A spore leaf of undaria pinnatifida-derived active peptide and a preparation method and application thereof

Abstract

The application belongs to the technical field of biology, and discloses a Undaria pinnatifida sporophyll-derived active peptide as well as a preparation method and application thereof. The Undaria pinnatifida sporophyll-derived active peptide is selected from at least one of peptides with amino acid sequences as shown in SEQ. ID. NO. 1-4. The Undaria pinnatifida sporophyll-derived active peptide is obtained by screening from Undaria pinnatifida sporophyll through bioinformatics tools and molecular docking. Experiments prove that the Undaria pinnatifida sporophyll-derived active peptide has antioxidant and anti-inflammatory activities, and can be used for preparing antioxidant or antioxidant-assisted products and anti-inflammatory products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an active peptide derived from the spores of Undaria pinnatifida, its preparation method, and its application. Background Technology

[0002] Antioxidant peptides are a class of bioactive peptides with antioxidant activity. They are easily absorbed, highly stable, and non-immunogenic, and possess multiple physiological functions such as nutritional fortification and immune regulation. Due to their natural origin, high safety, lack of toxic side effects, and outstanding nutritional value, antioxidant peptides are widely used in the food industry and have become one of the most sought-after natural active ingredients in the functional food sector. Therefore, developing antioxidant peptides that combine nutritional value and safety using low-cost biological resources has always been an important direction in the field of peptide research.

[0003] Anti-inflammatory peptides are a class of small molecule peptides with anti-inflammatory activity that can effectively alleviate related symptoms by regulating inflammatory responses. They are characterized by easy absorption, high stability, and non-immunogenicity, while also possessing nutritional value and various bioactive functions, such as immune regulation and tissue repair promotion. Due to their natural, safe, non-toxic, and high nutritional value, natural anti-inflammatory peptides have shown broad application prospects in the fields of medicine, cosmetics, and food, and have become one of the important natural products in functional foods and biomedicine. Therefore, developing anti-inflammatory peptides with high anti-inflammatory activity and good safety using low-cost biological resources has always been a research hotspot in the field of peptides.

[0004] Oxidative stress and inflammatory responses are closely intertwined and operate through specific mechanisms within organisms. On one hand, excessive reactive oxygen species (ROS) can activate multiple cellular signaling pathways (such as NF-κB), thereby promoting the expression of pro-inflammatory factors and triggering or exacerbating inflammatory responses. On the other hand, a persistent inflammatory state can also lead to the production of large amounts of ROS by immune cells, creating a vicious cycle. The Keap1-Nrf2 pathway is a core regulatory hub for cellular resistance to oxidative stress and inflammatory responses. Therefore, developing antioxidant and anti-inflammatory peptides with synergistic effects by targeting and activating the Nrf2 pathway has become an important strategy in functional foods, medical foods, and biomedicine, with application potential superior to that of single active peptides.

[0005] Wakame seaweed is an important large economic brown algae, rich in various nutrients and bioactive components. Although its reproductive organ—the sporophyll—has a high fiber content and poor palatability, it remains a valuable biological resource. Currently, major seaweed processing enterprises in Shandong and Liaoning provinces still have a high protein content in the byproducts obtained after extracting sporophyll polysaccharides, which remains unutilized. However, research on the deep enzymatic transformation of this protein resource is insufficient, especially in the systematic preparation, isolation, and identification of peptides with clear biological activity; effective high-value development has yet to be achieved.

[0006] Virtual screening of bioactive peptides based on bioinformatics tools and molecular docking technology refers to the simulation and prediction of the potential biological activity, safety, and structure-activity relationship of known bioactive peptides using relevant databases and specialized software, thereby identifying target peptides. This method further achieves rapid and precise targeted screening of target bioactive peptides by evaluating the interaction between ligands and receptors. Compared with traditional preparation methods, virtual screening using bioinformatics and molecular docking can significantly save time and experimental costs. In practice, combining the two can effectively improve screening efficiency and result reliability. Using this strategy to screen bioactive peptides from Undaria pinnatifida sporophytes provides important technical support for the development of products with auxiliary antioxidant and anti-inflammatory functions. Summary of the Invention

[0007] To address the shortcomings of the existing technology, this invention provides an active peptide derived from *Wakame seaweed* sporophylls, its preparation method, and its applications. The active peptide of this invention is prepared and screened from *Wakame seaweed* sporophylls and has been experimentally verified to possess antioxidant and anti-inflammatory activities.

