Pleurotus pulmonarius PpXN08 as well as mycelium, application and functional product of pleurotus pulmonarius PpXN08

Abstract

The invention belongs to the field of functional microorganisms, and particularly relates to Pleurotus pulmonarius PpXN08 as well as mycelia, application and a functional product thereof, and the preservation number of the Pleurotus pulmonarius PpXN08 is CCTCC NO: M 20242531. The pleurotus pulmonarius contains active ingredients such as selenoglycoprotein with a brand new structure, and has excellent activity and selectivity for resisting pleuroperitoneal cavity cancers and female cancers.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to a Pleurotus ostreatus technology for producing selenium glycopeptides. Background Technology

[0002] Selenoglycopeptides are a class of functional glycoproteins containing selenium, primarily obtained through natural extraction and artificial synthesis. In natural sources, edible fungi (such as oyster mushrooms, Ganoderma lucidum, Hericium erinaceus, and Cordyceps) and plants can accumulate selenium from the environment and integrate it into the glycopeptide structure, forming natural selenoglycopeptides. These organisms bind selenium to the glycopeptides in the form of selenocysteine ​​(Sec) or selenomethionine (SeMet) through their metabolic pathways. In terms of artificial synthesis, selenoglycopeptides can be prepared through chemical synthesis or genetic engineering techniques. Naturally derived selenoglycopeptides have attracted considerable attention due to their biocompatibility and diversity.

[0003] Selenium-enriched mycelium refers to fungal mycelium in which selenium is integrated into the mycelium through bioaccumulation. Its biosynthesis process mainly includes three key steps: selenium absorption, metabolism, and integration.

[0004] First, fungal mycelia absorb inorganic selenium (sodium selenite) from a selenium-rich liquid culture medium and enter the cell via transport proteins on the cell membrane. Subsequently, selenium undergoes a series of metabolic reactions within the cell, being reduced to selenocysteine ​​(Sec) or selenomethionine (SeMet). These two selenoamino acids are key precursors for selenium integration into proteins and glycopeptides. Finally, the selenoamino acids are integrated into the protein or polysaccharide structures of the mycelium through post-translational modifications or enzymatic reactions, forming selenium-enriched glycopeptides or other selenium-containing biomacromolecules. During the cultivation of selenium-enriched mycelia, the selenium concentration, pH, and temperature of the culture medium significantly affect the efficiency of selenium absorption and integration.

[0005] Different strains exhibit different metabolic behaviors and possess entirely different physiological activities. Existing technologies have also disclosed some *Pleurotus pulmonaryus* strains. For example, Chinese patent document CN115710554A discloses a *Pleurotus pulmonaryus* strain for decolorization and COD removal from wastewater and its applications. Patent document CN117327592A discloses a *Pleurotus pulmonaryus* strain and its applications. Patent document CN117487669A discloses a thermoresistant *Pleurotus pulmonaryus* strain and its applications.

[0006] In summary, while existing technologies have reported on some *Pleurotus pulmonarius* strains, research on selenium-enriched glycopeptides from edible and medicinal strains is currently lacking. Furthermore, the extraction process for selenium-enriched glycopeptides is complex and generates other byproducts, posing challenges to the uniformity and standardization of quality in actual production. Therefore, screening edible and medicinal strains with high selenium-enriched glycopeptide content and stable biomass, exploring their selenium absorption and metabolic mechanisms, and developing a complete production process are of theoretical significance for the development and utilization of selenium-enriched glycopeptide dietary foods and anticancer drugs. Summary of the Invention

[0007] The primary objective of this invention is to provide a novel *Pleurotus pulmonarius* PpXN08 (also referred to as *Pleurotus pulmonarius* or PpXN08), which aims to provide a new strain capable of producing special selenoglycopeptides and / or glycoproteins while also exhibiting excellent anticancer and other physiological activities.

[0008] A second objective of the present invention is to provide mycelium of the aforementioned *Pleurotus pulmonarius*.

[0009] This invention provides the preparation and application of the aforementioned Pleurotus pulmonale and its mycelium.

[0010] The fourth objective of this invention is to provide a functional article comprising Pleurotus pulmonale and its mycelium and extract.

[0011] A lung-shaped Pleurotus erythrorhizon PpXN08, with accession number CCTCC NO: M 20242531.

[0012] This invention provides a novel *Pleurotus pulmonale* species that can produce novel active ingredients such as selenoglycopeptides and / or glycoproteins, exhibiting excellent anticancer and other physiological activities as well as physiological safety. For example, components containing novel natural structures possess high activity, selectivity, and safety against thoracic and abdominal cancers, as well as female cancers.

[0013] The preservation information of *Pleurotus pulmonarius* described in this invention is as follows:

[0014] Strain: Pleurotus pulmonaryus (also known as selenium-enriched Pleurotus pulmonaryus).

