Spirulina immunomodulatory peptide, preparation method and application thereof

Abstract

This invention specifically relates to spirulina immunomodulatory peptides, their preparation methods, and applications, belonging to the field of protein bioactive peptide technology. This invention is the first to screen immunomodulatory peptides Ser-Pro-Ser-Trp-Tyr (SPSWY), Met-Phe-Asp-Ala-Phe (MFDAF), and Phe-Gly-Arg-Phe-Arg (FGRFR) from spirulina phycocyanin. The structure and immunomodulatory activity of peptides SPSWY, MFDAF, and FGRFR were clarified. Peptides SPSWY, MFDAF, and FGRFR can enhance macrophage cell viability and phagocytic capacity, and also increase the secretion of NO and cytokines by macrophages. Their immunomodulatory activity also exhibits good stability. Furthermore, peptides SPSWY, MFDAF, and FGRFR have the advantages of being safe, non-toxic, and having high biological activity.

Description

Technical Field

[0001] This invention belongs to the field of protein bioactive peptide technology, specifically relating to spirulina immunomodulatory peptides, their preparation methods, and applications. Background Technology

[0002] Currently, weakened immunity due to unhealthy lifestyles, stress, illness, and medication use is widespread. Immunomodulatory drugs are commonly used to enhance immunity. However, the vast majority of currently used immunomodulatory drugs are chemical drugs, which often have certain toxic side effects and pose potential risks to human health and safety. Therefore, the development of safe and effective natural immunomodulators is essential. Immunomodulatory peptides developed from food proteins have advantages such as safety, naturalness, and easy absorption, and are welcomed both domestically and internationally.

[0003] Immunomodulatory peptides are bioactive peptides with immunomodulatory effects. They can be used as functional ingredients in food, health products, pharmaceuticals, and cosmetics to enhance human immunity, and their application prospects are broad.

[0004] Spirulina (Spirulina platensis) is a nutrient-rich single-celled cyanobacteria, abundant in protein, vitamins, polysaccharides, minerals, and other nutrients, making it an ideal food and nutritional supplement. Phycocyanin (C-PC) is the main protein and active substance in Spirulina, accounting for up to 20% of its composition. Its amino acid composition is balanced, containing all eight essential amino acids. Phycocyanin is sky blue and can be used as a natural pigment in food additives and cosmetics. It also exhibits bright fluorescence, making it widely used as a fluorescent marker in molecular biology, immunology, and cell biology research. Numerous studies have shown that phycocyanin possesses a wide range of biological activities, such as immunomodulation, blood pressure reduction, anti-tumor activity, blood sugar reduction, anti-inflammatory and antibacterial effects, antioxidant activity, anti-aging activity, anti-radiation activity, cell growth promotion, nerve protection, DNA protection, and liver protection. Therefore, phycocyanin has broad application prospects in food, pharmaceuticals, cosmetics, and other fields.

[0005] Peptides lack biological activity within their parent proteins and must be released from them to exert their effects. Therefore, utilizing appropriate technologies to release peptides from food proteins is crucial for the development of bioactive peptides. Currently, the main methods for peptide preparation include chemical hydrolysis, enzymatic hydrolysis, microbial fermentation, gene recombination, and chemical synthesis. Among these, enzymatic hydrolysis is the most commonly used method. Different types of proteases, due to differences in their cleavage sites, result in peptides with varying molecular weights, amino acid compositions, and sequences when hydrolyzing the same protein. Furthermore, the reaction time, pH, temperature, and enzyme dosage also affect enzyme activity and hydrolysis results to varying degrees. Therefore, when preparing peptides, it is essential to select the appropriate protease type based on the specificity of the cleavage site and the enzyme activity level, and to optimize the hydrolysis conditions. Suitable proteases and optimal hydrolysis conditions are crucial for the development and utilization of peptides, directly affecting whether bioactive peptides with specific functions can be obtained, as well as the efficiency and quality of bioactive peptide preparation. Currently, proteases have been successfully used to prepare bioactive peptides from food protein raw materials. Enzymatic preparation of bioactive peptides is widely used due to its advantages such as controllable conditions, mild reaction, high safety, and lack of toxic side effects. Commonly used proteases include neutral proteases, alkaline proteases, pepsin, trypsin, chymotrypsin, papain, trypsin, heat-soluble enzymes, and flavor proteases, among which trypsin, alkaline proteases, papain, and pepsin are the most widely used. Currently, bioactive peptides have been prepared from food protein raw materials such as milk, eggs, fish, rice, soybeans, flaxseed, whey, spirulina, clams, and frogs. To date, reported peptides with immunomodulatory activity include: bovine casein peptides VEPIPY and LLY, human casein peptide GLF, rice protein peptide GYPMYPLP, ​​cod protein peptide PTGADY, shellfish protein peptide GVSLLQQFFL, and so on.

