Stropharia rugoso-annulata glycoprotein-chitosan nanoparticle as well as preparation method and application thereof
By preparing *Agaricus bisporus* glycoprotein-chitosan nanoparticles, the problem of poor water solubility of *Agaricus bisporus* glycoprotein was solved, and its stability and bioavailability in the fields of food, medicine and biomaterials were improved, and it has immunomodulatory function.
Patent Information
- Application Number
- CN202510988760.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-17
- Publication Date
- 2025-10-28
Smart Images
Figure BDA0005505218410000051 
Figure BDA0005505218410000061 
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bio-nanomaterials technology, specifically relating to a giant oyster mushroom glycoprotein-chitosan nanoparticle, its preparation method, and its application. Background Technology
[0002] Glycoproteins are a class of bound proteins in which polysaccharide or oligosaccharide groups are covalently linked to proteins. They are widely distributed in nature, and natural glycoprotein components can be extracted from animals, plants, and microorganisms. Glycoproteins possess various biological functions, including anti-tumor activity, immune enhancement, and blood sugar and lipid-lowering effects. Therefore, glycoproteins have broad development and application prospects in the fields of food, medicine, and biomaterials. *Stropharia spp.* glycoprotein is a type of natural bound protein extracted from the fruiting body of *Stropharia spp.* It possesses immunomodulatory and lipid-lowering activities. However, *Stropharia spp.* glycoprotein has poor water solubility and low bioavailability, which affects its in-depth and widespread application. Therefore, how to deeply develop and utilize *Stropharia spp.* glycoprotein and provide *Stropharia spp.* glycoprotein nanoparticles with high stability, good dispersibility, high bioavailability, and good biological activity is a technical problem that urgently needs to be solved in this field. Summary of the Invention
[0003] The purpose of this invention is to provide a giant oyster mushroom glycoprotein-chitosan nanoparticle, its preparation method, and its application.
[0004] To achieve the above objectives, the present invention provides the following technical solution:
[0005] This invention provides a giant oyster mushroom glycoprotein-chitosan nanoparticle, which is prepared from a chitosan solution and a giant oyster mushroom glycoprotein solution, and the particle size of the nanoparticle is less than 270 nm.
[0006] Preferably, the preparation method of the *Stropharia macrocarpa* glycoprotein solution includes: using *Stropharia macrocarpa* bodies as raw materials, pulverizing and sieving to obtain *Stropharia macrocarpa* powder; adding *Stropharia macrocarpa* powder to sodium hydroxide aqueous solution for extraction for 120 minutes, filtering, collecting the filtrate to obtain *Stropharia macrocarpa* extract; concentrating the *Stropharia macrocarpa* extract under reduced pressure to obtain a concentrate; centrifuging the concentrate to obtain the supernatant; acid precipitation of the supernatant to collect the precipitate, dissolving the precipitate in a solvent and dialyzing, collecting the dialysate and freeze-drying to obtain a freeze-dried crude glycoprotein sample; dissolving the freeze-dried glycoprotein sample to remove free protein, centrifuging to obtain the supernatant, and freeze-drying the supernatant to obtain *Stropharia macrocarpa* glycoprotein;
[0007] The degree of deacetylation of the chitosan is 90%, and the viscosity is <100 mPa·s.
[0008] The present invention also provides a method for preparing the aforementioned *Agaricus spp.* glycoprotein-chitosan nanoparticles, wherein a chitosan solution is added dropwise to a *Agaricus spp.* glycoprotein solution at a rate of 1-2 drops per second, and stirring is continued for 60 minutes after the addition is complete, followed by freeze-drying to obtain *Agaricus spp.* glycoprotein-chitosan nanoparticles.
[0009] Preferably, the chitosan solution is added to the sucrose protein solution at a rate of 1-2 drops per second under a stirring speed of 600 rpm / min.
[0010] Preferably, the volume ratio of the chitosan solution to the Pleurotus ostreatus glycoprotein solution is (1-4):(5-20).
[0011] Preferably, the concentration of the chitosan solution is 0.5–1 mg / mL, and the concentration of the Pleurotus ostreatus glycoprotein solution is 1–2 mg / mL.