[0008] The specific technical solution is as follows:

[0009] One objective of this invention is to provide an active peptide derived from the spores of *Wagwassa sporangioides*, wherein the active peptide is selected from at least one peptide with an amino acid sequence as shown in SEQ ID NO. 1 to 4.

[0010] Wherein, SEQ.ID.NO.1 is IFISDF.

[0011] Among them, SEQ.ID.NO.2 is LEGDPLM.

[0012] Among them, SEQ.ID.NO.3 is DFSDPILN.

[0013] Among them, SEQ.ID.NO.4 is ALFDTL.

[0014] Experiments have confirmed that the above-mentioned active peptides derived from *Undaria pinnatifida* sporophytes possess antioxidant and anti-inflammatory activities. Among them, the peptide with the amino acid sequence shown in SEQ.ID.NO.2 exhibits the best antioxidant and anti-inflammatory activities.

[0015] A second objective of this invention is to provide a method for preparing the above-mentioned active peptide derived from Undaria pinnatifida spores, comprising the following steps:

[0016] S1. Obtain Undaria sporophylla peptides;

[0017] S2. Sequence identification of Undaria sporophytes peptides;

[0018] S3. Molecular docking of the Wakame spore leaf peptide with the receptor Keap1 was performed to screen the peptides.

[0019] Furthermore, in step S1: Undaria sporophylla spore leaf protein raw material is enzymatically hydrolyzed to obtain Undaria sporophylla spore leaf peptide.

[0020] Specifically, in step S1: the protein raw material of Undaria pinnatifida spores is enzymatically hydrolyzed using a neutral protease.

[0021] More specifically, in step S1, the preferred working conditions for enzymatic hydrolysis include: adding neutral protease to the raw material to be treated, adjusting the pH to 6.0~7.0, and enzymatically hydrolyzing at 30~50℃ for 2~5 h to obtain the enzymatic hydrolysate.

[0022] The preferred amount of neutral protease added is 3000~5000 U / g based on the raw material to be treated.

[0023] Specifically, in step S1: the wakame spore leaf protein raw material is mixed with water and then enzymatically hydrolyzed. The preferred material-to-liquid ratio of wakame spore leaf protein raw material to water is 1g:(20~100)mL. After mixing the wakame spore leaf protein raw material with water, it is preferable to first treat it at 70~100℃ for 5~15 min and then cool it for enzymatic hydrolysis.

[0024] Specifically, in step S1: after enzymatic hydrolysis, enzyme inactivation is performed. The preferred enzyme inactivation conditions are 80~100℃ for 10~20 minutes.

[0025] Specifically, the protein raw material from *Wakame spore leaves* can be prepared by ultrasound-assisted alkaline extraction. The preparation method includes: drying the byproducts of sugar extraction from *Wakame spore leaves* at 50-80 ℃, pulverizing them through a 30-80 mesh sieve to obtain *Wakame spore leaf* byproduct powder; taking the *Wakame spore leaf* byproduct powder, mixing it evenly with a 0.1-0.5 mol / L NaOH aqueous solution at a material-to-liquid ratio of 1 g:(20-50) mL, and performing ultrasonic extraction at a power of 300-500 W, a temperature of 30-60 ℃, and a time of 30-80 min, followed by centrifugation; taking the supernatant after centrifugation, adjusting the pH to 3.0-4.0, allowing it to stand for 8-16 h, centrifuging again, collecting the crude protein precipitate, dissolving it in water, and then drying it.

[0026] In this process, after dissolving the crude protein in water, it is preferable to use a dialysis bag to desalinate it.

[0027] Furthermore, after enzymatic hydrolysis, the peptides are purified using membrane separation.

[0028] Specifically, ultrafiltration membranes can be used to retain peptides of different molecular weights. Peptides with a molecular weight cutoff of less than 1 kDa are preferred.

[0029] Further, in step S2: peptide sequence analysis is performed using LC-MS / MS.

[0030] Specifically, in step S2: it is preferable to desalt the product obtained in step S1 before performing peptide sequence analysis. A C18 StageTip column is preferably used for desalting.

[0031] Furthermore, in step S3: prior to molecular docking, it is preferable to use the PeptideRanker tool to predict the potential biological activity of the peptide. Specifically, peptides with a biological activity score > 0.5 are selected.