[0006] Phycocyanin has a balanced amino acid composition, making it a high-quality protein source for developing bioactive peptides. Literature reports the use of papain to hydrolyze phycocyanin to obtain antioxidant peptides, and the use of thermophilic bacteria protease to hydrolyze phycocyanin to obtain the ACE-inhibiting peptide VTY. Theoretically, phycocyanin contains a high proportion of hydrophobic amino acids, making it an excellent protein raw material for preparing immunomodulatory peptides. However, to date, few immunomodulatory peptides have been screened from Spirulina phycocyanin. Currently, only LDAVNR and MMLDF have been reported as immunomodulatory peptides from Spirulina phycocyanin.

[0007] The evaluation of immunomodulatory peptide activity primarily involves measuring immunomodulatory activity in cells, animal models, and even humans. In vitro immunomodulatory activity assays based on cells are widely used, while in vivo immunomodulatory activity assays based on animal models or humans can more realistically evaluate immunomodulatory efficacy; however, in vivo assays are costly, time-consuming, and challenging. Macrophages are widely used as cell models for evaluating immunomodulatory activity, with changes in cell viability, phagocytic capacity, and the secretion of nitric oxide, IL-6, and TNF-α reflecting the immunomodulatory activity of peptides. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides spirulina immunomodulatory peptides, their preparation methods, and applications. This invention is the first to screen previously unreported immunomodulatory peptides, Ser-Pro-Ser-Trp-Tyr (SPSWY), Met-Phe-Asp-Ala-Phe (MFDAF), and Phe-Gly-Arg-Phe-Arg (FGRFR), from spirulina phycocyanin. Mass spectrometry identified the structures of peptides SPSWY, MFDAF, and FGRFR. Bioinformatics analysis showed that peptides SPSWY, MFDAF, and FGRFR are safe, non-toxic, and have high biological activity. Macrophage assays confirmed that peptides SPSWY, MFDAF, and FGRFR possess immunomodulatory activity. Therefore, they have promising applications as functional components in foods, health products, cosmetics, and pharmaceuticals with immunomodulatory effects.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A spirulina immunomodulatory peptide, wherein the amino acid sequence of the immunomodulatory peptide is shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3;

[0011] The amino acid sequences of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are: Ser-Pro-Ser-Trp-Tyr (SPSWY), Met-Phe-Asp-Ala-Phe (MFDAF), and Phe-Gly-Arg-Phe-Arg (FGRFR), respectively.

[0012] A composition comprising the above-mentioned immunomodulatory peptide and pharmaceutically, food, or health product-acceptable excipients.

[0013] The use of the above-mentioned immunomodulatory peptides or the above-mentioned compositions in the preparation of cosmetics with immunomodulatory effects.

[0014] The use of the above-mentioned immunomodulatory peptides or the above-mentioned compositions in the preparation of drugs with immunomodulatory effects.

[0015] The use of the above-mentioned immunomodulatory peptides or the above-mentioned compositions in the preparation of food or health products with immunomodulatory effects.

[0016] The preparation and screening methods for the above-mentioned immunomodulatory peptides include the following steps:

[0017] (1) Enzymatic hydrolysis: Papain was added to the spirulina phycocyanin solution for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme activity was inactivated, the supernatant was collected, ultrafiltration and centrifugation were performed, the filtrate was collected, and the spirulina phycocyanin peptide lyophilized powder was prepared by freeze drying.

[0018] (2) Screening: The sequence of Spirulina phycocyanin peptide was identified by ultra-high performance liquid chromatography-tandem mass spectrometry. Then, peptides with potential immunomodulatory activity were screened according to the following conditions: 1) amino acid sequence length of 2-10 amino acid residues; 2) PeptideRanker prediction score greater than 0.5; 3) rich in hydrophobic or basic amino acid residues; 4) not reported in the BIOPEP database; 5) predicted to have no potential toxicity.

[0019] (3) Activity assay: Chemically synthesize polypeptides with potential immunomodulatory activity, measure the immunomodulatory activity of the polypeptides, and identify spirulina immunomodulatory peptides.

[0020] According to a preferred embodiment of the present invention, the amount of papain added in step (1) is 2000-2500 U / g phycocyanin.

[0021] According to a preferred embodiment of the present invention, the pH value of the enzymatic hydrolysis in step (1) is 7 ± 0.2.

[0022] According to a preferred embodiment of the present invention, the temperature of the enzymatic hydrolysis in step (1) is 55-60°C.

[0023] According to a preferred embodiment of the present invention, the enzymatic hydrolysis time in step (1) is 4-5 hours.

[0024] According to a preferred embodiment of the present invention, the ultrafiltration centrifugation in step (1) uses an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa.