[0012] Preferably, the solvent used to prepare the chitosan solution and the Pleurotus ostreatus glycoprotein solution is distilled water; the pH of the chitosan solution is 4.5.
[0013] Preferably, the pH of the chitosan solution is adjusted with acetic acid.
[0014] The present invention also provides the application of the nanoparticles or the preparation method of the *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles in the preparation of products with immunomodulatory activity.
[0015] Preferably, the product includes functional foods, health products, or pharmaceuticals.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The *Stropharia macrocarpa* glycoprotein-chitosan nanoparticles of this invention exhibit good stability, low toxicity to RAW264.7 macrophages, and excellent biocompatibility. Furthermore, these nanoparticles provide enhanced immunomodulatory effects and can be applied in functional foods, health products, or pharmaceuticals. The preparation process of the *Stropharia macrocarpa* glycoprotein-chitosan nanoparticles provided by this invention is simple, rapid, and convenient, facilitating practical application. Attached Figure Description
[0018] Figure 1 The image shows a transmission electron microscope (TEM) image of SRP-CTS NPs3 from Example 1, where 200 nm is the numerical value of the scale bar. SRP-CTS NPs3 is the Pleurotus ostreatus glycoprotein-chitosan nanoparticle 3 prepared in Example 4.
[0019] Figure 2The image shows the FT-IR spectra of SRP, CTS, and SRP-CTS NPs3 in Example 1, where SRP is serotonin from *Agaricus macrocarpa*; CTS is chitosan; and SRP-CTS NPs3 are *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles 3 prepared in Example 4.
[0020] Figure 3 The results of the effects of SRP, CTS and SRP-CTS NPs3 on the cell viability of macrophages RAW 264.7 in Experiment Example 2 are shown. SRP is Pleurotus ostreatus glycoprotein; CTS is chitosan; and SRP-CTS NPs3 is Pleurotus ostreatus glycoprotein-chitosan nanoparticles 3 prepared in Example 4.
[0021] Figure 4 The results of the effects of SRP, CTS and SRP-CTS NPs3 on NO release from RAW 264.7 macrophages in Experiment Example 2 are shown. SRP is serotonin from *Agaricus bisporus*; CTS is chitosan; and SRP-CTS NPs3 is *Agaricus bisporus* glycoprotein-chitosan nanoparticles prepared in Example 4. The data are mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences at the P < 0.01 level.
[0022] Figure 5 The results of the effects of SRP, CTS, and SRP-CTS NPs3 on the release of TNF-α (A), IL-1β (B), and IL-6 (C) from RAW 264.7 macrophages in Experiment Example 2 are shown. SRP is Pleurotus ostreatus glycoprotein; CTS is chitosan; and SRP-CTS NPs3 is Pleurotus ostreatus glycoprotein-chitosan nanoparticles 3 prepared in Example 4. The data are mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences at the P < 0.05 level. Detailed Implementation
[0023] This invention provides a giant oyster mushroom glycoprotein-chitosan nanoparticle, which is prepared from a chitosan solution and a giant oyster mushroom glycoprotein solution, and the particle size of the nanoparticle is less than 270 nm.