[0032] Furthermore, prior to molecular docking, it is preferable to use the Innovagen tool to predict the physical properties of the peptide.

[0033] Furthermore, prior to molecular docking, it is preferable to use the ProtParam tool to predict peptide stability.

[0034] Furthermore, prior to molecular docking, it is preferable to use the admetSAR tool to predict the solubility of the peptide.

[0035] Furthermore, prior to molecular docking, it is preferable to use the AllergenOnline tool to predict the sensitization potential of the peptide. Specifically, peptides without potential allergenicity are selected.

[0036] Furthermore, prior to molecular docking, it is preferable to use the ToxinPred tool to predict the potential toxicity of the peptide. Specifically, peptides without potential toxicity are selected.

[0037] A third objective of this invention is to provide the application of the above-mentioned *Wakame seaweed* sporophyll-derived active peptides in the preparation of antioxidant products or in contributing to the preparation of antioxidant products. Experiments have confirmed that the above-mentioned *Wakame seaweed* sporophyll-derived active peptides possess antioxidant activity.

[0038] A fourth objective of this invention is to provide the application of the above-mentioned *Undaria pinnatifida* spore-derived active peptides in the preparation of anti-inflammatory products. Experiments have confirmed that the above-mentioned *Undaria pinnatifida* spore-derived active peptides possess anti-inflammatory activity.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention screened four peptides (SEQ.ID.NO.1~4) from *Undaria pinnatifida* sporophytes using bioinformatics tools and molecular docking. Their docking energies with the receptor Keap1 were -8.7 kcal / mol, -7.6 kcal / mol, -7.9 kcal / mol, and -8.5 kcal / mol, respectively. Experiments confirmed that these *Undaria pinnatifida* sporophyte-derived bioactive peptides possess antioxidant and anti-inflammatory activities and can be used to prepare antioxidant or antioxidant-enhancing products, as well as anti-inflammatory products. Among them, the peptide with the amino acid sequence shown in SEQ.ID.NO.2 exhibited the best antioxidant and anti-inflammatory activity. Attached Figure Description

[0041] Figure 1 To test the ORAC values ​​of the peptides in 1 whose amino acid sequences are shown in SEQ.ID.NO.1~4;

[0042] Figure 2 To test the ABTS radical scavenging rate of the peptides with amino acid sequences as shown in SEQ.ID.NO.2 and SEQ.ID.NO.3 in step 2;

[0043] Figure 3 To test the effect of peptides with amino acid sequences as shown in SEQ.ID.NO.1~4 on NO secretion in RAW264.7 cells;

[0044] Figure 4 To test the effect of peptides with amino acid sequences as shown in SEQ.ID.NO.1~4 on mitochondrial ROS production in RAW264.7 cells;

[0045] Figure 5 To test the effect of the peptide with the amino acid sequence shown in SEQ.ID.NO.2 on the secretion of IL-1β in RAW264.7 cells. Detailed Implementation

[0046] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] Example

[0048] The steps for preparing Undaria pinnatifida spore-derived bioactive peptides are as follows:

[0049] S1. Preparation of Undaria sporophylla peptides:

[0050] (1) Raw material pretreatment: The by-products of sugar extraction from wakame spore leaves were dried at 50 ℃, then pulverized by ultra-fine grinding and passed through a 60-mesh sieve. The sieve-passing material was collected to obtain wakame spore leaf by-product powder; it was then sealed and stored at -30 ℃.

[0051] (2) Protein extraction: The Undaria pinnatifida spore leaf by-product powder obtained in step (1) was mixed with 0.2 mol / L NaOH solution at a material-to-liquid ratio of 1 g: 20 mL. The mixture was extracted at 50℃ and 400 W for 60 min, with the ultrasonic treatment paused for 2 min every 10 min to obtain a mixture. The mixture was centrifuged at 4℃ and 7000 rpm for 15 min. The supernatant was taken and the pH was adjusted to 3.5 with hydrochloric acid solution. The mixture was then allowed to stand for 12 h and centrifuged at 4℃ and 7000 rpm for 15 min to collect the crude protein precipitate. The precipitate was dissolved in ultrapure water, desalted using a 100 Da dialysis bag at 4℃, and then freeze-dried to obtain Undaria pinnatifida spore leaf protein freeze-dried powder.