[0025] Beneficial effects:

[0026] This invention is the first to screen immunomodulatory peptides Ser-Pro-Ser-Trp-Tyr (SPSWY), Met-Phe-Asp-Ala-Phe (MFDAF), and Phe-Gly-Arg-Phe-Arg (FGRFR) from spirulina phycocyanin. The structure and immunomodulatory activity of peptides SPSWY, MFDAF, and FGRFR were also clarified. Peptides SPSWY, MFDAF, and FGRFR can enhance macrophage cell viability and phagocytic capacity, as well as increase the secretion of NO and cytokines by macrophages. Their immunomodulatory activity also exhibits good stability. Furthermore, peptides SPSWY, MFDAF, and FGRFR are safe, non-toxic, and have high biological activity. Therefore, they have good potential and application prospects as functional ingredients in foods, health products, cosmetics, and pharmaceuticals with immunomodulatory effects. Attached Figure Description

[0027] Figure 1 This is a mass spectrometry chromatogram of the polypeptide SPSWY.

[0028] Figure 2 This is a mass spectrometry analysis of the peptide MFDAF.

[0029] Figure 3 This is a mass spectrometry analysis of the peptide FGRFR.

[0030] Figure 4 The bar chart shows the cell viability of RAW264.7 macrophages after peptide treatment; different letters in the figure indicate significant differences between groups based on biostatistical analysis, and the same applies below.

[0031] Figure 5 Bar chart showing the phagocytic capacity of RAW264.7 macrophages after peptide treatment.

[0032] Figure 6 A bar chart showing the NO release from RAW264.7 macrophages after peptide treatment.

[0033] Figure 7 The bar chart shows the amount of TNF-α secreted by RAW264.7 macrophages after peptide treatment.

[0034] Figure 8 A bar chart showing the amount of IL-6 secreted by RAW264.7 macrophages after peptide treatment.

[0035] Figure 9 Bar graph showing the cell viability of RAW264.7 macrophages after treatment with peptides that simulate in vitro gastrointestinal digestion.

[0036] Figure 10Bar graph showing the cell viability of RAW264.7 macrophages after peptide treatment at different pH incubation. Detailed Implementation

[0037] The technical solution of the present invention will be described in detail below. It should be understood that the embodiments presented herein are merely preferred examples for illustrative purposes and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents and instruments used in the following embodiments are all commercially available products.

[0038] Example 1: Extraction of phycocyanin from Spirulina

[0039] Phycocyanin was crudely extracted using a repeated freeze-thaw method. First, spirulina powder was added to 0.1 mol / L pH 7.0 PBS buffer at a ratio of 1:20 (w / v, g / mL), stirred until completely dissolved, and then frozen completely. The mixture was then thawed under running water or in a 37°C water bath, and this freeze-thaw cycle was repeated three times. The extract was then centrifuged at 6000×g for 20 min (4°C) to remove the precipitate, yielding a blue crude phycocyanin extract. The crude phycocyanin extract was fractionated with ammonium sulfate, first with 25% and then 60% saturated ammonium sulfate solutions, and centrifuged after each precipitation. The precipitate was dissolved in an appropriate amount of PBS buffer and dialyzed overnight with PBS buffer. The dialysate was purified using a DEAE-Sephadex Fast Flow anion exchange chromatography column (1.6×20 cm). The chromatography column was first pre-equilibrated with 20 mM PBS buffer (pH 7.0), then loaded with the sample and eluted with PBS buffer to remove contaminating proteins. Elution was then performed with 20 mM PBS buffer (pH 7.0) containing 0.3 M and 0.5 M NaCl, respectively, at a rate of 1 mL / min. -1 Collect the blue fraction and dialyze it overnight (4°C, 12h) through a 3500 Da semipermeable membrane to obtain a purified phycocyanin solution. Freeze-dry the solution to obtain purified phycocyanin lyophilized powder. Store at -20°C for later use.

[0040] The concentration, yield, and purity of phycocyanin solution were calculated using the following formula:

[0041]

[0042]

[0043]

[0044] In the formula: C C-PC Indicates phycocyanin concentration; A 620 Indicates absorbance at a wavelength of 620 nm; A 652The absorbance is measured at a wavelength of 652 nm; Yield represents the yield of phycocyanin; V represents the volume of the phycocyanin solution; M represents the mass of spirulina powder; Purity represents the purity of phycocyanin; A 280 This indicates the absorbance at a wavelength of 280 nm.