[0024] In this invention, the preparation method of the *Stropharia macrocarpa* glycoprotein solution includes: obtaining *Stropharia macrocarpa* extract, concentrating it to obtain a supernatant, subjecting the supernatant to acid precipitation to obtain crude glycoprotein, freeze-drying the crude glycoprotein and then dissolving it, and freeze-drying the supernatant after removing free protein to obtain *Stropharia macrocarpa* glycoprotein. The preparation of the *Stropharia macrocarpa* extract includes: (1) using dried *Stropharia macrocarpa* fruiting bodies as raw material, pulverizing them and passing them through a 100-mesh sieve to obtain *Stropharia macrocarpa* powder; (2) adding the *Stropharia macrocarpa* powder to 20 times its weight of sodium hydroxide aqueous solution, placing it on a shaker at 150 r / min, extracting at room temperature for 120 minutes, filtering, and obtaining the filtrate. Repeating step (2) on the filter residue, extracting again, and combining the filtrates to obtain the *Stropharia macrocarpa* extract. Preferably, the pH of the sodium hydroxide aqueous solution is 8.5. The room temperature is 20–25°C. When concentrating the extract of *Stropharia macrocarpa*, the extract is placed at 40°C and concentrated under reduced pressure until the mass-to-volume ratio of the extract to the liquid is 1:5, yielding a concentrated solution. This concentrated solution is then centrifuged at 9000 rpm for 15 minutes, and the supernatant is collected. For acid precipitation, 0.1 M hydrochloric acid is titrated into the supernatant at room temperature until the pH reaches 4.5. The solution is then allowed to stand at 4°C for 24 hours, centrifuged at 9000 rpm for 15 minutes, and the precipitate is collected. The precipitate is dissolved in 5 times its volume of distilled water (m:V = 1:5, mg:mL), and dialyzed against running water using a 5000 Da dialysis bag for 72 hours. The dialysate is collected and freeze-dried to obtain a freeze-dried crude glycoprotein sample. The freeze-dried crude glycoprotein sample is completely dissolved in distilled water, and the free protein is removed using the sevage method to obtain a supernatant. This process is repeated 3-4 times. The supernatant is then freeze-dried to obtain a gray glycoprotein solid sample, which is *Stropharia macrocarpa* glycoprotein.
[0025] In this invention, the degree of deacetylation of the chitosan is 90%, and the viscosity is <100 mPa·s.
[0026] This invention also provides a method for preparing the aforementioned *Agaricus spp.* glycoprotein-chitosan nanoparticles. A chitosan solution is added dropwise to a *Agaricus spp.* glycoprotein solution at a rate of 1-2 drops per second, and stirring is continued for 60 minutes after the addition is complete. Then, the mixture is freeze-dried to obtain the *Agaricus spp.* glycoprotein-chitosan nanoparticles. In this invention, the chitosan solution is added dropwise to the *Agaricus spp.* glycoprotein solution at a rate of 1-2 drops per second under a stirring speed of 600 rpm / min.
[0027] In this invention, the preferred volume ratio of the chitosan solution to the *Agaricus spp.* glycoprotein solution is (1-4):(5-20), and more preferably, the volume ratio is 1:5. The concentration of the chitosan solution is preferably 0.5-1 mg / mL, and more preferably 0.5 mg / mL; the concentration of the *Agaricus spp.* glycoprotein solution is preferably 1-2 mg / mL, and more preferably 2 mg / mL.
[0028] In this invention, the solvents used to prepare the chitosan solution and the *Pleurotus ostreatus* glycoprotein solution are preferably distilled water; the pH of the chitosan solution is preferably 4.5. As an optional embodiment, the chitosan solution is prepared as follows: 0.25 g of chitosan is dissolved in 500 mL of distilled water, and the initial pH of the chitosan solution is adjusted to 4.5 using acetic acid.
[0029] The Pleurotus ostreatus glycoprotein-chitosan nanoparticles prepared by the above preparation method of the present invention have a particle size of less than 270 nm, enhanced stability, and uniform nanoparticles.
[0030] The preparation process of the *Agaricus bisporus* glycoprotein-chitosan nanoparticles provided by this invention is simple, quick, and easy to operate, making it convenient for practical application.
[0031] This invention also provides the application of the aforementioned nanoparticles or the *Pleurotus ostreatus* glycoprotein-chitosan nanoparticles prepared by the aforementioned method in the preparation of products with immunomodulatory activity. These products include functional foods, health products, or pharmaceuticals. The *Pleurotus ostreatus* glycoprotein-chitosan nanoparticles of this invention exhibit good stability, low toxicity to RAW264.7 macrophages, and good biosafety. Simultaneously, the *Pleurotus ostreatus* glycoprotein-chitosan nanoparticles can promote the release of more NO and immune factors TNF-α, IL-1β, and IL-6 from mouse RAW264.7 macrophages, resulting in better immunomodulatory effects, and can be applied to functional foods, health products, or pharmaceuticals.
