Fish scale collagen immunostimulatory peptide, preparation method and application

Abstract

This invention relates to a fish scale collagen immunostimulating peptide, its preparation method, and its application. The amino acid sequence of the peptide is QVPGLYYF. The preparation method includes the following steps: S1, removing calcium and non-collagenous proteins from sardine scales; S2, enzymatically hydrolyzing the pretreated sardine scales with a protease, followed by enzyme inactivation, centrifugation, fractionation, and filtration to collect fish scale collagen peptides with a molecular weight <1 kDa; S3, performing mass spectrometry on the fish scale collagen peptides, and analyzing the results using mass spectrometry analysis software to obtain multiple non-repeating polypeptide sequences; S4, first screening for polypeptide sequences with short chains composed of 2-10 amino acids, and then importing the screened polypeptide sequences into software for bioactivity prediction, selecting polypeptide sequences with a score greater than 0.75; S5, virtual screening; S6, polypeptide synthesis. This invention can obtain the amino acid sequence of a fish scale collagen immunostimulating peptide using sardine scales as raw material, and the prepared fish scale collagen immunostimulating peptide has good immunostimulatory effects.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a fish scale collagen immunostimulatory peptide, its preparation method, and its application. Background Technology

[0002] As people age, they inevitably face various immunodeficiency diseases and weakened immune function, leading to increasing consumer interest in immunomodulatory drugs. Currently, some immunomodulatory drugs, such as imiquimod and levamisole, are already in clinical use. On the one hand, these immunomodulatory drugs can, to some extent, improve patients' immunity, reduce the risk of infection, and assist in the treatment of autoimmune diseases. On the other hand, their side effects and economic costs also raise concerns. Extracts with immunomodulatory activity developed from natural ingredients have become a research hotspot in recent years. For example, Jinyang Cai et al. extracted and isolated the derivative peptide BCCY-1 from human β-casein, which can promote macrophage proliferation and activate signaling pathways (NF-κB and MAPK) to exert immunomodulatory effects. Low molecular weight peptides (DHAV) isolated, purified, and identified from silkworm pupa protein hydrolysates can promote spleen cell proliferation and stimulate the expression of related immune factors (IL-6 and IL-12, NF-κB, cyclin D1, etc.).

[0003] Sardines (scientific name: *Sardina pilchardus*) are fish belonging to the family Clupeiformes and the genus *Sardina*. They are slender, silvery fish, measuring 15–30 cm in length, with scaleless heads and cycloid scales of varying sizes on their sides. They are distributed in marine areas between 14 and 68 degrees north latitude. Sardines are warm-water fish found in coastal waters and are generally not found in the open ocean. Sardines have high nutritional value and are extensively caught for food processing. They are also an important economic fish globally, often a significant product of deep-sea fishing. Sardines contain eicosapentaenoic acid (EPA), which can help prevent cardiovascular disease, as well as nucleic acids, taurine, selenium, and other nutrients, giving them high medicinal value. They are also rich in DHA, which is beneficial for cardiovascular health. Additionally, they are rich in phospholipids (Omega-3 fatty acids), protein, and calcium. However, due to their small size, high yield but low economic value, and difficulties in preservation and processing, they are mostly made into fish balls, fish rolls, fish sausages, canned fish, and other convenience foods. Current research on sardines mainly focuses on food processing, with few reports on the deep processing and bioactivity of sardines. Summary of the Invention

[0004] The purpose of this invention is to provide a fish scale collagen immunostimulating peptide, its preparation method, and its application. It can obtain the amino acid sequence of the fish scale collagen immunostimulating peptide using fish scales such as sardines as raw materials, and the prepared fish scale collagen immunostimulating peptide has a good immune-stimulating effect.

[0005] To achieve the above objectives, this invention discloses a fish scale collagen immunostimulating peptide with the amino acid sequence QVPGLYYF.

[0006] Preferably, its molecular weight is 986.14 Da.