[0045] Example 2: Enzymatic preparation of Spirulina phycocyanin peptides

[0046] Take the phycocyanin solution prepared in Example 1, adjust the pH of the phycocyanin solution to 7, and then add papain (papain enzyme activity is 1×10⁻⁶) at a ratio of 2000 U / g phycocyanin. 5 Mix the spirulina (U / g) solution thoroughly and place it in a shaker for enzymatic hydrolysis at 100-200 rpm for 5 hours at 55℃. After hydrolysis, boil the hydrolysate in a water bath for 10 minutes to inactivate any residual enzymes. Centrifuge at 6000×g for 20 minutes (4℃), collect the supernatant, and discard the precipitate. The supernatant is the spirulina phycocyanin peptide solution. Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa to ultrafilter and centrifuge the spirulina phycocyanin peptide solution at 6000×g for 20 minutes (4℃). Aspirate the solution retained in the ultrafiltration tube; this is the ultrafiltration fraction with a molecular weight <3 kDa. Freeze-dry the ultrafiltration fraction under vacuum to prepare spirulina phycocyanin peptide lyophilized powder.

[0047] Example 3: Structural Identification of Spirulina Phycocyanin Peptides

[0048] (1) Mass spectrometry identification

[0049] The lyophilized spirulina phycocyanin peptide powder with a molecular weight <3kDa prepared in Example 2 was dissolved in ultrapure water, and particles were removed by filtration using a 0.22μm aqueous syringe filter. The phycocyanin peptide was then identified by mass spectrometry using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-TOF-MS / MS) on a Thermo Fisher Q Exactive Focus. The mobile phase A of the UPLC was an aqueous solution containing 0.1% formic acid, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid. The detection wavelength was 220nm. The elution time was 60 min, and the gradient elution conditions are shown in Table 1. The injection volume was 5μL, and the flow rate was set to 300μL / min.

[0050] Table 1. Elution conditions for ultra-high performance liquid chromatography

[0051]

[0052] Tandem mass spectrometry was performed in positive ion mode, using secondary mass spectrometry analysis with a capillary voltage of 3.5 kV and a full MS scan of 100-2000 m / z at a resolution of 120000. Specific mass spectrometry parameter settings are shown in Table 2.

[0053] Table 2. Tandem Mass Spectrometry Analysis Conditions

[0054]

[0055] (2) Mass spectrometry data analysis

[0056] The raw files obtained from UPLC-ESI-MS / MS analysis were converted into MGF format mass spectrometry universal files using MM File Conversion software. The database was searched using the MASCOT search engine. The MS / MS data were analyzed using Peaks Viewer 4.5 software, and peptide sequencing was performed using manual de novo sequencing.

[0057] (3) Multiple polypeptide sequences were obtained by UPLC-ESI-TOF-MS / MS identification.

[0058] Example 4: Bioinformatics prediction of Spirulina phycocyanin peptides

[0059] Analysis of the active peptide sequences revealed that low molecular weight peptides have higher activity, and most of them contain 2-10 amino acid residues. Therefore, peptide sequences with <10 amino acid residues were selected for further analysis.

[0060] Use the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) to check whether the peptide sequence has been verified.

[0061] Next, the potential biological activity of the identified peptide sequences was predicted using the PeptideRanker online platform (http: / / distilldeep.ucd.ie / PeptideRanker / ). A prediction score >0.5 was considered to indicate potential biological activity. The peptide sequences with prediction scores >0.5 underwent physicochemical property analysis. PepDraw (https: / / www2.tulane.edu / ~biochem / WW / PepDraw / ) was used to analyze the physicochemical properties (such as molecular weight, isoelectric point, hydrophobicity, and charge) of the screened peptides.

[0062] Bioinformatics techniques were then used to predict the potential toxicity of peptides (http: / / crdd.osdd.net / raghava / toxinpred / ). The ToxinPrep platform (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php) was used, based on the SVM (Swiss-Port) algorithm, to predict the potential toxicity of peptides.

[0063] The immunomodulatory activity of bioactive peptides is closely related to the amino acid sequence composition, the ratio of hydrophobic amino acids to basic amino acid residues, etc. Peptides with potential immunomodulatory activity should meet the following conditions: (1) amino acid sequence length is 2-10 amino acid residues; (2) PeptideRanker prediction score is greater than 0.5; (3) rich in hydrophobic amino acid or basic amino acid residues; (4) not reported in the BIOPEP database; (5) predicted to have no potential toxicity.

[0064] The peptide sequences that simultaneously meet the above conditions are Ser-Pro-Ser-Trp-Tyr (SPSWY, SEQ ID NO.1), Met-Phe-Asp-Ala-Phe (MFDAF, SEQ ID NO.2), and Phe-Gly-Arg-Phe-Arg (FGRFR, SEQ ID NO.3). The PeptideRanker prediction scores for peptides SPSWY, MFDAF, and FGRFR are 0.848464, 0.956328, and 0.975761, respectively. The mass spectrometry analysis results are shown below. Figure 1 , Figure 2 and Figure 3 The molecular weight and hydrophobicity results are shown in Table 3. Bioinformatics prediction analysis results indicate that peptides SPSWY, MFDAF, and FGRFR have high potential biological activity.