[0032] The SRP-CTS NPs3 prepared in this invention exhibited significantly higher NO-stimulating activity in RAW264.7 macrophages compared to that of *S. spp.* glycoprotein within the concentration range of 5–50 μg / mL. Furthermore, at concentrations of 10–50 μg / mL, SRP-CTS NPs3 significantly stimulated the release of IL-6, IL-1β, and TNF-α from RAW264.7 macrophages compared to that of *S. spp.* glycoprotein.
[0033] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.
[0034] In the following embodiments, the freeze-drying instrument used is an Alpha 2-4LD plus freeze dryer (Martin Christ Freeze Dryers, Germany), with parameters set to a temperature of -50±5℃ and a vacuum of approximately 1mbar.
[0035] In the following examples, LPS represents lipopolysaccharide, purchased from Merck GmbH, Germany, catalog number P4516-1G.
[0036] In the following examples, the chitosan was purchased from Shanghai Titan Technology Co., Ltd., product number: 01123490, degree of deacetylation: 90%, viscosity <100mPa.s.
[0037] The RAW264.7 cells were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The colorless RPMI 1640 cell culture medium was purchased from Gibco, USA. The Alamar Blue reagent was purchased from Sigma-Aldrich, USA. The ELISA kit was purchased from Sizhengbai Biotechnology Co., Ltd.
[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.
[0039] Example 1
[0040] Preparation of Pleurotus ostreatus glycoprotein
[0041] (1) Extraction of crude glycoprotein from Pleurotus ostreatus
[0042] a) Using dried *Stropharia macrocarpa* bodies as raw material, pulverize and pass through a 100-mesh sieve to obtain *Stropharia macrocarpa* powder; b) Add the *Stropharia macrocarpa* powder to 20 times its weight of sodium hydroxide aqueous solution (pH=8.5), place on a shaker at 150 r / min, and extract at room temperature (20-25℃) for 120 minutes, then filter. Repeat step b) above with the filter residue for another extraction, combine the filtrates to obtain *Stropharia macrocarpa* extract.
[0043] (2) Concentration of Pleurotus ostreatus extract
[0044] The extract of *Stropharia carota* was concentrated under reduced pressure at 40°C until the material-to-liquid ratio (mass / volume) was 1:5. The concentrate was then centrifuged at 9000 rpm for 15 minutes, and the supernatant was collected.
[0045] (3) Acid precipitation of crude glycoprotein
[0046] At room temperature, hydrochloric acid (0.1M) was titrated into the above concentrated supernatant until the pH reached 4.5. The mixture was allowed to stand at 4°C for 24 hours, centrifuged at 9000 rpm / min for 15 minutes, the precipitate was collected, dissolved in distilled water, and dialyzed with running water through a 5000 Da dialysis bag for 72 hours. The dialysate was collected and lyophilized to obtain the lyophilized crude glycoprotein sample.
[0047] (4) Removal of free proteins
[0048] The lyophilized glycoprotein sample was completely dissolved in distilled water, and the free protein was removed by the Sevage method (chloroform: n-butanol = 4:1 (V / V)). The sample was centrifuged to obtain the supernatant. The supernatant was then lyophilized 3-4 times to obtain a gray glycoprotein solid sample, which is the glycoprotein of *Schefflera heptaphylla*.
[0049] Example 2
[0050] The preparation method of *Stropharia macrocarpa* glycoprotein-chitosan nanoparticles is as follows:
[0051] (1) Dissolve 0.25g of chitosan (CTS, purchased from Shanghai Titan Technology Co., Ltd., item number: 01123490) in 500mL of distilled water, and adjust the initial pH of the chitosan solution to 4.5 with acetic acid (1M);
[0052] Dissolve 0.5g of the saccharin (SRP) prepared in Example 1 above in 250mL of distilled water, and then dilute with distilled water to prepare a 2mg / mL saccharin solution.
[0053] (2) At room temperature, with a magnetic stirrer at a stirring speed of 600 rpm / min, 24 mL of chitosan solution (0.5 mg / mL) was added dropwise to 60 mL of Pleurotus ostreatus glycoprotein solution (2 mg / mL) using a dropping funnel at a rate of 1-2 drops per second. After the addition was complete, the mixture was stirred for 60 min under the above magnetic stirring conditions. Then, the mixture was freeze-dried to obtain Pleurotus ostreatus glycoprotein-chitosan nanoparticles 1 (SRP-CTS NPs1).