[0052] (3) Enzymatic hydrolysis: The freeze-dried Undaria pinnatifida spore leaf protein obtained in step (2) was mixed with ultrapure water at a ratio of 1 g: 40 mL and treated in a water bath at 85°C for 6 min. After cooling, the pH value was adjusted to 7.0 using 0.1 mol / L hydrochloric acid or sodium hydroxide solution. Then, 3500 U / g of neutral protease based on the freeze-dried Undaria pinnatifida spore leaf protein was added and enzymatically hydrolyzed at 40°C for 3 h. After the enzymatic hydrolysis was completed, the enzyme was inactivated in a water bath at 90°C for 20 min. Then, the mixture was centrifuged at 4°C and 7000 rpm for 10 min and the supernatant was collected to obtain the enzymatic hydrolysate.

[0053] (4) Ultrafiltration purification: The enzymatic hydrolysate obtained in step (3) was separated using a 1 kDa ultrafiltration membrane to obtain a sample with a molecular weight of less than 1 kDa. After freeze-drying, it was stored at -30℃ to obtain peptide powder.

[0054] S2. Sequence analysis of Undaria pinnatifida sporophylls polypeptides:

[0055] The peptide powder obtained in step S1 was desalted using a C18 StageTip column, and peptide sequence analysis was performed using LC-MS / MS: the sample was injected into the trapping column (100 μm × 20 mm, 5 μm, C18, Dr. Maisch GmbH) and then subjected to gradient separation using the analytical column (75 μm × 150 mm, 3 μm, C18, Dr. Maisch GmbH) at a flow rate of 300 nL / min; after peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF mass spectrometer; comparative analysis was performed using a database to obtain all peptide sequences; the mass spectrometry database search software used was MaxQuant 2.4.14.0, and the sample database used was the uniprot protein database. A total of 26 sequences were obtained from the by-products of Undaria pinnatifida spores through peptide sequencing.

[0056] S3. Bioinformatics tools assist in the screening of bioactive peptides derived from Undaria pinnatifida spores:

[0057] The PeptideRanker tool was used to predict the potential biological activity of peptides, with a score >0.5 indicating potential biological activity. The Innovagen tool was used to predict the physical properties of peptides. The ProtParam tool was used to predict the stability of peptides. The admetSAR tool was used to predict the solubility of peptides. The AllergenOnline tool was used to predict the sensitization potential of peptides. The ToxinPred tool was used to predict the potential toxicity of peptides. Sequences with potential biological activity and without potential sensitization or toxicity were selected for further screening and validation.

[0058] S4. Molecular docking virtual screening of bioactive peptides from Wrigley's sporophylla byproducts:

[0059] The peptides screened in step S3 were used as ligands, and Keap1 was used as the acceptor. Molecular docking techniques were employed to analyze the interaction sites and forces between the active peptides from *Undaria pinnatifida* sporophylls and Keap1. Using the crystal structure of the Keap1-Nrf2 complex (PDB ID: 2FLU) as a template, PyMOL was used to remove the Nrf2 peptide and water molecules and add hydrogen atoms to obtain the acceptor protein. The peptide ligands were plotted in two dimensions using ChemDraw, converted to a three-dimensional conformation using Chem3D, and subjected to energy minimization and hydrogen addition. AutoDock Vina software was used, with the docking box center coordinates set to x=11.031, y=18.753, z=10.876, covering the Keap1 active pocket. One hundred independent docking operations were performed, and the conformation with the lowest binding free energy was selected as the optimal result. Discovery Studio was used to analyze the interactions (hydrogen bonds, hydrophobic interactions, etc.) between the peptides and Keap1. The RMSD values ​​of the docking conformation and the original Nrf2 binding mode were calculated using PyMOL, with an RMSD < 2.0 Å requirement to verify docking reliability. Four peptides were screened, and their amino acid sequences are shown in SEQ.ID.NO.1~4. The amino acid sequences of the four peptides and the molecular docking results are shown in Table 1.

[0060] Table 1. Results of docking between Undaria pinnatifida spore-derived bioactive peptides and keap1 molecules.

[0061]

[0062] The bioactivity scores, solubility, potential allergenicity, potential toxicity, and physicochemical properties of the four peptides are shown in Table 2.