[0015] Preservation period: November 11, 2024;

[0016] China Center for Type Culture Collection (CCTCC);

[0017] Accession number: CCTCC NO: M 20242531;

[0018] Classified and named as: Pleurotus pulmonarius PpXN08 (also known as Pleurotus pulmonarius PpXN08);

[0019] Address: China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province.

[0020] In this invention, the genus of *Pleurotus pulmonaryis* belongs to the kingdom Fungi, phylum Basidiomycota, class Agaricomycetes, order Agaricales, family Pleurotaceae, and genus *Pleurotus*.

[0021] In this invention, the screening method for Pleurotus pulmonaryus PpXN08 is as follows: the mycelium is cultured in PDA medium with different selenium concentrations, and the variety PpXN08 with fast mycelial growth, good growth, short plate filling time and high selenium tolerance is screened.

[0022] The present invention also provides mycelium (also known as fermented mycelium) of the aforementioned Pleurotus pulmonae PpXN08, which is obtained by culturing Pleurotus pulmonae PpXN08 in a selenium-containing liquid culture medium.

[0023] The mycelium of *Pleurotus pulmonarius* PpXN08 is prepared by first culturing *Pleurotus pulmonarius* PpXN08 in a basal medium to obtain a mother culture, and then culturing the mother culture in a selenium-containing basal medium to obtain the mycelium.

[0024] The basic culture medium is an aqueous solution containing 150-250 g / L potato, 15-25 g / L glucose, 1-5 g / L yeast, 0.1-1 g / L magnesium sulfate and 0.5-1.5 g / L potassium dihydrogen phosphate.

[0025] In the selenium-containing basal culture medium, the selenium is sodium selenite, and its concentration can be 5~40 mg / L, or more specifically 10~20 mg / L;

[0026] The incubation temperature is 24~25℃, the stirring pressure is controlled at 0.1~0.15 Pa, and the incubation time is more than 5 days.

[0027] This invention also provides applications of Pleurotus pulmonale PpXN08 and its mycelium, directly preparing functional products from them, and / or preparing functional products after extracting active ingredients from them.

[0028] The present invention demonstrates that the *Pleurotus pulmonarius* PpXN08 and its mycelium contain novel functional components with good physiological activity.

[0029] For example, as an optional application, an extract containing at least one of selenoglycopeptides and / or selenoglycoproteins, comprising an anticancer active ingredient, can be extracted from the aforementioned *Pleurotus pulmonale* PpXN08 and its mycelium, and the extract can be used to prepare functional articles. Further, the anticancer active ingredient includes an active ingredient against at least one of gastric cancer, liver cancer, bronchial cancer, ovarian cancer, breast cancer, and cervical cancer.

[0030] In this invention, the functional product includes at least one of pharmaceuticals, health products, and food.

[0031] The present invention shows that the *Pleurotus pulmonarius* PpXN08 and its mycelium are rich in functional components with high anti-cancer activity. In addition, these components have low toxicity to normal cells and can be used to prepare pharmaceuticals, health products and food.

[0032] The present invention demonstrates that the *Pleurotus pulmonarius* PpXN08 possesses unique and highly functional active ingredients that can be used to prepare anticancer drugs and other products.

[0033] The present invention also provides a method for preparing an anticancer active glycoprotein (also referred to as selenoglycoprotein in the present invention) from the aforementioned Pleurotus pulmonale, wherein crude selenoglycoprotein is obtained by defatting, salt extraction and alcohol precipitation of Pleurotus pulmonale mycelium powder;

[0034] The crude selenium glycoprotein is separated by a cellulose exchange column. The separation process includes sequential elution with water, elution with a sodium chloride solution of 0.05~0.15 mol / L (preferably 0.08~0.12M), and elution with a sodium chloride solution of 0.25~0.35 mol / L (preferably 0.28~0.32M).

[0035] The eluent of 0.05~0.15 mol / L sodium chloride solution was collected as target eluent 1, and after desalting, concentration and dextran gel column purification, the anticancer active glycoprotein 1 (Pleurotus 93 selenium glycoprotein-Ⅰ, also known as Se-POGP-1a) was obtained.

[0036] The eluent of a 0.25-0.35 mol / L sodium chloride solution was collected as target eluent 2, and after desalting, concentration, and dextran gel column purification, the anticancer active glycoprotein 2 (Se-POGP-2b) was obtained.

[0037] In this invention, the solvent for the degreasing process is an aqueous alcohol-water solution, wherein the volume fraction of the alcohol is 75-85%. Further, the alcohol in the aqueous alcohol-water solution is ethanol. Further, the degreasing method is reflux degreasing.

[0038] In this invention, the defatted product is subjected to salt extraction in brine; wherein the brine used in the salt extraction process is a 0.05~0.15 mol / L sodium chloride solution. The salt extraction temperature is 85~95 °C.