[0054] Example 3
[0055] The difference from Example 2 is that 16 mL of chitosan solution (0.5 mg / mL) was added dropwise to 60 mL of Pleurotus ostreatus glycoprotein solution (2 mg / mL) using a dropping funnel at a rate of 1-2 drops per second to obtain Pleurotus ostreatus glycoprotein-chitosan nanoparticles 2 (SRP-CTS NPs2).
[0056] Example 4
[0057] The difference from Example 2 is that 12 mL of chitosan solution (0.5 mg / mL) was added dropwise to 60 mL of Pleurotus ostreatus glycoprotein solution (2 mg / mL) using a dropping funnel at a rate of 1-2 drops per second to obtain Pleurotus ostreatus glycoprotein-chitosan nanoparticles 3 (SRP-CTS NPs3).
[0058] Example 5
[0059] The preparation method of *Stropharia macrocarpa* glycoprotein-chitosan nanoparticles is as follows:
[0060] (1) Dissolve 0.25g of chitosan (CTS, purchased from Shanghai Titan Technology Co., Ltd., item number: 01123490) in 500mL of distilled water, and adjust the initial pH of the chitosan solution to 4.5 with acetic acid (1M);
[0061] Dissolve 0.5g of the saccharin (SRP) prepared in Example 1 above in 250mL of distilled water, and then dilute with distilled water to prepare a 1mg / mL saccharin solution.
[0062] (2) At room temperature, with a magnetic stirrer at a stirring speed of 600 rpm / min, 24 mL of chitosan solution (0.5 mg / mL) was added dropwise to 120 mL of Pleurotus ostreatus glycoprotein solution (1 mg / mL) using a dropping funnel at a rate of 1-2 drops per second. After the addition was complete, the mixture was stirred for 60 min under the above magnetic stirring conditions. Then, the mixture was freeze-dried to obtain Pleurotus ostreatus glycoprotein-chitosan nanoparticles 4 (SRP-CTS NPs4).
[0063] Example 6
[0064] The difference from Example 5 is that 16 mL of chitosan solution (0.5 mg / mL) was added dropwise to 120 mL of Pleurotus ostreatus glycoprotein solution (1 mg / mL) using a dropping funnel at a rate of 1-2 drops per second to obtain Pleurotus ostreatus glycoprotein-chitosan nanoparticles 5 (SRP-CTS NPs5).
[0065] Example 7
[0066] The difference from Example 5 is that 12 mL of chitosan solution (0.5 mg / mL) was added dropwise to 120 mL of Pleurotus ostreatus glycoprotein solution (1 mg / mL) using a dropping funnel at a rate of 1-2 drops per second to obtain Pleurotus ostreatus glycoprotein-chitosan nanoparticles 6 (SRP-CTS NPs6).
[0067] Experimental Example 1
[0068] Characterization of Pleurotus ostreatus glycoprotein-chitosan nanoparticles
[0069] (1) Determination of particle size, zeta potential and polydispersity index of Pleurotus ostreatus glycoprotein-chitosan nanoparticles
[0070] The particle size, zeta potential, and polymer polydispersity index of the different *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles prepared in Examples 2-7 were determined using a laser particle size analyzer (Zetasizer NanoZS90, Malvern, UK).
[0071] The results are shown in Table 1. The lower the concentration of *Stropharia macrocarpa* glycoprotein, the larger the particle size of the formed nanoparticles, the smaller the absolute value of the Zeta potential, the worse the stability, the larger the PDI value, and the less uniform the nanoparticles. At the same concentration of *Stropharia macrocarpa* glycoprotein, the larger the mass ratio of *Stropharia macrocarpa* glycoprotein to chitosan, the smaller the particle size of the formed nanoparticles, the larger the absolute value of the Zeta potential, the stronger the stability, the smaller the PDI value, and the more uniform the nanoparticles.