[0007] The preparation method of the above-mentioned fish scale collagen immunostimulating peptides includes the following steps:

[0008] S1, Preprocessing

[0009] Remove the calcium and non-collagenous proteins from the sardine scales, then dry them for later use;

[0010] S2. Preparation of fish scale collagen peptides

[0011] The pretreated sardine scales were enzymatically hydrolyzed using protease. After hydrolysis, the enzyme was inactivated to obtain the hydrolysate. The hydrolysate was centrifuged, and the supernatant was collected. The supernatant was then fractionated and filtered to collect fish scale collagen peptides with a molecular weight <1kDa, which were then freeze-dried.

[0012] S3, Sequence Identification

[0013] Fish scale collagen peptides were analyzed by LC-MS / MS, and the results were analyzed using mass spectrometry software to obtain multiple non-repeating peptide sequences.

[0014] S4, Pre-screening

[0015] First, peptide sequences with short chains consisting of 2 to 10 amino acids are screened out. Then, the screened peptide sequences are imported into software for bioactivity prediction, and peptide sequences with a score greater than 0.75 are selected.

[0016] S5, Virtual Filtering

[0017] The software is used to perform molecular docking between the peptide sequences screened in step S4 and the receptor, screen out peptide sequences with strong receptor binding ability, and perform toxicity and ADMET prediction.

[0018] S6, Polypeptide Synthesis

[0019] The polypeptide sequences screened in step S5 were synthesized in a solid phase to obtain sardine scale immunomodulatory peptides.

[0020] Preferably, in step S1, the removal of calcium is performed as follows: sardine scales are added to a lactic acid solution at a material-to-liquid ratio of 1:10-30 and soaked for 1-2 hours, wherein the concentration of the lactic acid solution is 0.5-1 mol / L. After soaking, the sardine scales are rinsed with water until neutral. The calcium-removed sardine scales are then added to a 1-5% NaCl solution and soaked for 12-24 hours at a material-to-liquid ratio of 1:10-30. After soaking, the sardine scales are rinsed with water until neutral, thereby filtering out the non-collagenous proteins in the sardine scales. The calcium-removed and non-collagenous protein-removed sardine scales are then dried at 40-50°C.

[0021] Preferably, in step S1, the dried sardine scales are first broken up before the calcium and non-collagen components are removed; the sardine scales are also subjected to ultrasound while soaking in lactic acid solution, with an ultrasound power of 100-200W.

[0022] Preferably, in step S2, the pretreated sardine scales are first added to distilled water at a material-to-liquid ratio of 1:40-60, then the pH is adjusted to 8.0-11.0, and preheated at 40-50°C for 10-60 min; then, protease is added for enzymatic hydrolysis at an amount of 4000-20000 U / g, and the hydrolysis is carried out for 3-7 h; after the hydrolysis is completed, the enzyme is inactivated at 95-100°C for 10-15 min; after the enzyme is inactivated, the hydrolysate is cooled to room temperature and then centrifuged at a speed of 3000-6000 r / min, a centrifugation temperature of 2-6°C, and a centrifugation time of 10-40 min.

[0023] Preferably, in step S2, the supernatant is sequentially passed through a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane for fractionation to obtain fish scale collagen peptides with a molecular weight of <1 kDa.

[0024] Preferably, in step S5, the receptor is the TLR4 protein.

[0025] Furthermore, the present invention also discloses the application of the above-mentioned fish scale collagen immunostimulating peptide in at least one of the following situations:

[0026] (1) Application in enhancing the phagocytic capacity of macrophages;

[0027] (2) Application in the preparation of macrophage phagocytic capacity enhancers;

[0028] (3) Application in promoting macrophage proliferation;

[0029] (4) Application in the preparation of macrophage proliferation promoters;

[0030] (5) Application in enhancing the release of NO, IL-6, TNF-α and IL-1β from macrophages;

[0031] (6) Application in the preparation of macrophage NO release, IL-6, TNF-α and IL-1β secretion promoters;

[0032] (7) Application in the preparation of drugs that enhance biological immunity;

[0033] (8) Application in the preparation of food ingredients that enhance biological immunity.

[0034] Preferably, the concentration of the fish scale collagen immunostimulating peptide is 50–200 μg / mL.

