CN116514957A - Spirulina phycocyanin active peptide with immunoregulatory activity, preparation method and application thereof - Google Patents

Abstract

The invention particularly relates to a spirulina phycocyanin active peptide with immunoregulatory activity, a preparation method and application thereof, and belongs to the technical field of protein active peptides. The invention screens the active peptide Tyr-Asn-Lys-Phe-Pro-Tyr (YNKFPY) and Gly-Tyr-Tyr-Leu-Arg-Met (GYYLRM) from spirulina phycocyanin for the first time, and defines the structure and immunoregulatory activity of the active peptide YNKFPY, GYYLRM, the active peptide YNKFPY, GYYLRM can improve the cell activity and phagocytic capacity of macrophages, can also improve the secretion of NO and cytokines of the macrophages, has good stability of the immunoregulatory activity, and meanwhile, the active peptide YNKFPY, GYYLRM also has the advantages of safety, NO toxicity and high biological activity, and has good potential and application prospect.

Description

Spirulina phycocyanin active peptide with immunomodulatory activity and preparation method thereof application

technical field

The invention belongs to the technical field of protein active peptides, and in particular relates to a spirulina phycocyanin active peptide with immunoregulatory activity and a preparation method and application thereof.

Background technique

At this stage, the phenomenon of low immunity of the population caused by unhealthy lifestyle, stress, disease and drug application is relatively common. Drugs with immunomodulatory effects are usually used to enhance immunity. Most of the currently used drugs with immunomodulatory effects are chemical drugs. These immunomodulatory drugs usually have certain toxic and side effects, and there are potential risks to human health and safety. Therefore, safe and efficient natural immune regulators have been developed. Dosage is very necessary.

Immunomodulatory peptides refer to active peptides with immunomodulatory effects, which can be used as functional ingredients in food, health care products, medicines and cosmetics to enhance human immunity, and have broad application prospects. Immunomodulatory peptides developed from food proteins are safe, natural and easy to absorb, and are welcomed at home and abroad.

Spirulina (Spirulina platensis) is a nutritious single-cell blue-green algae rich in protein, vitamins, polysaccharides, minerals and other nutrients, and is an ideal food and nutritional supplement. C-Phycocyanin (C-Phycocyanin, C-PC) is the main protein and active substance in spirulina, with a content of up to 20% in spirulina. Its amino acid composition is balanced and contains 8 kinds of essential amino acids for human body. Phycocyanin is sky blue and can be used as a natural pigment in food additives, cosmetics, etc. Phycocyanin also has bright fluorescence, so it is widely used as a fluorescent marker in molecular biology, immunology, and cell biology research. A large number of studies have shown that phycocyanin has a wide range of biological activities, such as immune regulation, lowering blood pressure, anti-tumor, lowering blood sugar, anti-inflammatory and antibacterial, anti-oxidation, anti-aging, anti-radiation, promoting cell growth, protecting nerves, and protecting DNA , liver protection, etc. Therefore, phycocyanin has broad application prospects in the fields of food, medicine, cosmetics and medicine.

Peptides have no biological activity in the parent protein and must be released from the parent protein to play their role. Therefore, using appropriate technology to release peptides from food proteins is crucial to the development of bioactive peptides. At present, the peptide preparation methods mainly include: chemical hydrolysis, enzymatic hydrolysis, microbial fermentation, genetic recombination and chemical synthesis. Among them, enzymatic hydrolysis is currently the most commonly used method for preparing peptides. Different types of proteases have different cleavage sites, resulting in the hydrolysis of the same protein to produce peptides with different molecular weights, different amino acid compositions and different sequences. In addition, the time, pH, temperature, and amount of enzyme added to the enzymatic hydrolysis reaction will also affect the enzyme activity and enzymatic hydrolysis results to varying degrees. Therefore, when preparing peptides, it is necessary to select the appropriate type of protease based on the specificity of the enzyme cleavage site and the level of enzyme activity, and optimize the enzymatic hydrolysis conditions. Appropriate proteases and their optimal enzymatic hydrolysis conditions are crucial to the development and utilization of peptides. Important, it directly affects whether the bioactive peptides with specific efficacy can be obtained and affects the preparation efficiency and quality of bioactive peptides. Currently, proteases have been successfully used to prepare bioactive peptides from food protein sources. Because the enzymatic preparation of biologically active peptides has the advantages of controllable conditions, mild reaction, high safety and no side effects, it is widely used. Commonly used proteases include neutral protease, alkaline protease, pepsin, trypsin, chymotrypsin, papain, pancreatin, thermolysin, flavor protease, etc. Among them, trypsin, alkaline protease, papain and pepsin most widely used. Currently, bioactive peptides have been prepared from food protein sources such as milk, eggs, fish, rice, soybeans, flaxseed, whey, spirulina, clams, and frogs. The peptides with immunomodulatory activity reported so far 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.