[0065] Table 3. Bioinformatics prediction results of phycocyanin peptides

[0066]

[0067] Example 5: Chemical synthesis and immunomodulatory activity assay of immunomodulatory peptides SPSWY, MFDAF, and FGRFR

[0068] The peptides SPSWY, MFDAF, and FGRFR were chemically synthesized using Fmoc amino acid solid-phase synthesis technology. The peptides were synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. The purity of the synthesized peptides was >95%. The synthesized peptides were subjected to quality control using HPLC and HPLC-MS.

[0069] The immunomodulatory activities of peptides SPSWY, MFDAF, and FGRFR were measured in vitro on RAW264.7 macrophages.

[0070] (1) Cell viability assay

[0071] Macrophage culture: RAW264.7 macrophages were seeded in DMEM medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) and cultured in a cell culture incubator at 37°C and 5% CO2. When the cells reached approximately 80% confluence at the bottom of the culture dish, they were passaged. The old medium was discarded, and the cells were washed twice with PBS buffer. Fresh medium was then added, and the cells were pipetted to form a cell suspension. The cell suspension was centrifuged at 800 rpm for 10 min, the old medium was discarded, fresh medium was added, and the cells were transferred to new cell culture dishes. Fresh medium was added again, and the cells were cultured in the incubator.

[0072] Once the cells have reached the logarithmic growth phase, they are seeded into 96-well plates, with the cell density adjusted to 5 × 10⁶ cells / well before seeding. 4 Cells were cultured at 100 μL / mL per well for 12-24 h until they were fully adhered and reached approximately 80% confluence. The supernatant was discarded, and peptide samples at different concentrations (50, 100, 200 μg / mL) prepared in DMEM complete medium were added. DMEM complete medium served as a blank control, and DMEM complete medium containing 1 μg / mL LPS served as a positive control. The plates were incubated for 24 h. Then, 20 μL of 5 mg / mL MTT was added, and the plates were incubated in the dark for 4 h. The cell culture medium was then discarded, and 150 μL of DMSO was added. The 96-well plate was placed in a microplate shaker and shaken until the crystals dissolved and no visible particles remained. The absorbance at 490 nm was measured using a microplate reader. Cell viability was calculated using the formula:

[0073] Cell viability = A 490(样品) / A 490(空白对照)

[0074] In the formula, A 490(样品) A represents the absorbance of the peptide sample group at 490 nm. 490(空白对照) This indicates the absorbance of the blank control group at 490 nm.

[0075] The effects of peptides on the viability of RAW264.7 macrophage cells are shown in the following results. Figure 4 Compared with the blank control group, except for peptide MFDAF, the other two peptides, FGRFR and SPSWY, significantly improved macrophage cell viability, and the differences between peptides FGRFR (200 μg / mL) and SPSWY (100 and 200 μg / mL) and the blank control group were statistically significant (p<0.05). Furthermore, peptides FGRFR and SPSWY exhibited a dose-response effect within the concentration range of 50-200 μg / mL. These results indicate that peptides FGRFR and SPSWY can both enhance the cell viability of RAW264.7 macrophages and promote cell proliferation.

[0076] (2) Measurement of phagocytic capacity

[0077] RAW264.7 macrophages were cultured as described previously. Once the cells reached the logarithmic growth phase, they were seeded into 96-well plates, with the cell density adjusted to 5 × 10⁶ cells / well before seeding. 4 Cells were cultured at 100 μL / well for 12-24 h until they were fully adhered and reached approximately 80% confluence. The supernatant was discarded. Different concentrations of peptide sample solutions (final concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, prepared in DMEM complete medium), a blank control (DMEM complete medium), and a positive control (LPS, 1 μg / mL, prepared in DMEM complete medium) were added to 96-well plates (100 μL / well). The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Then, 0.1% neutral red solution (100 μL / well) was added to each well, and the plates were incubated for another 1 h. Cells were then washed twice with PBS buffer to remove excess neutral red. Finally, a mixture of 0.1 M glacial acetic acid and ethanol (1:1, v / v) (100 μL / well) was added to each well, and the plates were incubated at 37°C for 2 h. Finally, the absorbance of each well at 540 nm was measured using a microplate reader. The phagocytic rate of macrophages phagocytosing neutral red cells was calculated using the formula:

[0078] Cell phagocytosis rate (%) = A 540(样品) / A 540(空白对照) ×100%.

[0079] In the formula: A 540(样品) A represents the absorbance of the polypeptide sample group at a wavelength of 540 nm. 540(空白对照) This indicates the absorbance of the blank control group at a wavelength of 540 nm.