[0035] The present invention has the following beneficial effects:

[0036] This invention uses fish scales from sardines and other fish as raw materials to obtain the amino acid sequence of fish scale collagen immunostimulating peptides. The prepared fish scale collagen immunostimulating peptides have a good immune-stimulating effect. This invention utilizes sardine scales as raw material, making use of waste materials and providing a new approach for the high-value processing and utilization of sardine by-products. It also expands resources for the development of natural immune-regulating products. Attached Figure Description

[0037] Figure 1 This is an UPLC-MS / MS image of the peptide QVPGLYYF.

[0038] Figure 2 A 3D model for docking of the peptide QVPGLYYF with the TLR4 protein molecule.

[0039] Figure 3 for Figure 2 Enlarged schematic diagram of part A in the middle.

[0040] Figure 4 This is a 2D structural diagram of the polypeptide QVPGLYYF.

[0041] Figure 5 This is the hydrophobic interface of the peptide QVPGLYYF binding site.

[0042] Figure 6 This is a graph showing the root mean square deviation (RMSD) of all atoms in a protein-ligand complex relative to its initial structure.

[0043] Figure 7 The RMSF value represents the change in the amino acid backbone atoms of the protein-ligand complex over time.

[0044] Figure 8 The RMSF value represents the change in the atomic index of a protein-ligand complex over time.

[0045] Figure 9 This is a schematic diagram illustrating the detailed interaction between ligand atoms and protein residues.

[0046] Figure 10 A statistical graph of protein-ligand complex interaction types (classified by amino acid participation).

[0047] Figure 11 The effect of peptide QVPGLYYF on the viability of RAW264.7 cells.

[0048] Figure 12 The effect of peptide QVPGLYYF on NO release in RAW264.7 cells.

[0049] Figure 13 The effect of peptide QVPGLYYF on phagocytosis in RAW264.7 cells.

[0050] Figure 14 The effect of peptide QVPGLYYF on cellular IL-6 secretion.

[0051] Figure 15 The effect of the polypeptide QVPGLYYF on cellular IL-1β secretion.

[0052] Figure 16 The effect of the polypeptide QVPGLYYF on cellular TNF-α secretion. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0054] This invention discloses a fish scale collagen immunostimulating peptide with the amino acid sequence QVPGLYYF, as shown in SEQ ID NO: 1 of the sequence listing. The molecular weight of the above-mentioned fish scale collagen immunostimulating peptide is 986.14 Da.

[0055] The preparation method of the above-mentioned fish scale collagen immunostimulating peptides includes the following steps:

[0056] S1, Preprocessing

[0057] Remove the calcium and non-collagenous proteins from the sardine scales and dry them for later use. First, break the dried (air-dried) sardine scales to destroy their net-like structure.

[0058] Sardinian scales were soaked in a 0.75 mol / L lactic acid solution at a ratio of 1:20 for 1 hour to remove calcium. To accelerate the removal of calcium from the scales, the lactic acid-soaked scales were subjected to ultrasonic treatment at 40°C and a power of 100W. After soaking for 1 hour, the scales were repeatedly rinsed with distilled water until neutral.

[0059] Sardinian scales, after calcium removal, were soaked in a 3% NaCl solution overnight (12 hours), with a scale-to-NaCl solution ratio of 1:20. After soaking, the sardine scales were rinsed with distilled water until neutral, and the non-collagenous proteins were filtered out. The calcium- and non-collagenous protein-removed sardine scales were then dried in an oven at 40–50°C for later use.

[0060] S2. Preparation of crude fish scale collagen peptides

[0061] Weigh a certain amount of pretreated fish scales into a beaker, add distilled water at a material-to-liquid ratio of 1:50, and adjust the pH to the optimal pH for enzymatic hydrolysis (10.5) using hydrochloric acid or sodium hydroxide solution. Preheat in a 40℃ constant temperature water bath for 30 minutes, then add protease and mix thoroughly for enzymatic hydrolysis. The amount of protease added is 7526 U / g, and the hydrolysis time is 4 hours. During the enzymatic hydrolysis, continuously adjust the pH with sodium hydroxide solution (stabilizing at 10.5) until the fish scale solids are almost completely eliminated. After the enzymatic hydrolysis is completed, inactivate the protease at 100℃ for 15 minutes to terminate the reaction.