Phycocyanin has a wide range of biological activities and a balanced amino acid composition. It is a high-quality protein source for the development of bioactive peptides. It has been reported in the literature that the antioxidant peptide was obtained by hydrolyzing phycocyanin with papain, and the ACE inhibitory peptide VTY was obtained by hydrolyzing phycocyanin with thermolysin. Theoretically, phycocyanin contains a relatively high proportion of hydrophobic amino acids, which is an excellent protein raw material for the preparation of immunomodulatory active peptides. However, up to now, there are few immunomodulatory peptides screened from spirulina phycocyanin. Currently, only LDAVNR and MMLDF are the only immunomodulatory active peptides of spirulina phycocyanin reported.

The evaluation of the activity of immunomodulatory peptides is mainly to measure the immunomodulatory activity on cells, animal models and even human body. Cell-based in vitro immunomodulatory activity assays are widely used, while in vivo immunomodulatory activity assays based on animal models or humans can more truly evaluate immune regulatory efficacy, but in vivo assays are costly, time-consuming, and difficult. Macrophages are widely used as a cell model to evaluate immunomodulatory activity, and the immunomodulatory activity of peptides is reflected by changes in cell viability, phagocytosis, and secretion of nitric oxide, IL-6, and TNF-α.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a spirulina phycocyanin active peptide with immunomodulatory activity and its preparation method and application. The present invention obtains unreported immunomodulatory activity from spirulina phycocyanin for the first time Peptides Tyr-Asn-Lys-Phe-Pro-Tyr(YNKFPY), Gly-Tyr-Tyr-Leu-Arg-Met(GYYLRM), the structures of the active peptides YNKFPY and GYYLRM were identified by mass spectrometry, and the bioinformatic analysis showed that the active peptide YNKFPY And GYYLRM have the advantages of safety, non-toxicity, high biological activity, etc., and the active peptides YNKFPY and GYYLRM have immunomodulatory activity determined by macrophage test, so YNKFPY and GYYLRM can be used as functional ingredients in foods and health products with immunomodulatory effects , cosmetics and pharmaceuticals, has a good application prospect.

To achieve the above object, the present invention adopts the following technical solutions:

A spirulina phycocyanin active peptide with immunomodulatory activity, the amino acid sequence of the active peptide is shown in SEQ ID NO.1 or SEQ ID NO.2;

The amino acid sequences of said SEQ ID NO.1 and SEQ ID NO.2 are respectively Tyr-Asn-Lys-Phe-Pro-Tyr (YNKFPY) and Gly-Tyr-Tyr-Leu-Arg-Met (GYYLRM).

A composition comprising the above active peptide and pharmaceutically, food or health product acceptable excipients.

Use of the above-mentioned active peptide or the above-mentioned composition in the preparation of cosmetics with immunoregulatory effect.

Application of the above-mentioned active peptide or the above-mentioned composition in the preparation of medicines with immunoregulatory effect.

Application of the above-mentioned active peptide or the above-mentioned composition in the preparation of food or health products with immunoregulatory effect.

The preparation and screening method of the above-mentioned active peptide comprises the following steps:

(1) Enzymolysis: Add papain to the spirulina phycocyanin solution for enzymolysis, inactivate the enzyme activity after enzymolysis, collect the supernatant, ultrafiltration and centrifugation, collect the filtrate, freeze-dry to make spirulina phycocyanin Peptide freeze-dried powder;

(2) Screening: use ultra-high performance liquid chromatography-tandem mass spectrometry to identify the sequence of Spirulina phycocyanin peptides by mass spectrometry, and then screen the peptides with potential immunomodulatory activity according to the following conditions: 1) The length of the amino acid sequence is 2-10 amino acid residues; 2) PeptideRanker prediction score greater than 0.5; 3) rich in hydrophobic amino acids or basic amino acid residues; 4) BIOPEP database has not been reported; 5) predicted no potential toxicity;

(3) Activity measurement: chemically synthesize a polypeptide with potential immunomodulatory activity, measure the immunomodulatory activity of the polypeptide, and determine the spirulina phycocyanin active peptide with immunomodulatory activity.

Preferably according to the present invention, the amount of papain added in step (1) is 2000-2500U/g phycocyanin.

Preferably according to the present invention, the pH value of the enzymatic hydrolysis in step (1) is 7±0.2.