[0080] Macrophages are important cells in the human immune system. They can recognize and destroy foreign invaders through innate, adaptive, or adaptive immunity, and can engulf and kill intracellular parasites, bacteria, and tumor cells. The effect of peptides on the phagocytic capacity of RAW264.7 macrophages was evaluated using neutral red staining. The results are as follows: Figure 5 As shown in the figure, compared with the blank control group, the phagocytic capacity of macrophages was significantly enhanced after LPS treatment (p<0.05); the phagocytic rate of macrophages was significantly increased after peptide treatment (p<0.05) in a dose-dependent manner. These results indicate that the peptides SPSWY, MFDAF, and FGRFR can enhance the phagocytic capacity of RAW264.7 macrophages, thereby activating the immunomodulatory activity of the cells.

[0081] (3) Measurement of NO and cytokine secretion

[0082] The effect of synthetic peptides on NO release from RAW264.7 macrophages was analyzed by determining nitrite levels in macrophage supernatants using the Griess reaction. The specific method was as follows: macrophages were cultured and treated with different samples according to the cell viability assay procedure described above. After incubation for 24 h, cell supernatants from each treatment group were collected, centrifuged at 1000×g for 10 min, and 50 μL of the supernatant was transferred to a 96-well plate. 50 μL of Griess reagent I and II were added to each well, and the mixture was incubated at room temperature for 10 min. The absorbance at 540 nm was measured using a microplate reader. A standard curve was constructed using sodium nitrite (NaNO2) concentration, and the NO concentration in the supernatant of each treatment group was calculated based on the standard curve. Cytokine (IL-6 and TNF-α) levels were measured using an ELISA kit.

[0083] The effect of peptides on NO release from RAW264.7 macrophages is as follows: Figure 6 As shown in the figure, compared with the blank control group, the NO release from macrophages increased to varying degrees after treatment with LPS and peptides, with the most significant increase observed in the LPS (1 μg / mL) group. Peptide FGRFR at concentrations of 100 and 200 μg / mL, and peptide SPSWY at a concentration of 200 μg / mL, also significantly increased NO release from macrophages (p<0.05). These results indicate that peptides SPSWY, MFDAF, and FGRFR can enhance NO release from RAW264.7 macrophages.

[0084] Macrophages produce cytokines and participate in immune regulation. Increased secretion of TNF-α and IL-6 is considered a marker of immune stimulation. Therefore, the concentrations of TNF-α and IL-6 in the cell supernatant after peptide treatment of RAW264.7 macrophages can be used to verify the immunostimulatory activity of the peptide. The results of the effect of the peptide on TNF-α secretion in RAW264.7 macrophages are as follows: Figure 7 As shown in the figure, compared with the blank control group, macrophages treated with 1 μg / mL LPS showed a significant increase in TNF-α secretion (p<0.05), reaching a concentration of 568.52±24.14 pg / mL. Treatment with peptides also increased TNF-α secretion in macrophages, exhibiting a dose-response effect within the concentration range of 50-200 μg / mL. Specifically, the peptide SPSWY significantly increased TNF-α secretion in macrophages at concentrations of 50-200 μg / mL (p<0.05).

[0085] The effects of peptides on IL-6 secretion in RAW264.7 macrophages are as follows: Figure 8As shown in the figure, compared with the blank control group, the IL-6 secretion of macrophages was significantly increased after LPS treatment (p<0.05); after 24 h of peptide treatment, the IL-6 secretion of macrophages also increased to varying degrees, and peptides FGRFR, SPSWY, and MFDAF all showed dose-response effects. Among them, the IL-6 secretion of macrophages at a concentration of 200 μg / mL of peptides FGRFR and SPSWY was significantly different from that of the blank control group (p<0.05). It is worth mentioning that the TNF-α and IL-6 secretion of macrophages in different peptide groups were significantly different from those in the LPS group (p<0.05), indicating that peptides SPSWY, MFDAF, and FGRFR can induce immune stimulation, but do not lead to excessive inflammation.

[0086] Example 6: Stability Study of Immunomodulatory Peptides SPSWY, MFDAF, and FGRFR

[0087] (1) Gastrointestinal digestion is one of the important factors affecting the activity of bioactive peptides in vivo. To study the effects of gastrointestinal digestion on peptides SPSWY, MFDAF, and FGRFR, a computer simulation of gastrointestinal digestion (PeptideCutter, https: / / web.expasy.org / cgi-bin / peptide_cutter / peptidecutter.pl) was first used. The changes in the sequence structure of peptides SPSWY, MFDAF, and FGRFR are shown in Table 4. Peptides SPSWY, MFDAF, and FGRFR were all hydrolyzed to varying degrees by pepsin during gastric digestion. Then, peptide FGRFR was further hydrolyzed by trypsin during intestinal digestion. However, peptides SPSWY and MFDAF were not hydrolyzed during intestinal digestion because they do not have specific trypsin cleavage sites.