[0039] In this invention, alcohol is added to the extract after salt extraction for alcohol precipitation, followed by solid-liquid separation to obtain the alcohol precipitate. Further, the alcohol is ethanol. Additionally, during the alcohol precipitation process, to save on the amount of alcohol used, the extract can be moderately concentrated as needed. The amount of alcohol can be adjusted appropriately as needed; for example, the volume ratio of extract (or its concentrate) to alcohol can be 1:1 to 10; more specifically, it can be 1:3 to 5.

[0040] In this invention, the alcohol precipitate is dialyzed to obtain crude selenium glycoprotein (labeled as (Se-POGP)).

[0041] In this invention, the cellulose exchange column is a DEAE-52 cellulose ion exchange column;

[0042] Preferably, the target eluent is desalted by dialysis;

[0043] Preferably, the dextran gel column is a G-100 dextran gel column.

[0044] This invention also provides an application of the aforementioned pleurotus 93 selenoglycoprotein-I (Se-POGP-1a), using it as an anticancer active ingredient in the preparation of drugs against thoracic and abdominal visceral cancers. A preferred application is its use as an anticancer active ingredient in the preparation of drugs that inhibit thoracic and abdominal visceral cancers caused by at least one of HepG2 cells, MGC-803 cells, and A549 cells. In a preferred application, the thoracic and abdominal visceral cancers include at least one of gastric cancer, liver cancer, and bronchial cancer. In this invention, the pleurotus 93 selenoglycoprotein-I is used to prepare an anticancer drug that induces apoptosis in HepG2 cells, MGC-803 cells, and A549 cells without damaging normal liver cells, gastric cells, and bronchial cells. The pleurotus 93 selenoglycoprotein-I prepared by this invention can effectively kill cancer cells without damaging the function of normal cells. Research on pleurotus 93 selenoglycoprotein-I as an anticancer active ingredient is of great significance to the development of the anticancer field. In this invention, the normal liver cells can be the Lx2 cell line, the normal gastric cells can be the GES-1 cell line, and the normal bronchial cells can be the 16HBE cell line. For example, when the concentration of Pleurotus ostreatus 93 selenoglycoprotein-I described in this invention is 600 μg / mL, the cancer cell survival rate is approximately 39%, and the tumor inhibition rate is approximately 61%. However, at the same drug concentration, the survival rate of normal cells can reach over 95%. Therefore, while maintaining an inhibitory effect on cancer cells, there is no significant damage to normal cells.

[0045] This invention also provides the use of the aforementioned Se-POGP-2b in the preparation of a drug for treating female tumors. The application uses it as an anticancer active ingredient to inhibit female tumors caused by at least one cancer cell in Skov3, MDA-231, and HeLa cells. The female tumors described in this invention include at least one cell type from the ovary, breast, and cervical glands.

[0046] The present invention also provides a functional article comprising at least one of Pleurotus pulmonaryus PpXN08 and its mycelium and extract.

[0047] The aforementioned functional product is a functional product containing the active ingredients described in this invention (such as selenium polypeptides and / or selenium glycoproteins).

[0048] The aforementioned functional product is at least one of a drug, health product, or food.

[0049] Beneficial effects

[0050] This invention provides a novel *Pleurotus pulmonarius* species, which can metabolize to produce novel selenoglycopeptides and / or glycoproteins, exhibiting excellent anticancer and other physiological activities. In addition, it has very low toxicity to normal cells, demonstrating excellent anticancer selectivity and physiological safety.

[0051] The present invention also shows that the *Pleurotus pulmonarius* PpXN08 contains Se-POGP-1a and Se-POGP-2b active ingredients with special novel structures, and that it can exhibit excellent activity and selectivity against thoracic and abdominal tumors as well as female tumors. Attached Figure Description

[0052] Figure 1 This is a diagram showing mycelial growth under different selenium concentrations in Example 1;

[0053] Figure 2 This is a graph showing the total selenium content of the mycelium in Example 1;

[0054] Figure 3 Figure 2 shows liquid fermentation at different selenium concentrations in Example 2;

[0055] Figure 4 The graphs show the molecular weight and monosaccharide composition of the 93-selenoglycoprotein-I (Se-POGP-1a) component from Pleurotus ostreatus. In the graph, a is the molecular weight graph and b is the monosaccharide composition graph.

[0056] Figure 5 The FT-IR infrared spectrum of pleurotus 93 selenogenetic protein-I component;

[0057] Figure 6The image shows a one-dimensional NMR spectrum of the 93-selenoglycoprotein-I component of Pleurotus ostreatus, where a is the H spectrum and b is the C spectrum.