Preferably according to the present invention, the temperature of the enzymolysis in step (1) is 55-60°C.

Preferably according to the present invention, the enzymatic hydrolysis time in step (1) is 4-5 hours.

Preferably according to the present invention, the ultrafiltration centrifugation described in step (1) adopts an ultrafiltration centrifuge tube with a molecular weight cut off of 3kDa.

Beneficial effect:

The present invention first screened the active peptides Tyr-Asn-Lys-Phe-Pro-Tyr (YNKFPY) and Gly-Tyr-Tyr-Leu-Arg-Met (GYYLRM) from Spirulina phycocyanin, and clarified the active peptides The structure and immunoregulatory activity of YNKFPY and GYYLRM. The active peptides YNKFPY and GYYLRM can improve the cell viability and phagocytosis of macrophages, and can also increase the secretion of NO and cytokines in macrophages. Their immunoregulatory activity also has good stability. At the same time, the active peptides YNKFPY and GYYLRM have the advantages of safety, non-toxicity and high biological activity. Therefore, they have good potential and application prospects as functional ingredients in foods, health care products, cosmetics and pharmaceuticals with immunomodulatory effects.

Description of drawings

Fig. 1 is a mass spectrometry analysis diagram of polypeptide YNKFPY.

Fig. 2 is a mass spectrometry analysis chart of polypeptide GYYLRM.

Figure 3 is a histogram of cell viability of macrophage RAW264.7 treated with polypeptides; in the figure, different letters indicate that the results between groups are significantly different by biostatistical analysis, the same below.

Fig. 4 is a histogram of the phagocytosis ability of macrophage RAW264.7 after polypeptide treatment.

Fig. 5 is a histogram of NO release amount of macrophage RAW264.7 treated with polypeptide.

Fig. 6 is a histogram of TNF-α secretion of macrophage RAW264.7 treated with polypeptide.

Fig. 7 is a bar graph of IL-6 secretion of macrophage RAW264.7 treated with polypeptide.

Fig. 8 is a histogram of cell viability of macrophage RAW264.7 treated with a polypeptide simulated in vitro digested by the gastrointestinal tract.

Fig. 9 is a bar graph of cell viability of macrophage RAW264.7 treated with polypeptides incubated at different pHs.

Detailed ways

The technical solution of the present invention will be described in detail below. It should be understood that the embodiments presented here are only preferred embodiments for the purpose of illustration, and are not intended to limit the protection scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

Embodiment 1: Extraction of spirulina phycocyanin

Phycocyanin was crudely extracted by repeated freeze-thaw method. First, take spirulina powder, add 0.1mol/LpH 7.0 PBS buffer solution at a ratio of 1:20 (w/v, g/mL), stir until completely dissolved, freeze in the refrigerator until completely frozen, then run water or 37°C Thaw in water bath, repeat freeze-thaw 3 times. Then centrifuge at 6000×g for 20 min (4° C.) to remove the precipitate, and obtain a blue crude phycocyanin extract. The crude phycocyanin extract is precipitated by ammonium sulfate fractionation, successively precipitated by ammonium sulfate with a saturation of 25% and 60%, centrifuged, and the precipitate is taken. The pellet was dissolved in an appropriate amount of PBS buffer, and dialyzed against PBS buffer overnight. The dialysate was purified by DEAE-Sephadex Fast Flow anion exchange chromatography column (1.6×20cm). The chromatographic column was pre-equilibrated with PBS buffer containing 20mM pH7.0, then loaded, eluted with PBS buffer to remove foreign proteins, and then washed with PBS buffer containing 0.3M and 0.5M NaCl (20mM , pH 7.0) at a rate of 1 mL·min ^-1 . The blue fraction was collected and dialyzed overnight (4°C, 12h) with a 3500Da semi-permeable membrane to obtain a purified phycocyanin solution, which was lyophilized to obtain a purified phycocyanin lyophilized powder. Store at -20°C for later use.

The phycocyanin concentration, yield and purity determination of the phycocyanin solution are calculated using the following formula:

In the formula: C _C-PC represents the concentration of phycocyanin; A ₆₂₀ represents the absorbance at a wavelength of 620nm; A ₆₅₂ represents the absorbance at a wavelength of 652nm; Yield represents the yield of phycocyanin; V represents the volume of phycocyanin solution; Quality of spirulina powder; Purity means phycocyanin purity; A ₂₈₀ means absorbance at 280nm wavelength.