[0072] Table 1. Particle size, zeta potential, and polymer polydispersity index (PDI) of different *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles (SRP-CTS NPs) (n=3)
[0073]
[0074] Note: SRP stands for strophanthidin glycoprotein; CTS stands for chitosan; SRP-CTS NPs are strophanthidin glycoprotein-chitosan nanoparticles; m:m is the mass ratio.
[0075] As shown in Table 1 above, the *Pleurotus ostreatus* glycoprotein-chitosan nanoparticles 3 (SRP-CTSNPs3) prepared in Example 4 have the smallest particle size. Therefore, the SRP-CTS NPs3 prepared in Example 4 was selected for structural characterization and in vitro bioactivity experiments.
[0076] (2) Morphological observation of SRP-CTS NPs3 under transmission electron microscopy
[0077] The surface morphology of the *Symplocos macrantha* glycoprotein-chitosan nanoparticles 3 (SRP-CTS NPs3) prepared in Example 4 was observed using a bio-type field emission transmission electron microscope (Talos F200C G2).
[0078] Transmission electron microscopy results as follows Figure 1 As shown, the S. agaricus glycoprotein-chitosan nanoparticles 3 (SRP-CTS NPs3) are basically spherical or subspherical, with relatively uniform size and very few particles aggregated.
[0079] (3) Fourier transform infrared spectroscopy analysis of SRP-CTS NPs3
[0080] 2.0 mg of the freeze-dried *Agaricus bisporus* glycoprotein-chitosan nanoparticles 3 (SRP-CTSNPs3) prepared in Example 4, along with a purchased chitosan solid sample and the *Agaricus bisporus* glycoprotein solid sample prepared in Example 1, were placed at designated positions on an infrared spectrometer for scanning. The spectral acquisition range was 600–4000 cm⁻¹. -1 The resolution is set to 4cm. -1 Infrared absorption spectra of strophanthidin glycoprotein, chitosan, and strophanthidin glycoprotein-chitosan nanoparticles were collected.
[0081] The results are as follows Figure 2 As shown, it is located between 3200 and 3550 cm. -1 The broad characteristic peak at 2923.07 cm⁻¹ is attributed to the stretching vibration of OH or NH. This peak is relatively broad because the absorption bands of the stretching vibrations of OH and NH overlap. -1 The peak at 1645.95 cm⁻¹ is an absorption peak due to the stretching vibration of the CH bond. -1 The peak at 1556.75 cm is an absorption peak due to the stretching vibration of the C=O bond. -1 and 1078.41cm -1 The peak at this point is an absorption peak due to the CH bending vibration. Compared with *S. macrocarpa* glycoprotein and chitosan, *S. macrocarpa* glycoprotein-chitosan nanoparticles 3 (SRP-CTS NPs3) have a peak at 3277.91 cm⁻¹. -1 The blue shift at the location is 2923.07cm. -1 The redshift at the peak indicates that the glycoprotein of *S. macrocarpa* is bound to chitosan via hydrogen bonds. Therefore, the hydrogen bonds between *S. macrocarpa* glycoprotein and chitosan may be one of the main forces promoting the formation of *S. macrocarpa* glycoprotein-chitosan nanoparticles 3 (SRP-CTS NPs3).
[0082] Experimental Example 2
[0083] Evaluation of the in vitro immunomodulatory activity of SRP-CTS NPs3 prepared in Example 4
[0084] (1) Cytotoxicity against RAW264.7 macrophages
[0085] The Alamar blue assay was used to determine the cytotoxicity of *Agaricus macrocarpa* glycoprotein, chitosan, and the *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles prepared in Example 4 against RAW264.7 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). RAW264.7 cell suspensions were inoculated into 96-well plates at a cell count of 5 × 10⁶ cells / mL. 5 180 μL of each sample was incubated at 37°C in a 5% CO2 incubator for 24 h. The colorless RPMI 1640 cell culture medium (purchased from Gibco, USA) was removed from the wells and replaced with 200 μL of colorless RPMI 1640 cell culture medium containing different concentrations of SRP-CTS NPs3 sample (final concentrations: 2.5, 5, 10, 25, 50 μg / mL). After incubation at 37°C for 48 h, 20 μL of Alamar Blue reagent (purchased from Sigma, USA) was added to each well for further incubation. The effect of the selected concentration range of samples on the viability of RAW264.7 cells was evaluated by the Alamar Blue method. Each sample was tested in triplicate.