[0058] Figure 7 For Pleurotus 93 selenium glycoprotein-I component 1 H- 1 H COSY diagram;

[0059] Figure 8 HSQC diagram of Pleurotus ostreatus 93 selenium glycoprotein-I component;

[0060] Figure 9 HMBC diagram of Pleurotus ostreatus 93 selenium glycoprotein-I component;

[0061] Figure 10 NOSEY diagram of Pleurotus ostreatus 93 selenium glycoprotein-I component;

[0062] Figure 11 The structural diagram of the protein obtained by identifying Pleurotus ostreatus 93 selenyl glycoprotein-I (Se-POGP-1a); including proteins with kinesin motility domains, actin, and valine-tRNA synthetase;

[0063] Figure 12 A docking simulation diagram of Pleurotus ostreatus 93 selenogen-I component;

[0064] Figure 13 The results of the CCK-8 assay for Pleurotus ostreatus 93 selenoglycoprotein-I fraction are shown below. a) is the result for HepG2, b) is the result for Lx2, c) is the result for MGC-803, d) is the result for GES-1, e) is the result for A549, and f) is the result for 16HBE.

[0065] Figure 14 The results of the CCK-8 assay for the crude fraction of Pleurotus ostreatus 93 selenogenetic glycoprotein (Se-POGP) are shown below. Among them, a) is the result of HepG2, b) is the result of Lx2, c) is the result of MGC-803, d) is the result of GES-1, e) is the result of A549, and f) is the result of 16HBE.

[0066] Figure 15 Diagram showing the monosaccharide composition of Se-POGP-2b;

[0067] Figure 16 Two-dimensional nuclear magnetic resonance of Se-POGP-2b 1 H- 1 H COSY diagram;

[0068] Figure 17 HSQC diagram for Se-POGP-2b;

[0069] Figure 18 HMBC diagram of Se-POGP-2b;

[0070] Figure 19 NOSEY plot of Se-POGP-2b;

[0071] Figure 20 The images show the protein structures identified by Se-POGP-2b; (a) is a diagram of ribosomal protein S12 (protein ID A0A8H7DQ59); (b) is a diagram of nucleolar proribosome-associated protein 2 (protein ID A0A067NRL7); (c) is a diagram of RNA-dependent RNA polymerase (protein ID A0A067NMT3); and (d) is a diagram of peroxidase domain protein (protein ID A0A067N671).

[0072] Figure 21 The diagram shows a partial docking simulation of Se-POGP-2b; (a) is the optimal conformation diagram of the docking of the peroxidase domain protein and Se-POGP-2b using Autodock software; (b) is the optimal conformation diagram of the docking of nucleolar proribosome-associated protein 2 and Se-POGP-2b using Autodock software.

[0073] Figure 22 The diagram shows a partial docking simulation of Se-POGP-2b; (a) is the optimal conformation diagram of ribosomal protein S12 and Se-POGP-2b docking using Autodock software; (b) is the optimal conformation diagram of RNA-dependent RNA polymerase and Se-POGP-2b docking using Autodock software.

[0074] Figure 23 The figures show the CCK-8 experimental results for Se-POGP-2b; where a) is the result for Skov3, b) is the result for IOSE, c) is the result for MDA-231, d) is the result for MCF-10A, e) is the result for hela, and f) is the result for VK2.

[0075] Figure 24 Figures show the experimental results of CCK-8 for Se-POGP; where a) is the result for Skov3, b) is the result for IOSE, c) is the result for MDA-231, d) is the result for MCF-10A, e) is the result for hela, and f) is the result for VK2.

[0076] Figure 25 Anatomical diagram of tumor size in mice after administration of crude selenium glycoprotein (Se-POP) and Se-POGP-2b of Pleurotus pulmonaryis mycelium. Detailed Implementation

[0077] Part 1: Mycelial Selection, Culture, and Results

[0078] Example 1:

[0079] Two culture methods, solid-state and liquid-state, were used to screen selenium-enriched bacterial strains and determine the optimal selenium concentration range. PDA medium was used, with selenium concentrations of 0, 10, 20, 30, and 40 mg / L on the solid medium. The same amount of mycelial blocks were inoculated into PDA medium at different selenium concentrations (see Table 1). Cultures were conducted under the same conditions, and mycelial growth rate, vigor, and time to full plate were recorded to screen the optimal selenium concentration range and maximum selenium tolerance range for different strains. At a selenium concentration of 40 mg / L, compared to 0 mg / L, the average biomass of each strain showed some difference (P<0.05). The strain with the highest average biomass among the 15 strains was XN08, with an average biomass per plate of 0.581 ± 0.105 g. Referring to the national food safety standards GB5009.268-2016 "Determination of Multiple Elements in Food" and GB5009.93-2017 "Determination of Selenium Content in Food," ICP-MS has the advantages of being rapid, accurate, and having low limits, making it suitable for experiments involving small sample weights and large quantities. The total selenium content of 15 lung-shaped pleural auricles at selenium concentrations of 0 and 40 mg / L was determined using inductively coupled plasma mass spectrometry (ICP-MS). The results showed... Figure 3 When the selenium addition was 0 mg / L, the total selenium content in each strain was relatively low. At a selenium addition of 40 mg / L, XN08 had the highest selenium content among the 15 strains, reaching 206 ± 0.01 mg / kg. Therefore, considering the biomass yield, growth rate, and total selenium content after selenium addition, strain XN08, with high selenium content, was selected. It was then deposited, with the accession number CCTCC NO: M 20242531.