[0088] Table 4. Results of PeptideCutter simulated enzyme digestion of peptides

[0089]

[0090] Computer simulations theoretically model the cleavage sites of pepsin and trypsin and idealize the hydrolysis process. However, in actual hydrolysis, protease hydrolysis is probabilistic. Therefore, the impact of gastrointestinal digestion on peptide activity needs to be further verified through experiments.

[0091] The peptides SPSWY, MFDAF, and FGRFR (10 mg) were dissolved in 10 mL of distilled water. The pH was adjusted to 2.0 with 1 M HCl, and then 2% (w / w) pepsin was added. The mixture was incubated at 37 °C for 2 h to simulate gastric digestion. The pH of the digestive solution was then adjusted to 5.3 with 0.9 M NaHCO3 solution, and further adjusted to 7.5 with 1 M NaOH solution. Subsequently, 2% (w / w) trypsin was added, and the mixture was incubated at 37 °C for 2 h to simulate intestinal digestion. After digestion, the digestive solution was inactivated in a 100 °C water bath for 10 min, centrifuged at 10,000 rpm for 10 min, and the supernatant was freeze-dried to obtain the gastrointestinal digestive solution of the peptides.

[0092] The effect of peptides after in vitro simulated gastrointestinal digestion on the viability of RAW264.7 macrophages was determined according to the steps in Example 5. The results are as follows: Figure 9 As shown in the figure, compared with the untreated group, the cell viability of RAW264.7 macrophages decreased to varying degrees after digestion with pepsin and trypsin, but both still maintained high cell viability. The cell viability of macrophages after digestion with pepsin for peptides FGRFR and MFDAF was not significantly different from that of the untreated group.

[0093] Computer simulation analysis revealed that the new sequences generated after simulated gastrointestinal digestion still have high potential biological activity. For example, the PeptideRanker prediction scores of peptide sequences such as GRF, GR, and SPSW are still greater than 0.5, and most of them have high hydrophobicity and isoelectric point, which may be the reason why these peptide sequences can maintain high immunostimulatory activity.

[0094] (2) pH value is also one of the important factors affecting peptide activity. To analyze the pH stability of peptides SPSWY, MFDAF, and FGRFR, peptide solutions (1 mg / mL) were incubated at pH 3.0, 5.0, 7.0, and 9.0 for 30 min, the pH was adjusted to 7.0, the peptides were lyophilized, and cell viability was measured. Peptides that were not subjected to pH treatment served as controls, and cell viability was measured.

[0095] The effects of peptides treated at different pH levels on the viability of RAW264.7 macrophage cells are shown in the following results. Figure 10As shown, the relative cell viability of macrophages treated with peptides SPSWY, MFDAF, and FGRFR did not show a significant decrease within a pH range of 3-9, all reaching over 80% of the cell viability of the untreated group. In the peptide groups treated under neutral conditions, macrophage cell viability remained essentially unchanged, with only minor fluctuations. Although the cell viability of macrophages treated under acidic or alkaline conditions was affected, they still exhibited good cell activity. Specifically, peptide FGRFR increased the relative cell viability of macrophages to 1.08 ± 0.01 at pH 3.0. This is presumably because peptide FGRFR carries more positive charge under acidic conditions, and the positive charge decreases as the pH increases. Studies have shown that positively charged immunomodulatory peptides have stronger chemotactic activity, binding to receptors on the surface of immune cell membranes and stimulating immune responses. These results indicate that peptides SPSWY, MFDAF, and FGRFR possess good pH stability and can be used in food and health product processing systems at pH 3-9 while maintaining good immune activity.

[0096] Example 7: Molecular docking of immunomodulatory peptides SPSWY, MFDAF, and FGRFR

[0097] Toll-like receptor proteins TLR2 (PDB ID: 1FYW) and TLR4 / MD2 (PDB ID: 5IJD) were used as protein receptors. Protein crystal structures were downloaded from the RCSB PDB database (http: / / www.rcsb.org / ). Preprocessing of the receptor protein crystal structures included hydrogenation, water molecule removal, amino acid modification, energy optimization, and force field parameter adjustment to achieve a low-energy conformation for ligand binding.

[0098] Molecular docking of the peptide with the binding pockets of Toll-like receptor proteins TLR2 (PDB ID: 1FYW) and TLR4 / MD2 (PDB ID: 5IJD) was performed using AutoDock Vine software, and the interaction mechanism between the peptide and receptor proteins was analyzed using PYMOL software. The binding energy between the peptide and receptor protein can be used as a reference value for predicting the interaction. The lower the binding energy, the higher the binding affinity between the peptide and the Toll-like receptor protein, thus making it easier to form a more stable molecular binding conformation. The binding energies of the peptide with Toll-like receptor proteins TLR2 and TLR4 / MD2 are shown in Table 5.