Example 2: Preparation of Spirulina Phycocyanin Peptide by Enzymolysis

Take the phycocyanin solution prepared in Example 1, adjust the pH value of the phycocyanin solution to 7, and then add papain according to the ratio of 2000 U/g phycocyanin (the enzyme activity of papain is 1×10 ⁵ U/g) , after mixing, put it in a shaker for oscillating enzymolysis, the rotation speed of the shaker is 100-200rpm, the enzymolysis time is 5h, and the enzymolysis temperature is 55°C. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis solution is boiled in water for 10 minutes to inactivate the residual enzyme. Centrifuge at 6000×g for 20 min (4° C.), collect the supernatant, discard the precipitate, and the supernatant is the spirulina phycocyanin peptide solution. Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 3kDa to ultrafilter and centrifuge the spirulina phycocyanin peptide solution, centrifuge at 6000×g for 20min (4°C), and absorb the solution intercepted in the ultrafiltration tube, which is the ultrafiltration fraction with a molecular weight of <3kDa . Freeze and vacuum-dry the ultrafiltration fraction to prepare spirulina phycocyanin peptide freeze-dried powder.

Example 3: Structural Identification of Spirulina Phycocyanin Peptides

(1) Identification by mass spectrometry

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

Table 1. Ultra High Performance Liquid Chromatography Elution Conditions

The tandem mass spectrometer adopts positive ion mode, the secondary mass spectrometry analysis, the capillary voltage is 3.5kV, and the full MS scan of 100-2000m/z is performed at a resolution of 120000. The specific mass spectrometry parameter settings are shown in Table 2.

Table 2. Tandem mass spectrometry analysis conditions

(2) Analysis of mass spectrometry data

Use MM File Conversion software to convert the original files obtained from UPLC-ESI-MS/MS analysis into mass spectrometer general files in MGF format, use the MASCOT search engine to search the database, use Peaks Viewer 4.5 software to analyze MS/MS data, and combine Peptide sequencing was performed by manual de novo sequencing.

After identification by UPLC-ESI-TOF-MS/MS, multiple polypeptide sequences were obtained.

Example 4: Bioinformatics Prediction of Spirulina Phycocyanin Peptides

According to the analysis of active peptide sequences, it was found that low molecular weight peptides had higher activity, and most of them contained 2-10 amino acid residues. Therefore, peptide sequences with <10 amino acid residues were selected for further analysis.

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

Afterwards, the PeptideRanker online platform (http://distilldeep.ucd.ie/PeptideRanker/) was used to predict the potential biological activity of the identified polypeptide sequences. Prediction scores >0.5 were considered potentially biologically active. Analyze the physical and chemical properties of the peptide sequences with a prediction score > 0.5, and use PepDraw (https://www2.tulane.edu/~biochem/WW/PepDraw/) to analyze the physical and chemical properties of the screened peptides (such as molecular weight, isoelectric points, hydrophobicity, charge, etc.).

Then use bioinformatics to predict whether the peptide has potential toxicity (http://crdd.osdd.net/raghava/toxinpred/). ToxinPrep (https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php) platform is used to predict the potential toxicity of peptides based on the SVM (Swiss-Port) algorithm.

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

The polypeptide sequences meeting the above conditions at the same time are YNKFPY (Tyr-Asn-Lys-Phe-Pro-Tyr, SEQ ID NO.1) and GYYLRM (Gly-Tyr-Tyr-Leu-Arg-Met, SEQ ID NO.2), the polypeptide The PeptideRanker prediction scores of YNKFPY and GYYLRM are 0.695966 and 0.827762, respectively. The results of mass spectrometry are shown in Figure 1 and Figure 2, respectively. The results of molecular weight and hydrophobicity are shown in Table 3. The results of bioinformatics predictive analysis showed that the potential biological activities of polypeptides YNKFPY and GYYLRM were high.

Table 3. Bioinformatics prediction results of phycocyanin active peptides

Example 5: Chemical synthesis of active peptides YNKFPY and GYYLRM and assay of their immunomodulatory activity

Using Fmoc amino acid solid-phase synthesis technology, chemically synthesized polypeptides YNKFPY and GYYLRM. Peptides were synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. Synthetic polypeptides were >95% pure. The synthetic peptides were checked for quality by HPLC and HPLC-MS.

The immunomodulatory activity of polypeptide YNKFPY and GYYLRM was determined in vitro on macrophage RAW264.7.