[0080]

[0081] Example 2: Preparation of selenium-enriched glycopeptides from liquid mycelia of strain XN08

[0082] (1) Preparation of primary mother seeds

[0083] The mycelium balls fermented in the liquid culture medium were inoculated into the cultivation substrate; after the mycelium had fully grown the bags in the dark at 24℃ in the cultivation room, the bags were opened and the temperature was increased to 24℃ and the humidity to 80% or higher for fruiting management. After the fruiting bodies grew, the fruiting bodies were taken out and obtained as primary mother culture after tissue separation and transfer.

[0084] Liquid culture medium formula: potato 200 g / L, glucose 20 g / L, yeast 3 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L;

[0085] (2) Preparation of liquid bacterial strains

[0086] The primary mother culture blocks were transferred to selenium-containing liquid culture medium. 10-12 blocks with a diameter of 5 mm were inoculated and cultured on a shaker at 24℃ and 135 rpm / min for 8-10 days to obtain selenium-enriched liquid mycelium.

[0087] Selenium-containing liquid culture medium formula: potato 200 g / L, glucose 20 g / L, yeast 3 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 1 g / L, sodium selenite concentration 15 mg / L

[0088] Liquid mycelium production

[0089] The liquid culture was inoculated into a sterilized liquid fermenter containing selenium (15 mg / L), the temperature was 24-25℃, the stirring pressure was controlled at 0.1-0.15 Pa, and after 10 days of cultivation, the bacterial balls were filtered, rinsed 3 times with pure water, soaked and stirred for 15 min, filtered and dried, and selenoglycopeptides were extracted for determination.

[0090] Example 3: Screening of selenium concentration addition during liquid fermentation of strains

[0091] Compared with Example 2, the only difference is that the selected XN08 strain was inoculated into liquid culture medium with selenium concentrations of 0, 5, 10, 15, 20, and 25 mg / L, cultured at 135 rpm / min and 24°C for 10 days, and the biomass yield was measured. Based on the biomass yield, 15 mg / L was determined to be the maximum selenium concentration to be added for liquid fermentation.

[0092]

[0093] Detection of selenoglycopeptide content in strain XN08

[0094] Weigh 2 g of XN08 bacterial powder and place it in a round flask. Add a stir bar and 40 mL of 0.1 mol / L NaCl solution. Heat the mixture at 70 °C for 1 h using a magnetic stirrer. After 1 h, remove the mixture and pour it into a vacuum filtration flask. Filter the mixture and pour it back into the round flask. Transfer the solution to a 3500 molecular weight dialysis bag using a pipette. Dialyze the bag in pure water for 1 day. Then, pour the solution into a rotary evaporator and evaporate to about 10 mL. Add 40 mL of anhydrous ethanol to precipitate the crude glycopeptides. Centrifuge the precipitate and collect the supernatant using a dropper. Dissolve the precipitate in deionized water and continue dialysis in the dialysis bag for 1 day. Pour the solution into a lyophilization bottle, seal it with filter paper, refrigerate it for one day, and then perform liquid lyophilization for 2-3 days. Weigh 5 mg of the lyophilized powder and determine its selenoglycopeptide content. The results show that the selenoglycoprotein content reaches 260 mg / kg. The active ingredient is selenoglycoprotein-III.

[0095] Detection of beneficial metabolites of strain XN08 after selenium enrichment

[0096] Non-targeted metabolomics was used to detect metabolites in the hyphae, and anti-cancer related metabolites such as Carnosine, 2-trans,6-trans-Farnesal, N7-Methylguanosine, and Perillyl alcohol were found.

[0097] XN08 has a higher content of selenoside polysaccharides from Pleurotus ostreatus compared to the patents ZL202011051402.6, ZL202011251224.1, ZL202011251768, ZL202011253503.1, ZL202011252716.2, ZL202011051265.6, and ZL202011055176.9, and its tumor inhibition rate is higher than those five patents.

[0098] In summary, 1. In a selenium-rich environment, the survival rate is high. Compared with the existing method of extracting selenoglycopeptides from edible fungi fruiting bodies (3 months), the present invention directly extracts selenoglycopeptides from fermentation mycelium, and the preparation of selenoglycopeptides is faster, more efficient, and has a higher selenium content (10 days).

[0099] 2. The optimal selenium concentration for liquid fermentation of strain XN08 obtained through screening was 15 mg / L, with a biomass yield of 1.462±0.021 g / L and a selenoglycopeptide content of 260 mg / kg.

[0100] 2. When the concentration of the active ingredient selenoglycoprotein-III, an anticancer active ingredient in XN08 bacterial powder, is 600 μg / mL, the survival rate of cancer cells is approximately 29%, and the tumor inhibition rate is approximately 71%. However, at the same dosage concentration, the survival rate of normal cells can reach over 95%. Therefore, while maintaining an inhibitory effect on cancer cells, it does not cause significant damage to normal cells.