[0063] Table 2. Scores of bioactive peptides derived from Wakame spores and leaf extracts, and predictions of allergenicity and toxicity.

[0064]

[0065] Four peptides with amino acid sequences as shown in SEQ.ID.NO.1~4 were chemically synthesized using a solid-phase synthesis method for the verification of antioxidant and anti-inflammatory activities.

[0066] Test 1

[0067] The ORAC (oxygen radical scavenging capacity) of four peptides with amino acid sequences as shown in SEQ.ID.NO.1~4 was tested. The test method is as follows:

[0068] Phosphate buffer (75 mmol / L), fluorescein solution (0.63 μmol / L), AAPH radical initiator solution (18.29 mmol / L), and Trolox standard solution (100 μmol / L) were prepared at pH 7.4. In a 96-well plate, 20 μL of the target peptide solution, 20 μL of phosphate buffer, and 20 μL of fluorescein solution were added sequentially. After shaking and mixing, the mixture was incubated at 37 °C for 5 min, followed by the rapid addition of 140 μL of AAPH solution to initiate the reaction. The fluorescence intensity decay curve was dynamically monitored at 37 °C for 140 min, with data collected every 2 min. The excitation wavelength was 485 nm, and the emission wavelength was 538 nm. A standard curve was plotted using Trolox as the standard, and the ORAC value was determined by calculating the relative fluorescence intensity. The results are expressed as µmol TE / mg. The test results are shown in [Figure number missing]. Figure 1 . Figure 1 In the x-axis: IF6 is SEQ.ID.NO.1, LM7 is SEQ.ID.NO.2, DN8 is SEQ.ID.NO.3, AL6 is SEQ.ID.NO.4, and GSH is glutathione. Figure 1 In the figure, each value is the mean ± SD of three parallel experiments (n=3); different lowercase letters (a, b, c, d) indicate significant differences in ORAC values ​​among different peptide components (P < 0.05). The results show that the peptides with amino acid sequences as shown in SEQ.ID.NO.1~4 have ORAC values ​​of 0.20, 1.22, 0.007, and 0.48 µmol TE / mg, respectively, all exhibiting a certain oxygen free radical absorption capacity and showing potential application in the preparation of antioxidant or antioxidant-enhancing products, such as antioxidant health foods. Among them, SEQ.ID.NO.2 has the highest ORAC value, indicating that it has a better oxygen free radical absorption capacity.

[0069] Test 2

[0070] The free radical scavenging ability of peptides with amino acid sequences as shown in SEQ.ID.NO.2 and SEQ.ID.NO.3 was tested. The test method is as follows:

[0071] Prepare 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution, mix equal volumes of both, and react at room temperature in the dark for 12–16 h to obtain the ABTS stock solution. Before use, dilute the stock solution to an absorbance of 0.7 ± 0.02 at 734 nm. Add 50 µL of different concentrations of the sample solution and 100 µL of the diluted ABTS solution sequentially to a 96-well plate, and measure the absorbance at 734 nm after 70 min. A control group was set up using 50 µL of ultrapure water instead of the sample solution; a blank group was set up using 100 µL of ultrapure water instead of the ABTS working solution. The ABTS free radical scavenging rate was calculated using the following formula:

[0072] .

[0073] Test results are available Figure 2 Each value is derived from the mean ± SD of three parallel experiments (n=3). Figure 2 In the x-axis: LM7 is SEQ.ID.NO.2, and DN8 is SEQ.ID.NO.3. Figure 2 In the diagram, different lowercase letters (a, b) indicate significant differences in ABTS radical scavenging rates among different peptide components (P < 0.05). The results show that SEQ.ID.NO.2 and SEQ.ID.NO.3 exhibited ABTS radical scavenging rates of 24.11% and 13.86%, respectively, demonstrating a certain ABTS radical scavenging ability. This suggests that the antioxidant activity of these two peptides may primarily derive from their interaction with antioxidant pathway proteins.