[0086]
[0087] Test results as follows Figure 3 As shown, at concentrations of 2.5, 5, 10, 25, and 50 μg / mL, the lowest viability of RAW264.7 macrophages treated with *S. macrophages* glycoprotein (SRP), chitosan (CTS), and *S. macrophages* glycoprotein-chitosan nanoparticles 3 (SRP-CTS NPs3) was 89.67%. These results indicate that *S. macrophages* glycoprotein, chitosan, and *S. macrophages* glycoprotein-chitosan nanoparticles 3 did not exhibit cytotoxicity at concentrations ranging from 2.5 to 50 μg / mL.
[0088] (2) Effects on NO release from RAW264.7 macrophages
[0089] RAW264.7 cells were diluted with colorless RPMI 1640 cell culture medium (purchased from Gibco, USA) to a concentration of 5 × 10⁶ cells per milliliter. 5 A suspension of cells was added to 96-well plates, 200 μL per well. Cells were cultured at 37°C until complete adhesion. The culture medium was then removed from the wells and replaced with 200 μL of fresh, colorless RPMI 1640 cell culture medium containing different concentrations of *S. macrocarpa* glycoprotein (SRP), chitosan (CTS), and *S. macrocarpa* glycoprotein-chitosan nanoparticles (SRP-CTS NPs3) (final concentrations set at 2.5, 5, 10, 25, and 50 μg / mL). A blank control group (PBS) and a 1 μg / mL LPS group were also included. After 48 h of culture at 37°C, 100 μL of the supernatant was added to 50 μL of Griess reagent (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0021S). The reaction was allowed to proceed for 10 min, and the absorbance at 543 nm was measured to determine the NO release.
[0090] Test results as follows Figure 4 As shown, within the concentration range of 2.5–50 μg / mL, *Pseudomonas aeruginosa* glycoprotein, chitosan, and *Pseudomonas aeruginosa* glycoprotein-chitosan nanoparticles all stimulated NO release from RAW264.7 macrophages in a concentration-dependent manner. Chitosan showed weaker activity in stimulating NO release from RAW264.7 macrophages within this concentration range. Within the concentration range of 5–50 μg / mL, SRP-CTS NPs3 showed significantly higher activity in stimulating NO release from RAW264.7 macrophages than *Pseudomonas aeruginosa* glycoprotein.
[0091] (3) Effects on the release of immune factors TNF-α, IL-1β and IL-6 from macrophage RAW264.7
[0092] RAW264.7 cells were diluted with colorless RPMI 1640 cell culture medium (purchased from Gibco, USA) to a concentration of 5 × 10⁶ cells per milliliter. 5 A suspension of cells was added to 96-well plates, 200 μL per well. Cells were cultured at 37°C until fully adherent. The culture medium was then removed from the wells and replaced with 200 μL of fresh RPMI 1640 cell culture medium containing different concentrations of S. pv. magna glycoprotein (SRP), chitosan (CTS), and S. pv. magna glycoprotein-chitosan nanoparticles (SRP-CTS NPs3) (final concentrations set at 2.5, 5, 10, 25, and 50 μg / mL). A blank control group (PBS) and a 1 μg / mL LPS group were also included. After 48 h of culture at 37°C, the supernatant was collected and centrifuged at 12000g for 10 min. The levels of inflammatory factors IL-6, IL-1β, and TNF-α were strictly measured according to the ELISA kit requirements (purchased from Sizhengbai Biotechnology Co., Ltd., catalog numbers CME0006, CME0005, and CME0004, corresponding to the detection of IL-6, IL-1β, and TNF-α in sequence).