[0101] Part Two: Extraction and Testing of Novel Selenoglycoproteins

[0102] 2.1: Extraction and purification

[0103] Example 4: Extraction and purification of active anticancer polysaccharides

[0104] (1) Defatting:

[0105] Weigh an appropriate amount of *Pleurotus pulmonarius* mycelium powder (prepared in Example 2), pour it into a round-bottom flask, add 80 v% ethanol solution (liquid-to-solid ratio of 5 mL / g), and reflux for defatting for 1 hour;

[0106] (2) Salt extraction: Pour the defatted mycelium into a round-bottom flask, add 0.1M sodium chloride at a ratio of 1:20~25 (w / v, g / mL), and extract twice in a water bath at 85~95°C for 1.5 hours each time.

[0107] (3) Alcohol precipitation: The extract collected in (2) was concentrated (to 50% of the original volume), and then the concentrated extract was slowly poured into pre-cooled anhydrous ethanol at a ratio of 1:4, with constant stirring. After precipitation at 4°C overnight, the product was dialyzed, collected, and freeze-dried to obtain crude selenium glycoprotein (Se-POGP).

[0108] (4) Column chromatography purification: The crude selenium glycoprotein was purified by cellulose exchange column. The steps were as follows: 100 mg of lyophilized crude selenium glycoprotein was dissolved in 3 mL of water and slowly loaded along the edge. Gradient elution was performed with deionized water, 0.1, 0.3, 0.5, and 0.7 mol / L sodium chloride solutions. The sugar content was monitored by the phenol-sulfuric acid method (200 μL of eluent was mixed with 100 μL of 0.5% phenol and 500 μL of concentrated sulfuric acid, cooled for 10 min, and the ultraviolet absorption at 490 nm was measured).

[0109] The fraction eluted with 0.1 mol / L sodium chloride solution was collected, and the target eluent was subjected to dialysis desalting. Subsequently, a second purification was performed using a dextran gel column (G-100 dextran gel column) to obtain the target active ingredient, named Se-POGP-1a. The selenium content in Se-POGP-1a was 67.4 ug / g.

[0110] The fraction eluted with 0.3 mol / L sodium chloride solution was collected, and the target eluent was subjected to dialysis desalting. Subsequently, a second purification was performed using a dextran gel column (G-100 dextran gel column) to obtain the target active ingredient, named Se-POGP-2b. The selenium content in Se-POGP-2b was 52.1 ug / g.

[0111] The cellulose exchange column is a DEAE-52 cellulose ion exchange column.

[0112] 2.2: Structural Identification of Se-POGP-1a

[0113] Example 5: Identification of the structure of Se-POGP-1a (Results are shown in...) Figures 4-12 ):

[0114] Example 5-1: Methylation Experiment:

[0115] Methylation experiment of selenoglycoprotein (Se-POGP-1a) from *Pleurotus pulmonarius* mycelium (prepared in Example 1):

[0116] Methylation: Weigh 3 mg of Se-POGP-1a sample into a glass reaction flask, add 1 mL of anhydrous DMSO, then add anhydrous alkaline solution A, dissolve by sonication, and then add iodomethane solution B. Stir magnetically in a water bath at 30°C for 60 min, and finally add 2 mL of ultrapure water to terminate the methylation reaction.

[0117] Acid hydrolysis: The methylated polysaccharide was hydrolyzed in 1 mL of 2M trifluoroacetic acid (TFA) for 90 min, and then concentrated by rotary evaporation until no liquid remained.

[0118] Reduction: Add 2 mL of double-distilled water and 60 mg of sodium borohydride for 8 hours, neutralize with glacial acetic acid, rotary evaporate, and dry in an oven at 101°C.

[0119] Acetylation: Add 1 mL of acetic anhydride and react at 100 °C for 1 h, then cool. Add 3 mL of toluene, concentrate under reduced pressure and evaporate to dryness. Repeat 4-5 times to remove excess distilled water. After thorough shaking, remove the supernatant. Repeat this process 4 times. Separate the CH₂Cl₂ layer with an appropriate amount of anhydrous acetic anhydride. Dissolve the acetylated product in 3 mL of CH₂Cl₂, transfer to a separatory funnel, add a small amount of sodium sulfate, dry, concentrate to 1 mL, and transfer to a liquid chromatography vial. Analyze the acetylated product sample using a Thermo Scientific 1300-7000 gas chromatograph-mass spectrometer.

[0120] GC-MS conditions: HP-INNOWAX column 30mm×0.32mm×0.25um; temperature program conditions: initial temperature 140 °C, increased to 230 °C at 1 °C / min; injection port temperature 250 °C, detector temperature 250 °C, carrier gas helium, flow rate 1 mL / min.