[0062] After the enzyme hydrolysate has cooled to room temperature, it is centrifuged at a speed of 5000 r / min, a temperature of 4℃, and a time of 20 min. The supernatant is then collected.

[0063] The supernatant was sequentially passed through a ceramic membrane, an ultrafiltration membrane, and a nanofiltration membrane for fractionation to remove impurities and desalination, yielding fish scale collagen peptides with a molecular weight <1kDa, which were then freeze-dried.

[0064] S3, Sequence Identification

[0065] The structure of fish scale collagen immunostimulatory peptides was determined using LC-MS / MS analysis on a Q-Exactive Plus mass spectrometer from Thermo Fisher Scientific, Inc. First, an appropriate amount of sardine scale collagen peptides (<1 kDa) was weighed and analyzed using a LC-MS / MS instrument. 18Desalting was performed using a desalting column (Acclaim PepMap C18). The sample loading volume was 3 μL, and the sample was separated using a gradient at a column temperature of 40℃ and a flow rate of 300 nL / min for 60 min. Mobile phase A was set as 0.1% formic acid solution, and mobile phase B as 0.1% formic acid-acetonitrile solution. Chromatographic parameters: electrospray voltage 2 kV, gradient starting with 2% phase B, increasing non-linearly to 35% over 47 min, increasing to 100% within 1 min, and holding for 12 min. Mass spectrometry was performed using ESI positive and negative ion scanning. The mass spectrometer was operated in data-dependent acquisition mode, with automatic switching between MS and MS / MS acquisition. The MS main scan range, resolution, AGC target, and residence time (m / z) were 200-2000, 70,000, 3e6, and 50 ms, respectively. The secondary scan resolution, AGC target, and maximum injection time were 17,500, 1e5, and 45 ms, respectively. In addition, the collision energy was 28 s, and the dynamic exclusion time was 30 s. The raw tandem mass spectrometry file was subjected to qualitative and quantitative analysis of sardine scale collagen peptides using PEAKS Studio version 10.6 software (Bioinformatics Solutions Inc., Waterloo, Canada), yielding multiple non-repeating polypeptide sequences. A total of 2914 polypeptide sequences were obtained in this step, a relatively large number.

[0066] S4, Pre-screening

[0067] Based on the characteristics of short peptide chains, generally composed of 2 to 10 amino acids, peptide sequences with short chains and composed of 2 to 10 amino acids were first screened. Then, the selected peptide sequences were further screened according to the conditions (-10lgP>20, Mass<1000, Length<10.), resulting in 486 peptide sequences, which is still a large number.

[0068] The peptide sequences selected from the secondary screening were imported into the PeptideRanker program on the Bioware website (http: / / distilldeep.ucd.ie / PeptideRanker / ) for bioactivity prediction. Bioactivity probability scores range from 0 to 1, with higher scores indicating a higher likelihood of bioactivity. Therefore, peptides were ranked, and sequences with a probability score greater than 0.75 were selected. Then, the water solubility of potentially functional active peptide fragments was predicted using online software (http: / / pepcalc.com / ). Peptides with good water solubility were selected for further screening to better assess their bioactivity.

[0069] A total of 84 polypeptide sequences were screened in this step, as shown in Table 1.

[0070] Table 1. Predicted water solubility and bioactivity of fish scale collagen peptides

[0071]

[0072]

[0073] S5, Virtual Filtering

[0074] The peptide sequences screened in step S4 were molecularly docked with the receptor using software to identify peptide sequences with strong receptor binding affinity. Specifically, the 3D structure of the active peptide was constructed using Discovery Studio 2019 as a ligand, and energy minimization was performed using the CHARMM force field. The TLR4 (PDB ID: 5IJD) crystal structure was downloaded from the RCSB PDB protein database (http: / / www.rcsb.org / ), and then dehydrated and hydrogenated using Discovery Studio 2019 software to remove protein multiple conformations, and optimized and minimized energy to produce a geometrically stable structure. Molecular docking was performed using the LibDock scheme in the DockLigand module of the software to screen peptides that bind tightly to TLR4. The binding site was defined based on the software prediction method to find potential active sites in the protein cavity and the results of literature search. In the algorithm, a semi-flexible docking was used, with the TLR4 receptor set to rigid and the peptide set to flexible. Each peptide output contained 20 conformations and corresponding scores, and other parameters were set to default values. The LibdockScore is a parameter that comprehensively evaluates energy, geometry, and chemical environment. A higher LibdockScore indicates greater activity of the small molecule in binding to the receptor, facilitating interaction. Conversely, a lower score indicates weaker interaction. Using the software's proprietary scoring function "LibdockScore" as the evaluation criterion, the 20 peptides with the strongest molecular docking affinity were screened for synthesis (see Table 2).