(1) Determination of cell viability

Cultivation of macrophages: macrophages RAW264.7 were inoculated in DMEM medium (containing 10% fetal calf serum and 1% penicillin and streptomycin), and cultured in a cell culture incubator at 5% CO ₂ at 37°C. Subculture the cells when the bottom of the culture dish is about 80% full, discard the old medium, and wash the cells twice with PBS buffer, then add fresh medium and blow the cells down with a pipette to make a cell suspension. Centrifuge the cell suspension at 800rpm for 10min, discard the medium, add fresh medium and transfer to a new cell culture dish, supplement with fresh medium, and continue culturing in the incubator.

When the cells grow to the logarithmic growth phase, inoculate them in a 96-well plate, adjust the cell density to 5× ¹⁰⁴ cells/mL before inoculation, 100 μL per well, and culture for 12-24 hours. When it reached about 80%, the upper medium was discarded, and polypeptide samples of different concentrations (50, 100, 200 μg/mL) prepared with DMEM complete medium were added. The DMEM complete medium of LPS was used as the positive control group and incubated in the incubator for 24h. Afterwards, 20 μL of MTT with a concentration of 5 mg/mL was added, and after incubation in the dark for 4 h, the cell culture medium was discarded, 150 μL of DMSO was added, and the 96-well plate was shaken on a microplate shaker until the crystals were dissolved and no particles were visible to the naked eye. The absorbance at a wavelength of 490 nm was measured with a microplate reader. Cell viability was calculated according to the following formula:

Cell viability=A _{490 (sample)} /A _{490 (blank control)}

In the formula, A _{490 (sample)} is the absorbance of the polypeptide sample group at 490 nm, and A _{490 (blank control)} represents the absorbance of the blank control group at 490 nm.

The effect of polypeptides on the cell viability of macrophage RAW264.7 is shown in Figure 3. Compared with the blank control group, both polypeptide YNKFPY and GYYLRM can significantly improve cell viability, and the effect of polypeptide YNKFPY is more significant. There was a significant difference between polypeptide YNKFPY (100 and 200μg/mL) and the blank control group (p<0.05), and the cell viability of macrophages was the highest at the concentration of polypeptide YNKFPY at 200μg/mL, which could reach 1.47±0.20. In addition, the polypeptide YNKFPY showed a dose effect in the concentration range of 50-200 μg/mL. It is worth noting that the effect of polypeptide YNKFPY on the cell viability of macrophages at concentrations of 100 and 200 μg/mL was comparable to that of LPS at 1 μg/mL, and there was no significant difference between the two (p>0.05). The above results showed that both polypeptide YNKFPY and GYYLRM could improve the viability of macrophage RAW264.7 cells and promote cell proliferation.

(2) Phagocytosis determination

Macrophages RAW264.7 were cultured as previously described. When the cells grow to the logarithmic growth phase, inoculate them in a 96-well plate, adjust the cell density to 5× ¹⁰⁴ cells/mL before inoculation, 100 μL per well, and culture for 12-24 hours. When it reaches about 80%, the upper culture medium is discarded, and different concentrations of polypeptide sample solutions (final concentration 50 μg/mL, 100 μg/mL and 200 μg/mL, DMEM complete medium configuration) and blank control (DMEM complete medium) and Positive controls (LPS, 1 μg/mL, DMEM complete medium configuration) were added to 96-well plates (100 μL/well), incubated at 37 ° C, 5% CO ₂ incubator for 24 h, and then each well was added with a concentration of 0.1% medium Neutral red solution (100 μL/well), and after further incubation for 1 h, the cells were washed twice with PBS buffer to remove excess neutral red. Afterwards, a mixture (100 μL/well) of glacial acetic acid (0.1 M) and ethanol (1:1, v/v) was added to each well, and incubated at 37° C. for 2 h. Finally, a microplate reader was used to detect the absorbance of each well at a wavelength of 540 nm. The phagocytosis rate of macrophages phagocytizing neutral red was calculated according to the following formula:

Cell phagocytosis rate (%)=A _{540 (sample)} /A _{540 (blank control)} ×100%.

In the formula: A _{540 (sample)} represents the absorbance of the polypeptide sample group at a wavelength of 540 nm, and A _{540 (blank control)} represents the absorbance of the blank control group at a wavelength of 540 nm.

Macrophages are important cells in the human immune system, which can recognize and destroy foreign invaders through innate immunity, adaptive immunity or acquired immunity, and can engulf and kill intracellular parasites, bacteria and tumor cells. Neutral red staining was used to evaluate the effect of the polypeptide on the phagocytosis of macrophage RAW264.7, and the results are shown in Figure 4. Compared with the blank control group, after the macrophages were treated with LPS, the phagocytosis ability of the cells was significantly enhanced (p<0.05); after the macrophages were treated with polypeptide YNKFPY and GYYLRM, the cell phagocytosis rate was significantly increased (p<0.05), and It is dose-dependent, and the phagocytosis rate of polypeptide YNKFPY can reach up to 159.83±1.45% at the concentration of 200 μg/mL. The above results indicated that the polypeptides YNKFPY and GYYLRM could enhance the phagocytic ability of macrophage RAW264.7, thereby activating the immune regulation activity of the cells.