[0121] The methylated sugar residues obtained in the experiment were compared with the mass spectra of standard PMAA (partially methylated sugar alcohol acetyl esters), and the assignment and proportion of each sugar residue were determined based on retention time and mass-to-nucleus ratio. The analysis results of the GC-MS data are shown in Table 3. The structure is shown in Equation 1:

[0122]

[0123] Example 5-2: Nuclear magnetic resonance experiment of selenoglycoprotein (Se-POGP-1a) from *Pleurotus pulmonae* mycelium (prepared in Example 1):

[0124] Take 50 mg of dried Se-POGP-1a sample, dissolve it in 500 μL of D2O, freeze-dry it three times, and then analyze the sample using nuclear magnetic resonance spectroscopy. The detection spectra include 1D NMR (…). 1 H NMR,13 C NMR) and 2D NMR ( 1 H 1 H-COSY, HSQC, HMBC, NOESY).

[0125] Analysis of the molecular structure of selenoglycoprotein (Se-POGP-1a) from *Pleurotus pulmonaryus* mycelium (prepared in Example 1):

[0126] according to 1 H and 13 The number of anomeric hydrogens and anomeric carbons is determined by the anomeric regions in the C-chromatic spectroscopy. Based on the chemical shifts of the anomeric hydrogens and anomeric carbons mentioned above, in... 1 H 1 In the H-COSY spectrum, identify the chemical shifts of other hydrogen atoms associated with the anomeric proton. Based on the proton's chemical shift, identify the corresponding carbon chemical shifts in the HSQC spectrum. (This is followed by a list of sugar residues.) 1 H, 13 The C chemical shifts are shown in Table 4. After assigning the carbon and hydrogen chemical shifts, the carbon-hydrogen correlation signal peaks were identified using the HMBC spectrum to determine the linkage mode and sequence of sugar residues, thus completing the NMR spectrum analysis of the selenoglycoprotein (Se-POGP-1a) component of Pleurotus pulmonale mycelium.

[0127]

[0128] Formula 1;

[0129] Example 5-3: Glycoprotein docking

[0130] Protein identification and docking experiment of selenoglycoprotein (Se-POGP-1a) from *Pleurotus pulmonarius* mycelium:

[0131] In this invention, protein identification is mainly achieved by extracting proteins from samples, followed by reduction and alkylation, enzymatic digestion, desalting, and detection using LC-MS / MS.

[0132] After obtaining the protein structure, search for available protein structures in the database, use docking software to dock the polysaccharide with the protein to predict its optimal structure, and then use visualization software such as PyMOL to visualize the structure.

[0133] The results show that the hydroxyl groups in polysaccharides are linked to at least one of the amino acids in proteins, namely aspartic acid, valine, glutamic acid, arginine, lysine, and serine, through hydrogen bonds.

[0134] 2.3: Anticancer activity of Se-POGP-1a (results are shown in...) Figures 13-14 )

[0135] Example 6

[0136] Efficacy studies of selenoglycoprotein from *Pleurotus pulmonaryus* mycelium (Se-POGP-1a) (prepared in Example 1) against liver cancer, gastric cancer, and lung cancer cells:

[0137] Hepatocellular carcinoma, gastric cancer, and lung cancer cells in the logarithmic growth phase were harvested and digested with trypsin until the cells became rounded. Cell culture medium was then added to terminate the digestion, and the cells were centrifuged at 1200 rpm for 5 min. The cell suspension was appropriately diluted, and cells were counted using a hemocytometer. 5 × 10⁶ cells were seeded per well in a 96-well plate. 3 Cells were cultured in a 37°C incubator for 24 h.

[0138] The culture medium in the 96-well plate was then replaced with serum-free cell culture medium, and the plate was incubated in a cell culture incubator for 24 hours.

[0139] Se-POGP-1a samples were dissolved in basal culture medium to prepare Se-POGP-1a solutions of different concentrations (0, 100, 200, 300, 400, 500, 600 μg / mL), and then sterilized by filtration through a 0.22 μm filter membrane.

[0140] Continue culturing for 24 h, wash once with PBS, add 100 μL of 10% CCK-8 diluted with serum-free culture medium to each well, incubate at 37℃ for 30 min, and then measure the absorbance (OD) at 450 nm using a microplate reader to calculate the cell viability.

[0141] Cell viability = (OD) 实验组 -OD 阴性对照组 ) / (OD 空白组 -OD 阴性对照组 )

[0142] To investigate the effect of Se-POGP-1a on the growth status of cancer cells, cancer cells were prepared at a density of 1 × 10⁶ cells / mL in each confocal glass dish. 5 Cells were seeded at a density of 1000 μL and incubated at 37 °C. After 24 h, the old culture medium was discarded, and the cell surface was gently washed with PBS. 1 mL of fresh culture medium containing different concentrations of Se-POGP-1a was added to each dish. After another 24 h of incubation, following the AO / EB kit instructions, 120 μL of a 10-fold diluted solution C, 15 μL of AO, and 15 μL of EB were added to each dish. The dishes were incubated at room temperature in the dark for 1–5 min, the dye was removed, the cells were gently washed once with PBS, and then soaked in 500 μL of PBS. Confocal microscopy was used for observation (AO excitation 488 nm, emission 515 nm; EB excitation 518 nm, emission 605 nm).