[0099] Table 5. Binding energies of peptides to receptor proteins TLR2 and TLR4 / MD2.

[0100]

[0101] In terms of binding energy, the interaction between peptides and the receptor protein TLR4 / MD2 is stronger than that between peptides and TLR2 itself. Peptides FGRFR, SPSWY, and MFDAF exhibit strong interactions with TLR4 / MD2, with binding energies of -8.9 kcal / mol, -8.2 kcal / mol, and -8.4 kcal / mol, respectively; while peptides FGRFR and SPSWY show strong interactions with TLR2, with binding energies of -8.3 kcal / mol and -7.4 kcal / mol, respectively. Peptide MFDAF has the lowest binding energy with TLR2, at -6.9 kcal / mol. The trend in binding energy is consistent with previous experimental results. Furthermore, the mechanism of action of phycocyanin immunomodulatory peptides can be further elucidated: the peptides bind to Toll-like receptor proteins TLR2 and TLR4 / MD2 on the surface of immune cell membranes, thereby activating cellular immune responses and exerting immunomodulatory activity.

[0102] The interaction sites between the peptide and the receptor protein were analyzed using PYMOL. The interaction between the peptide and the receptor protein TLR4 / MD2 is mediated through hydrogen bonds or hydrophobic contacts of residues. The binding pocket of the receptor protein TLR4 / MD2 is predominantly hydrophobic, with a relatively large cavity, allowing the peptide to bind stably within the cavity and be occupied by complementary structures. The peptide FGRFR forms two hydrogen bonds with the Glu-694 and Ser-696 residues of the receptor protein TLR2, with lengths of [missing information]. and It forms 9 hydrogen bonds with the receptor protein TLR4 / MD2 at Asn-129, Ser-182, Asp-208, Asn-70, Lys-72, and Asp-100 residues, with lengths of [missing information]. and The polypeptide SPSWY forms six hydrogen bonds with four amino acid residues of the receptor protein TLR2: Lys-751, Lys-754, Ala-732, and Ser-784. The lengths of these bonds are as follows: and Nine hydrogen bonds were formed with the receptor protein TLR4 / MD2 at the residues Try-454, Asn-456, Gln-505, Thr-457, Ser-180, and Gln-505, with lengths of [missing information]. and The peptide MFDAF forms six hydrogen bonds with the receptor protein TLR2 at residues Ser-636, Ile-639, His-697, and Ile-693, with lengths of [missing information]. and It forms six hydrogen bonds with the receptor protein TLR4 / MD2 at residues Asn-129, Asp-180, Try-202, Asp-100, and Asp-99, with lengths of [missing information]. and

[0103] Generally, the difference in the number of hydrogen bonds may be one of the reasons for the differences in ligand-receptor interactions. The groups on the peptide side chains (aromatic amino acids and amino acids with carboxyl groups generally form hydrogen bonds more easily) are influencing factors in hydrogen bond formation in the system. Overall, the peptides FGRFR and SPSWY form the most hydrogen bonds with the receptor protein TLR4 / MD-2, which contributes to strong interactions, and these peptides often exhibit better immunomodulatory activity. This result is consistent with actual results from in vitro cell experiments.

[0104] Studies have shown that hydrogen bonding (with a greater influence than charge interactions and π-π bond interactions) plays a crucial role in stabilizing the crystal structure of peptide-receptor complexes and has a positive effect on substrate inhibition or activation. Phycocyanin immunomodulatory peptides interact with TLRs by forming hydrogen bonds with binding sites in the binding pockets of Toll-like receptor proteins TLR2 and TLR4 / MD2, triggering a series of signal transduction pathways, stimulating macrophages, and activating and regulating the body's immune response.

[0105] The above experimental results show that Spirulina phycocyanin peptides SPSWY, MFDAF, and FGRFR have immunomodulatory activity, are safe and non-toxic, and can be applied to the development of immune-modulatory cosmetics, food, health products, and pharmaceuticals.

Claims

1. A spirulina immunomodulatory peptide, characterized in that, The amino acid sequence of the immunomodulatory peptide is shown in SEQ ID NO. 3; The amino acid sequence of SEQ ID NO.3 is: Phe-Gly-Arg-Phe-Arg.

2. A composition, characterized in that, The composition comprises the immunomodulatory peptide of claim 1 and pharmaceutically or food-acceptable excipients.

3. Use of an immunomodulatory peptide of claim 1 or a composition of claim 2, characterized in that, It is used in the preparation of drugs that help enhance immunity, wherein the enhancement of immunity is to improve the phagocytic capacity and cell viability of macrophages.

4. The use of an immunomodulatory peptide of claim 1 or a composition of claim 2, characterized in that, It can be used to prepare foods that help boost immunity.

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