[0075] Table 2. Top 20 Fish Scale Collagen Peptides by Molecular Docking Score

[0076]

[0077] Good water solubility and non-toxicity are important criteria for screening commercially available bioactive peptides. The toxicity and ADMET properties of the peptides were predicted using the toxicity prediction tool in Discovery Studio 2019 software. The TOPKAT model was used to predict Ames mutagenicity, developmental toxicity potential, and skin sensitization. Finally, the bioactive peptides screened were imported into the BIOPEP database (https: / / biochemia.uwm.edu.pl / en / biopep-uwm-2 / ) for novelty checks (Minkiewicz, Iwaniak, & Darewicz, 2019), and further research was conducted on unreported bioactive peptides. The results are shown in Table 3.

[0078] Table 3. Toxicity of Fish Scale Collagen Peptides and ADMET Prediction

[0079]

[0080]

[0081] S6, Polypeptide Synthesis

[0082] Twenty peptides obtained from virtual screening were synthesized using a solid-phase synthesis method. Their purity was determined / verified by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with LC-MS / MS. The synthesized peptides had a purity ≥98%. After drying at -20℃, they were stored for later use, yielding fish scale collagen immunostimulatory peptides. Figure 1 As shown, the amino acid sequence of this polypeptide was determined to be Gln-Val-Pro-Gly-Leu-Tyr-Tyr-Phe (QVPGLYYF), with a molecular weight of 986.14 Da. The polypeptide consists of eight amino acids, and the C-terminal phenylalanine is a hydrophobic amino acid.

[0083] The structure-activity relationship of fish scale collagen immunostimulatory peptide QVPGLYYF was studied. Specifically, AutoDock was used to perform molecular docking between TLR4 and QVPGLYYF. The QVPGLYYF ligand was set flexibly, and other parameters were left as default. Molecular docking was performed using the Vina program, and the structure-activity relationship between QVPGLYYF and TLR4 was analyzed.

[0084] like Figures 2-5As shown, QVPGLYYF forms hydrogen bonds, hydrophobic interactions, and charge interactions with the amino acids Phe236, Gly234, Asn235, Pro213, Pro112, Tyr183, yr185, Glu134, Lys136, His158, Asn159, and Phe160 in TLR4. Specifically, QVPGLYYF forms conventional hydrogen bonds with Phe236, Gly234, Asn235, Tyr185, Glu134, and Lys136, respectively. His158 and Asn159 in TLR4 bind to QVPGLYYF via carbon-hydrogen bonds. Furthermore, attractive charges are observed between QVPGLYYF and Lys136 and Glu134 in TLR4. Pro213, Tyr183, Pro112, His158, Phe160 form hydrophobic interactions with QVPGLYYF.

[0085] Molecular dynamics simulations of the fish scale collagen immunostimulatory peptide QVPGLYYF were performed. MD simulations have been used to determine the accuracy of binding affinity predictions based on molecular docking. Molecular dynamics simulations of TLR4 (5IJD) and fish scale collagen peptides were further investigated using Desmond / Maestronon commercial version 2022.1 software, building upon molecular docking. Molecular dynamics simulations use Newton's classical equations of motion, calculating the changes in atomic motion over time. In contrast to the molecular docking strategy, which provides a static picture of the active molecules in the protein's active binding region and predicts the ligand binding state, molecular dynamics simulations predicting ligand binding modes were performed in a physiological context. Using the TLR4 protein preparation wizard, the ligand-protein binding complex was first pretreated, then optimized and minimized. The protein-ligand complex was then attached to a rhomboid box in a pre-defined TIP3P water model, and the system was equilibrated with 0.15M sodium chloride solution. Considering pressure, timescale, and number of atoms, molecular dynamics simulations were performed for 100 ns using an isothermal and isobaric system at 300 K and 1 bar after minimizing and relaxing the system. Trajectory coordinates were recorded every 100 ps. Molecular dynamics analysis was performed using Desmond's simulation interaction plot to further explore the mechanism of binding between fish scale collagen peptides and TLR4.