(3) Determination of NO and cytokine secretion

The Griess reaction was used to measure the nitrite level in the macrophage supernatant, so as to analyze the effect of the peptide on the NO release of macrophage RAW264.7. The specific method is as follows. According to the above-mentioned steps of cell viability determination, macrophage culture and different sample processing were carried out. After incubation in the incubator for 24 hours, the cell supernatant of each treatment group was collected, centrifuged at 1000×g for 10 minutes, and 50 μL was centrifuged. The final supernatant was placed in a 96-well plate, mixed with 50 μL Griess reagent I and II, incubated at room temperature for 10 min, and the absorbance at 540 nm was measured using a microplate reader. A standard curve was made with the content of sodium nitrite (NaNO ₂ ), and the NO concentration of the supernatant in each treatment group was calculated according to the standard curve. Cytokines (IL-6 and TNF-α) levels were determined using ELISA kits.

The results of the effect of the polypeptide on the NO release of macrophage RAW264.7 are shown in Figure 5. Compared with the blank control group, after the macrophages were treated with LPS and peptides, the release of NO increased in varying degrees, and the LPS (1 μg/mL) group significantly increased the release of NO (p<0.05), and the polypeptide YNKFPY increased at 100 And 200μg/mL concentration can significantly increase the release of macrophage NO (p<0.05). The above results indicated that the peptides YNKFPY and GYYLRM could enhance the release of NO from macrophage RAW264.7 in a dose-dependent manner.

Macrophages can produce cytokines and participate in immune regulation. Increased secretion of TNF-α and IL-6 is considered a hallmark of immune stimulation. Therefore, after the polypeptide acts on macrophage RAW264.7, the concentrations of TNF-α and IL-6 in the cell supernatant can be used to verify the immunostimulatory activity of the polypeptide. The effect of the peptide on the secretion of TNF-α in macrophage RAW264.7 is shown in Figure 6. Compared with the blank control group, after the macrophages were treated with 1 μg/mL LPS, the secretion of TNF-α in the macrophages was significantly increased (p<0.05), and the concentration could reach 568.52±24.14pg/mL; , the polypeptide YNKFPY can also significantly increase the secretion of TNF-α in macrophages (p<0.05), and exhibit a dose effect within the concentration range of 50-200 μg/mL.

The effect of the polypeptide on the secretion of IL-6 by macrophage RAW264.7 is shown in Figure 7. Compared with the blank control group, after macrophages were treated with LPS, the secretion of macrophage IL-6 was also significantly increased (p<0.05). After the macrophage RAW264.7 was treated with the polypeptide for 24 hours, the secretion of IL-6 also increased in different degrees, and the polypeptide YNKFPY showed a dose effect. It is worth mentioning that the TNF-α and IL-6 secretion of macrophages in different concentrations of polypeptide treatment groups were significantly different from those in LPS group (p<0.05), indicating that polypeptides YNKFPY and GYYLRM can cause immune stimulation, but not lead to excessive inflammation.

Embodiment 6: Stability study of active peptide YNKFPY and GYYLRM

(1) Gastrointestinal digestion is one of the important factors affecting the activity of bioactive peptides in vivo. In order to study the effect of gastrointestinal digestion on the active peptides YNKFPY and GYYLRM, the gastrointestinal digestion was first simulated by computer (PeptideCutter, https://web.expasy.org/cgi-bin/peptide_cutter/peptidecutter.pl), the active peptide YNKFPY and GYYLRM sequence structure changes are shown in Table 4. The active peptides YNKFPY and GYYLRM were hydrolyzed to varying degrees by pepsin during gastric digestion, and then further hydrolyzed by trypsin during intestinal digestion.

Table 4 PeptideCutter simulated digestion results of polypeptides

Due to the computer simulation theorized the enzymatic cleavage sites of pepsin and trypsin, and idealized the hydrolysis process, but in the actual hydrolysis process, there is a certain probability of protease hydrolysis, so the gastrointestinal digestion has a great influence on the activity of active peptides. The impact needs to be further verified through experiments.