[0074] Test 3

[0075] The effects of four peptides with amino acid sequences shown in SEQ.ID.NO.1~4 on LPS (lipopolysaccharide)-induced NO secretion in RAW264.7 cells were tested. The Griess assay was used to detect LPS-induced NO release levels, thereby assessing the anti-inflammatory activity of the four active peptides. The test methods are as follows:

[0076] RAW264.7 cells in the logarithmic growth phase were harvested and cultured at a concentration of 5 × 10⁻⁶ cells / year. 4Cells were seeded into each well of a 96-well plate and incubated for 24 h. Active peptides of corresponding concentrations (SEQ.ID.NO.2, 1, 0.5, 0.25 mg / mL), SEQ.ID.NO.1 (0.5 mg / mL), SEQ.ID.NO.3 (0.5 mg / mL), and SEQ.ID.NO.4 (0.5 mg / mL) were added and incubated for 2 h. LPS (1 μg / mL) was then added for 24 h of stimulation. After incubation, the plates were removed, and the supernatant from each well was aseptically transferred to another 96-well plate, 100 μL per well. The NO content was determined by detecting the nitrite level in the supernatant using Griess reagent. The absorbance (OD540) of the sample at 540 nm was then measured using a microplate reader (SpectraMax M3). The obtained OD values ​​were substituted into the NO content standard curve to obtain the corresponding NO concentration.

[0077] The results of the determination of the effect of four peptides on NO secretion are as follows: Figure 3 As shown. Figure 3 In the x-axis: IF6 is SEQ.ID.NO.1, LM7 is SEQ.ID.NO.2, DN8 is SEQ.ID.NO.3, and AL6 is SEQ.ID.NO.4. Figure 3 In the mean ± SD of three parallel experiments (n=3), compared with the blank group, ###p<0.001; compared with the control group, *p<0.05, **p<0.01, ***p<0.001. The results show that the NO content in the control group was significantly increased compared with the blank group, indicating the successful construction of the RAW264.7 cell inflammation model. After peptide intervention, the NO level was significantly reduced, indicating that these four peptides can effectively inhibit LPS-induced inflammatory responses. Based on the above test results, all four active peptides have anti-inflammatory activity and can be used in the preparation of anti-inflammatory products, especially anti-inflammatory drugs.

[0078] Test 4

[0079] The effects of four peptides with amino acid sequences (SEQ.ID.NO.1~4) on LPS-induced mitochondrial ROS production in RAW264.7 cells were detected using an enzyme-linked immunosorbent assay (ELISA) reader. The assay method is as follows:

[0080] RAW264.7 cells in the logarithmic growth phase were harvested and their concentration adjusted to 5 × 10⁻⁶ cells / year. 5Cells were cultured at a density of 100 μL / well in black 96-well plates and incubated for 4 h to ensure cell adhesion. The control group received no treatment. The control group received an equal volume of DMSO. The sample groups received SEQ.ID.NO.2 (1, 0.5, 0.25 mg / mL), SEQ.ID.NO.2 (0.5 mg / mL), SEQ.ID.NO.3 (0.5 mg / mL), and SEQ.ID.NO.4 (0.5 mg / mL) for 2 h of pre-protection. Then, LPS (1 μg / mL) was added, and the plates were incubated for another 30 min. The 96-well plates were removed aseptically, the supernatant was discarded, and the plates were washed twice with pre-warmed PBS (100 μL / wash) at 37°C. Then, 50 μL of 5 μM mitoSOX probe was added, and the plates were incubated in the dark for 15 min. Remove the glass plate and, under sterile conditions protected from light, discard the mitoSOX probe. Wash twice with PBS preheated to 37°C (100 μL each time). Finally, add 50 μL of PBS and use a microplate reader to detect its fluorescence intensity. The excitation wavelength is 540 nm and the emission wavelength is 570 nm.

[0081] The MitoSOX fluorescence intensity detection results of the four peptides are as follows: Figure 4 As shown. Figure 4 In the Chinese dictionary: IF6 is SEQ.ID.NO.1, LM7 is SEQ.ID.NO.2, DN8 is SEQ.ID.NO.3, and AL6 is SEQ.ID.NO.4. Figure 4 In the study, compared with the blank group, #p<0.05; compared with the control group, *p<0.05. The results indicate that LPS treatment induced the generation of MtROS. All four peptides significantly inhibited LPS-induced MtROS generation. This suggests that the inhibition of LPS-triggered oxidative stress is closely related to the antioxidant activity of the aforementioned peptides.

[0082] Test 5

[0083] The effect of the peptide with the amino acid sequence shown in SEQ.ID.NO.2 on LPS-induced IL-1β secretion in RAW264.7 cells was tested.