[0086] During the simulation, the protein's RMSD stabilized at... Meanwhile, after fluctuations, the RMSD of the ligand eventually stabilizes at... Left and right, such as Figure 6 As shown in the figure. This indicates that the two have relatively small fluctuations relative to their initial conformations, suggesting that their initial conformations have high stability. After dynamic simulation, a more stable combined conformation is formed on the original basis.

[0087] like Figure 7 and Figure 8 As shown, the fluctuations in the RMSF values ​​of the protein and small molecule are within the normal range. Therefore, we believe that the protein-small molecule complex has formed a more stable conformation after kinetic simulation.

[0088] like Figure 9 and Figure 10 As shown, during the simulation, the protein and small molecule formed multiple sets of forces. For example, the hydrogen bond formation frequencies of PHE262 were 76% and 81%, respectively, with a total formation frequency as high as 157%. This indicates that the amino acid of PHE262 played a crucial role in the binding process. In addition, multiple hydrophobic interactions and water bridges were formed to help the two bind together.

[0089] In summary, the QVPGLYYF small molecule and protein were optimized based on their original binding conformation through kinetic simulation, resulting in a more stable conformation in which the two molecules exhibit higher affinity.

[0090] To verify the in vitro activity of the fish scale collagen immunostimulating peptide of the present invention, the following experiments were conducted. The experiments were divided into a normal group, an LPS group (1 μg / mL), a dexamethasone treatment group (50 μg / mL), and fish scale collagen immunostimulating peptide treatment groups (50 μg / mL, 100 μg / mL, and 200 μg / mL), with 6 replicates in each group.

[0091] Experiment 1: Determination of RAW264.7 macrophage cell viability

[0092] RAW264.7 macrophage cell viability was assessed using the MTS assay. Logarithmically growing RAW264.7 cells were collected and cultured in DMEM medium at a concentration of 1×10⁻⁶ cells / cells. 5 100 μL of cell suspension per 1 mL was seeded into a 96-well plate and incubated at 37°C for 24 hours. The sample was then dissolved in DMEM medium and added to the 96-well plate for another 24 hours of incubation. The supernatant was discarded, and 20 μL of MTS / PMS working solution was added to each well. After incubation at 37°C in the dark for 1–4 hours, the absorbance at 490 nm was measured using a microplate reader. The formula for determining macrophage viability is as follows:

[0093]

[0094] The effect of QVPGLYYF on the viability of RAW264.7 cells was investigated using the MTS assay. Figure 11As shown, compared with the normal group, different concentrations of QVPGLYYF had a certain impact on the viability of RAW264.7 cells, but none of them were cytotoxic. Furthermore, at concentrations of 50–200 μg / mL, it significantly promoted cell growth. Therefore, three concentrations of 50, 100, and 200 μg / mL were selected for subsequent experiments to further investigate its immunomodulatory effects.

[0095] Experiment 2: Determination of NO content

[0096] Similar to the culture method described above, when the cells have grown to 70%-80%, they are injected with 1×10⁻⁶ cells / mL. 5 After seeding cells at a density of [number] cells / mL until they adhered and grew stably, the cells were divided into groups and incubated for another 24 hours. 50 μL of the supernatant was transferred to each well of a new 96-well plate, and 50 μL of Griess I and Griess II solutions were added to each well. After thorough mixing, the absorbance at 540 nm was measured using a microplate reader. The concentration of NO was calculated by substituting the values ​​into a standard curve.

[0097] NO is an active mediator; macrophages can resist pathogen infection by releasing NO. Therefore, NO release levels were used to assess the immune-stimulating activity of QVPGLYYF. Figure 12 It can be seen that after treatment with QVPGLYYF, the amount of NO released in RAW264.7 macrophages was significantly increased, indicating that QVPGLYYF can resist foreign body infection by increasing the amount of NO released.