The active peptides YNKFPY and GYYLRM (10mg) were dissolved in 10mL of distilled water, and the pH was adjusted to 2.0 with 1M HCl, then 2% (w/w) pepsin was added and incubated at 37°C for 2h to simulate gastric digestion. Then the pH of the digested solution of simulated gastric digestion was adjusted to 5.3 with 0.9M NaHCO ₃ solution, and the pH was further adjusted to 7.5 with 1M NaOH solution. Then add 2% (w/w) trypsin and incubate at 37°C for 2h to simulate intestinal digestion. After digestion, the digestive juice was inactivated in a water bath at 100°C for 10 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was freeze-dried to obtain the gastrointestinal digestive juice of the active peptide.

According to the steps of Example 5, the effect of the active peptide after in vitro simulated gastrointestinal digestion on the viability of macrophage RAW264.7 cells was measured, and the results are shown in FIG. 8 . Compared with the untreated group, the pepsin and trypsin digestion of active peptides reduced the cell viability of macrophage RAW264.7 to varying degrees, but still maintained a high level of cell viability. After the active peptide GYYLRM was digested by pepsin, the cell viability was not significantly different from that of the untreated group.

Through computer simulation analysis, it is found that the new sequences produced after simulated gastrointestinal digestion still have high potential biological activities. Hydrophobicity and isoelectric point, which may be the reason why these peptide sequences can maintain high immunostimulatory properties.

(2) The pH value is also one of the important factors affecting the activity of the polypeptide. In order to analyze the pH stability of the active peptides YNKFPY and GYYLRM, the active peptide solution (1 mg/mL) was incubated at pH 3.0, 5.0, 7.0 and 9.0 for 30 min, adjusted to pH 7.0, lyophilized, and cell viability was measured. The active peptide without pH treatment was used as a control to measure cell viability.

The effect of active peptides treated with different pH on the viability of macrophage RAW264.7 cells is shown in Figure 9. When the active peptide is in the range of pH 3-9, the relative cell viability of the macrophages does not decrease significantly, and both reach more than 80% of the cell viability of the non-pH treatment group. In the active peptide group treated in a neutral environment, the cell viability of macrophages remained basically unchanged, with only small fluctuations. Although the cell viability of the active peptide group macrophages treated under acidic or alkaline conditions was affected, they still showed good cell viability. Studies have shown that positively charged immunomodulatory peptides have stronger chemotactic effects and can bind to receptors on the surface of immune cell membranes to stimulate immune responses. The above results show that the active peptides YNKFPY and GYYLRM have good pH stability and can be used in the processing system of food, medicine and health care products at pH 3-9, while maintaining good immune activity.

Example 7: Molecular docking of active peptides YNKFPY and GYYLRM

Using Toll-like receptor proteins TLR2 (PDB ID: 1FYW) and TLR4/MD2 (PDB ID: 5IJD) as protein receptors, protein crystal structures were downloaded from the RCSB PDB database (http://www.rcsb.org/). Pretreatment of the receptor protein crystal structure includes hydrogenation, removal of water molecules, modification of amino acids, optimization of energy and adjustment of force field parameters, etc., to meet the low-energy conformation of ligand binding.

Molecular docking was carried out between the active peptide and the binding pockets of Toll-like receptor proteins TLR2 (PDB ID: 1FYW) and TLR4/MD2 (PDB ID: 5IJD) by AutoDock Vine software, and the interaction between the active peptide and the receptor protein was performed using PYMOL software analysis of the interaction mechanism. The size of the binding energy between the active peptide sequence and the 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 active peptide and the Toll-like receptor protein, making it easier to form a more stable molecular binding conformation. The binding energies of active peptides to Toll-like receptor proteins TLR2 and TLR4/MD2 are shown in Table 5.

Binding energy between table 5 active peptide and receptor protein TLR2 and TLR4/MD2

From the perspective of binding energy, the interaction between the active peptide and the receptor protein TLR4/MD2 is stronger than the interaction with the receptor protein TLR2. The interaction between the active peptide YNKFPY and the receptor protein TLR4/MD2 is strong, and its binding energy is -9.1kcal/mol; the interaction between the active peptide YNKFPY and the receptor protein TLR2 is strong, and its binding energy is - 7.3kcal/mol. Through comparison, it is found that the trend of binding energy is basically consistent with the data trend obtained from the previous experimental results. In addition, it can be further clarified that the mechanism of action of the phycocyanin immunomodulatory active peptide is through the binding of the active peptide to the Toll-like receptor proteins TLR2 and TLR4/MD2 on the surface of immune cell membranes, thereby activating the immune response of immune cells and exerting immune regulatory activity.

