peptides and DHA molecules

[0129] To clarify the molecular interaction mode and binding stability between the Atlantic salmon-derived antioxidant peptides and DHA in this invention, molecular docking technology was used to analyze the binding sites, interaction types, and binding free energies of the antioxidant active peptides and DHA, providing a possible molecular mechanism for the antioxidant active peptides to stabilize DHA.

[0130] Molecular docking results as follows Figure 23 As shown, the three antioxidant peptides represented by SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.8 can form relatively stable complexes with DHA, with intermolecular binding free energies of -7.15 kcal / mol, -6.74 kcal / mol, and -7.09 kcal / mol, respectively. They all exhibit strong binding affinity and can inhibit the oxidative degradation of DHA through intermolecular interactions, demonstrating potential application value in DHA stabilization. The remaining five peptides did not produce a synergistic effect with DHA and were not docked.

[0131] This application explores the specific binding mode and stabilization mechanism of Atlantic salmon-derived antioxidant peptides to DHA through molecular docking, providing a molecular-level theoretical basis for the application of antioxidant peptides in the field of DHA stabilization.

Claims

1. An Atlantic salmon peptide with antioxidant activity, characterized in that, The amino acid sequence of the Atlantic salmon peptide is selected from one or more combinations of the polypeptides shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.

8.

2. A nucleic acid encoding the Atlantic salmon peptide as described in claim 1.

3. An expression vector containing the nucleic acid as described in claim 2.

4. Recombinant cells containing the expression vector as described in claim 3, characterized in that, The recombinant cells include Escherichia coli, yeast, and mammalian cell lines.

5. The use of the Atlantic salmon peptide with antioxidant activity as described in claim 1 in the preparation of antioxidant agents or cell protection agents, characterized in that, The preparations include pharmaceuticals, health foods, cosmetics, and functional food additives.

6. The application according to claim 5, characterized in that, The Atlantic salmon peptide with antioxidant activity can be used alone or in combination with other antioxidant active substances.

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