[0093] ELISA test results as follows Figure 5 As shown, compared with the PBS group, LPS stimulation of RAW264.7 macrophages significantly increased the levels of IL-6, IL-1β, and TNF-α. At concentrations of 2.5–50 μg / mL, both *Pseudomonas aeruginosa* glycoprotein and *Pseudomonas aeruginosa* glycoprotein-chitosan nanoparticles 3 stimulated the release of IL-6, IL-1β, and TNF-α from RAW264.7 macrophages in a concentration-dependent manner. Chitosan exhibited weaker activity in stimulating the release of IL-6, IL-1β, and TNF-α from RAW264.7 macrophages. At concentrations of 10–50 μg / mL, the activity of *Pseudomonas aeruginosa* glycoprotein-chitosan nanoparticles 3 in stimulating the release of IL-6, IL-1β, and TNF-α from RAW264.7 macrophages was significantly higher than that of *Pseudomonas aeruginosa* glycoprotein. These results indicate that *Pseudomonas aeruginosa* glycoprotein-chitosan nanoparticles possess better immunomodulatory activity than *Pseudomonas aeruginosa* glycoprotein.
[0094] The results in summary indicate that the *Pleurotus ostreatus* glycoprotein-chitosan nanoparticles have better immunomodulatory activity than *Pleurotus ostreatus* glycoprotein, and have certain application potential and prospects in the preparation of *Pleurotus ostreatus* glycoprotein-related functional foods, health products or pharmaceuticals.
[0095] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A type of Pleurotus ostreatus glycoprotein-chitosan nanoparticle, characterized in that, The nanoparticles were prepared from chitosan solution and Pleurotus ostreatus glycoprotein solution, and the particle size of the nanoparticles was less than 270 nm.
2. The *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles according to claim 1, characterized in that, The preparation method of the *Agaricus bisporus* glycoprotein solution includes: using *Agaricus bisporus* bodies as raw materials, crushing and sieving to obtain *Agaricus bisporus* powder; adding *Agaricus bisporus* powder to sodium hydroxide aqueous solution for extraction for 120 minutes, filtering, collecting the filtrate to obtain *Agaricus bisporus* extract; concentrating the *Agaricus bisporus* extract under reduced pressure to obtain a concentrated solution; centrifuging the concentrated solution to obtain the supernatant; acid precipitation of the concentrated supernatant to collect the precipitate, dissolving the precipitate in a solvent and dialyzing, collecting the dialysate and freeze-drying to obtain a freeze-dried crude glycoprotein sample; dissolving the freeze-dried glycoprotein sample to remove free protein, centrifuging to obtain the supernatant, and freeze-drying the supernatant to obtain *Agaricus bisporus* glycoprotein; The degree of deacetylation of the chitosan is 90%, and the viscosity is <100 mPa·s.
3. The method for preparing *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles as described in claim 1, characterized in that, Chitosan solution was added dropwise to the serotonin solution of *Agaricus bisporus* at a rate of 1-2 drops per second. After the addition was complete, stirring was continued for 60 minutes. Then, the mixture was freeze-dried to obtain *Agaricus bisporus* glycoprotein-chitosan nanoparticles.
4. The preparation method according to claim 3, characterized in that, The chitosan solution was added dropwise to the sucrose protein solution of *Agaricus bisporus* at a rate of 1-2 drops per second, while stirring at a speed of 600 rpm / min.
5. The preparation method according to claim 3, characterized in that, The volume ratio of the chitosan solution to the Pleurotus ostreatus glycoprotein solution is (1-4):(5-20).
6. The preparation method according to claim 3, characterized in that, The concentration of the chitosan solution is 0.5–1 mg / mL, and the concentration of the *Pleurotus ostreatus* glycoprotein solution is 1–2 mg / mL.
7. The preparation method according to claim 6, characterized in that, The solvent used to prepare the chitosan solution and the Pleurotus ostreatus glycoprotein solution is distilled water; the pH of the chitosan solution is 4.
5.
8. The preparation method according to claim 7, characterized in that, The pH of the chitosan solution was adjusted using acetic acid.
9. The application of the nanoparticles as described in claim 1 or 2, or the preparation method of any one of claims 3 to 8, of the *Agaricus macrocarpa* glycoprotein-chitosan nanoparticles in the preparation of products with immunomodulatory activity.
10. The application according to claim 9, characterized in that, The products include functional foods, health products, or pharmaceuticals.