The interaction sites between active peptides and receptor proteins were analyzed by PYMOL. The interaction between the active peptide and the receptor protein TLR4/MD2 is through the hydrogen bond or the hydrophobic contact of the residue between the active peptide and the receptor protein. The binding pocket of the receptor protein TLR4/MD2 is dominated by the hydrophobic region with a large cavity, and the active peptide can be stably bound in the hydrophobic cavity and occupied by the complementary structure. The active peptide YNKFPY forms 5 hydrogen bonds with the 4 amino acid residues of Asp-726, Glu-727, Ala-732 and Lys-754 of the receptor protein TLR2, the lengths of which are respectively and /> The active peptide YNKFPY forms two hydrogen bonds with the Glu-122 and Ile-124 residues of the receptor protein TLR4/MD2, the length of which is /> The active peptide GYYLRM forms 9 hydrogen bonds with 7 residues of Asn-777, Ser-734, Lys-698, Ala-732, Leu-734, Glu-727 and Tyr-715 of the receptor protein TLR2, and the lengths are respectively /> and /> The active peptide GYYLRM forms two hydrogen bonds with the Ser-413 and Glu-122 residues of the receptor protein TLR4/MD2, the lengths of which are respectively /> and />

In general, differences in the number of hydrogen bonds may be one of the reasons for the differences in ligand-receptor interactions. The groups of polypeptide side chains (generally aromatic amino acids and amino acids with carboxyl groups are easier to form) are the influencing factors of hydrogen bond formation in the system. Studies have shown that hydrogen bond interaction forces (influenced more than charge interactions and π-π bond interactions) play an important role in stabilizing the crystal structure of the polypeptide-receptor complex and have a positive effect on substrate inhibition or activation. Phycocyanin immunomodulatory active peptides interact with TLRs by forming hydrogen bonds with the 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 regulatory The body's immune response.

The above experimental test results show that the spirulina phycocyanin active peptides YNKFPY and GYYLRM have immunoregulatory activity, are safe and non-toxic, and can be applied to the development of cosmetics, food, health products and medicines related to immune regulation.

Claims (10)

1. The spirulina phycocyanin active peptide is characterized by having immunoregulatory activity, and the amino acid sequence of the active peptide is shown as SEQ ID NO.1 or SEQ ID NO. 2;

the amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2 are Tyr-Asn-Lys-Phe-Pro-Tyr, gly-Tyr-Tyr-Leu-Arg-Met respectively.

2. A composition comprising the active peptide of claim 1 and pharmaceutically, food or nutraceutical acceptable excipients.

3. Use of an active peptide according to claim 1 or a composition according to claim 2 for the preparation of a cosmetic having an immunomodulatory effect.

4. The use of an active peptide according to claim 1 or a composition according to claim 2 for the preparation of a medicament having an immunomodulatory effect.

5. The use of an active peptide according to claim 1 or a composition according to claim 2 for the preparation of a food or health care product having an immunomodulatory effect.

6. The method for preparing and screening an active peptide according to claim 1, comprising the steps of:

(1) Enzymolysis: adding papain into spirulina phycocyanin solution for enzymolysis, inactivating enzyme after enzymolysis, collecting supernatant, ultrafiltering and centrifuging, collecting filtrate, and freeze-drying to obtain spirulina phycocyanin peptide freeze-dried powder;

(2) Screening: performing mass spectrometry identification on the sequence of the spirulina phycocyanin peptide by utilizing an ultra-high performance liquid chromatography-tandem mass spectrometry, and then screening the polypeptide with potential immunoregulatory activity according to the following conditions: 1) The amino acid sequence is 2-10 amino acid residues in length; 2) The PeptideRanker predictive score is greater than 0.5; 3) Is rich in hydrophobic amino acid or basic amino acid residues; 4) The bipep database is not reported; 5) Predicting no potential toxicity;

(3) Activity measurement: chemically synthesizing polypeptide with potential immunoregulatory activity, determining immunoregulatory activity of the polypeptide, and determining spirulina phycocyanin active peptide with immunoregulatory activity.

7. The method of claim 6, wherein papain is added in step (1) in an amount of 2000 to 2500U/g phycocyanin.

8. The method of claim 6, wherein the enzymatic hydrolysis in step (1) has a pH of 7±0.2.

9. The method of claim 6, wherein the temperature of the enzymatic hydrolysis in step (1) is 55-60 ℃; the enzymolysis time in the step (1) is 4-5h.

10. The method of claim 6, wherein the ultrafiltration centrifugation in step (1) is performed using an ultrafiltration centrifuge tube having a molecular weight cut-off of 3 kDa.