[0084] RAW264.7 cells in the logarithmic growth phase were harvested and their concentration adjusted to 5 × 10⁻⁶ cells / year. 5Cell suspension was prepared in black 96-well plates at 100 μL per well and incubated for 4 h to ensure cell adhesion. The control group received no treatment, the control group received an equal volume of DMSO, and the sample group received SEQ.ID.NO.2 (1, 0.5, 0.25 mg / mL) for 2 h of pre-protection, followed by the addition of LPS (1 μg / mL) and incubation for another 24 h. Afterward, the 96-well plates were removed, and under aseptic conditions, 100 μL of supernatant was transferred from each well to another 96-well plate. The IL-1β content in the supernatant was detected using enzyme-linked immunosorbent assay (ELISA). The absorbance (OD450) of the sample at 450 nm was then measured using a SpectraMax M3 microplate reader. The OD values ​​were then substituted into the IL-1β concentration standard curve to obtain the corresponding IL-1β concentration.

[0085] The results of the determination of the effect of the four peptides on IL-1β levels are as follows: Figure 5 As shown. Figure 5 In the mean ± SD of three parallel experiments (n=3), each value is from the control group; ##p<0.01 compared to the blank group; **p<0.01 compared to the LPS-stimulated group. The results show that the IL-1β inflammatory factor content was significantly increased in the control group compared to the blank group, indicating the successful construction of the RAW264.7 cell inflammation model; compared to the control group, the secretion of IL-1β inflammatory factors in the sample group was significantly reduced, showing a concentration-dependent effect. This indicates that the peptide with the amino acid sequence shown in SEQ.ID.NO.2 can inhibit the release of IL-1β inflammatory factors, exhibiting significant anti-inflammatory activity.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Wakame spore-derived bioactive peptide, characterized in that, The amino acid sequence is shown in SEQ.ID.NO.

2.

2. The method for preparing the active peptide derived from *Undaria pinnatifida* spores as described in claim 1, characterized in that, Includes the following steps: S1. Obtaining wakame spore leaf peptides by enzymatic hydrolysis of wakame spore leaf protein raw material; The protein raw material of *Wakame spore leaves* was prepared by ultrasound-assisted alkaline extraction. The preparation method includes: drying the by-product of sugar extraction from *Wakame spore leaves* at 50-80 ℃, pulverizing it into ultrafine powder and passing it through a 30-80 mesh sieve to obtain *Wakame spore leaf* by-product powder; taking the *Wakame spore leaf* by-product powder, mixing it with 0.1-0.5 mol / L NaOH aqueous solution at a material-to-liquid ratio of 1g:(20-50)mL, and performing ultrasonic extraction at an ultrasonic power of 300-500 W, an ultrasonic temperature of 30-60℃, and an ultrasonic time of 30-80 min, followed by centrifugation; taking the supernatant after centrifugation, adjusting the pH to 3.0-4.0, then letting it stand for 8-16 h, centrifuging again, collecting the crude protein precipitate, dissolving it in water, and then drying it. The working conditions for enzymatic hydrolysis include: adding neutral protease to the raw material to be treated, adjusting the pH to 6.0~7.0, and enzymatic hydrolysis at 30~50℃ for 2~5 h to obtain the enzymatic hydrolysate; After enzymatic hydrolysis, the peptides were purified by membrane separation; ultrafiltration membranes were used to retain peptides with a content below 1 kDa. S2. Sequence identification of Undaria sporophytes peptides; S3. Molecular docking of the Wakame spore leaf peptide with the receptor Keap1 was performed to screen the peptides.

3. The preparation method according to claim 2, characterized in that, In step S3: Before molecular docking, the PeptideRanker tool is used to predict the potential biological activity of the peptide.

4. The preparation method according to claim 3, characterized in that, Peptides with a bioactivity score >0.5 were selected.

5. The preparation method according to claim 2, characterized in that, In step S3: Before molecular docking, the physical properties of the peptide were predicted using the Innovagen tool; Before molecular docking, the stability of the peptide was predicted using the ProtParam tool; Before molecular docking, the solubility of the peptide was predicted using the admetSAR tool; Prior to molecular docking, the AllergenOnline tool was used to predict the sensitization potential of the peptide. Before molecular docking, the ToxinPred tool was used to predict the potential toxicity of the peptide.

6. The application of the wakame spore leaf-derived active peptide as described in claim 1 in the preparation of antioxidant products.

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