[0098] Experiment 3: Measurement of Phagocytosis

[0099] Phagocytosis is an important indicator of the activation status of RAW264.7 macrophages. Neutral red staining was used to measure the effect of collagen immunostimulatory peptides from different fish scales on the phagocytic activity of macrophages. RAW 264.7 cells (1×10⁻⁶) 5 Cells (number / mL) were seeded into 96-well plates and incubated for 24 h. After intervention according to experimental groups, the cells were cultured for another 24 h. To rule out potential interference from drugs in the DMEM culture medium, the cells were washed 1-2 times with DPBS solution, followed by the addition of 20 μL of neutral red staining solution to each 200 μL of cell culture medium. The cells were then incubated at 37°C. After 2-4 hours, the DMEM culture medium containing neutral red staining solution was removed, the cells were washed 1-2 times with DPBS, lysis buffer (200 μL) was added, and the cells were incubated on a shaker at room temperature for 10 min to completely dissolve the neutral red in the cells. The absorbance at 540 nm was measured using a microplate reader, and the phagocytic rate of each sample was calculated using the following formula:

[0100]

[0101] like Figure 13 As shown, QVPGLYYF enhances the phagocytic capacity of RAW264.7 cells for neutral red in a dose-dependent manner. The results indicate that QVPGLYYF can appropriately enhance the phagocytic capacity of macrophages to exert an immunomodulatory effect.

[0102] Experiment 4: Cytokine Secretion Assay

[0103] Similar to the culture method described above, after intervention according to experimental groups, cell culture supernatant was collected, and the levels of cytokines (IL-6, IL-1β, and TNF-α) were detected using an ELISA kit according to the kit instructions.

[0104] IL-6 has multiple biological functions and can participate in immune regulation. Figure 14 The results showed that RAW264.7 macrophages treated with different concentrations of QVPGLYYF exhibited a significant increase in IL-6 secretion compared to the control group. However, the difference was not significant compared to cells treated with LPS (1 μg / mL) alone. These experimental results indicate that QVPGLYYF can enhance the immunomodulatory activity of RAW264.7 macrophages by stimulating IL-6 secretion.

[0105] Depend on Figure 15 The results showed that increasing QVPGLYYF concentration significantly increased the production of IL-1β in macrophages in a dose-dependent manner. However, this increase was significantly different from that observed with LPS (1 μg / mL) alone. These experimental results indicate that QVPGLYYF exerts its immunomodulatory effect on RAW264.7 macrophages by stimulating IL-1β secretion.

[0106] Depend on Figure 16 It was found that with increasing QVPGLYYF concentration, the production level of TNF-α in macrophages increased significantly in a dose-dependent manner. However, this was significantly different from that of cells treated with LPS (1 μg / mL) alone. The experimental results indicate that QVPGLYYF exerts its immunomodulatory effect on RAW264.7 macrophages by stimulating TNF-α secretion.

[0107] In summary, this invention also discloses the application of the above-mentioned fish scale collagen immunostimulating peptide in at least one of the following situations:

[0108] (1) Application in enhancing the phagocytic capacity of macrophages;

[0109] (2) Application in the preparation of macrophage phagocytic capacity enhancers;

[0110] (3) Application in promoting macrophage proliferation;

[0111] (4) Application in the preparation of macrophage proliferation promoters;

[0112] (5) Application in enhancing the release of NO, IL-6, TNF-α and IL-1β from macrophages;

[0113] (6) Application in the preparation of macrophage NO release, IL-6, TNF-α and IL-1β secretion promoters;

[0114] (7) Application in the preparation of drugs that enhance biological immunity;

[0115] (8) Application in the preparation of food ingredients that enhance biological immunity.

[0116] The concentration of the fish scale collagen immunostimulating peptide is 50–200 μg / mL.

[0117] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A fish scale collagen immunostimulatory peptide, characterized in that, Its amino acid sequence is: QVPGLYYF.

2. The application of the fish scale collagen immunostimulating peptide as described in claim 1 in the preparation of food ingredients that enhance biological immunity.

3. Use according to claim 2, wherein: The concentration of the fish scale collagen immunostimulating peptide is 50–200 μg / mL.

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