[0143] To further demonstrate whether Se-POGP-1a can induce apoptosis in cancer cells, cells were collected and seeded into six-well plates at a density of 200,000 cells per well, and cultured at 37 °C. After 24 h, the cell surface was washed with PBS, and fresh medium containing different concentrations of Se-POGP-1a was added to each well for further culture. After 24 h, the old medium was discarded, and the cells were washed again with PBS. 500 μL of EDTA-free trypsin was added to each well for digestion for 1-5 min until the cells rounded. The cells were centrifuged, and the supernatant was discarded. 500 μL of prepared staining solution (500 μL 1×BB, 5 μL Annexin V-FITC, and 10 μL PI) was added to each tube, and the cells were resuspended. The cells were incubated at room temperature in the dark for 10 min, centrifuged, and the supernatant was discarded. 1 mL of PBS was added for resuspending. The cells were analyzed by flow cytometry within one hour using Flowjo software.

[0144] The anticancer activity of crude glycoprotein Se-POGP was simultaneously verified using the method described above.

[0145] Furthermore, the safety of Se-POGP-1a in normal cells was further evaluated using normal liver cells (Lx2), normal gastric cells (GES-1), and normal bronchial cells (16HBE) in the logarithmic growth phase.

[0146] 3.1: Example 6 - Se-POGP-2b test (results see...) Figures 15-22 )

[0147] Example 7-1: Se-POGP-2b (prepared in Example 1) methylation experiment:

[0148] Similar to Example 5-1, the analysis results of GC-MS data are shown in Table 5.

[0149]

[0150] Example 7-2: Se-POGP-2b (prepared in Example 1) NMR experiment:

[0151] Take 50 mg of dried Se-POGP-2b sample, dissolve it in 500 μL of D2O, freeze-dry it three times, and then analyze the sample using nuclear magnetic resonance spectroscopy. The detection spectra include 1D NMR (…). 1 H NMR, 13 C NMR and 2D NMR (HH-COSY, HSQC, HMBC, NOESY).

[0152] Example 7-3 Analysis of the molecular structure of Se-POGP-2b (prepared in Example 1):

[0153] according to 1 H and13 The anomeric region of the C-chromatic spectroscopy determines the number of anomeric hydrogens and carbons. Based on the chemical shifts of the anomeric hydrogens and carbons, the chemical shifts of other hydrogens associated with the anomeric proton are identified in the HH-COSY spectrum. Based on the proton's chemical shift, the chemical shifts of the carbons corresponding to the proton's chemical shift are identified in the HSQC spectrum. All sugar residues... 1 H, 13 The C chemical shifts are shown in Table 6. After assigning the carbon and hydrogen chemical shifts, the carbon-hydrogen correlation signal peaks were identified using the HMBC spectrum to determine the linkage mode and sequence of sugar residues, thus completing the analysis of the NMR spectrum of the Se-POGP-2b component.

[0154]

[0155] The sugar structural unit in Se-POGP-2b is shown in Equation 2:

[0156] Formula 2

[0157] Example 7-4

[0158] Se-POGP-2b protein identification and docking experiments:

[0159] In this invention, protein identification is mainly achieved by extracting proteins from samples, followed by reduction and alkylation, enzymatic digestion, desalting, and detection using LC-MS / MS.

[0160] After obtaining the protein structure, a database was searched for available protein structures. Docking software was used to dock the polysaccharide with the protein to predict its optimal structure, and then visualization software such as PyMOL was used to visualize the structure. The results showed that the hydroxyl groups in the polysaccharide are linked to at least one amino acid from the protein, namely aspartic acid, valine, glutamic acid, arginine, lysine, and serine, via hydrogen bonds.

[0161] 3.2: Anticancer activity (results are shown in...) Figures 23-25 )

[0162] Example 8: Anticancer activity of Se-POGP-2b or Se-POGP

[0163] Efficacy studies of Se-POGP-2b or Se-POGP (prepared in Example 1) against cancer cells (ovarian cancer, breast cancer, and cervical cancer) and normal cells (normal cells of the ovary, breast, and cervix):

[0164] Log-phase cancer cells (ovarian, breast, and cervical cancer) and normal cells (ovarian, breast, and cervical normal cells) were collected and digested with trypsin until the cells became rounded. Cell culture medium was then added to stop the digestion, and the cells were centrifuged at 1200 rpm for 5 min. The cell suspension was appropriately diluted, and cells were counted using a hemocytometer. 5 × 10⁶ cells were seeded per well in a 96-well plate. 3 Cells were cultured in a 37°C incubator for 24 hours.

[0165] Dissolve Se-POGP-2b or Se-POGP samples in basal culture medium to prepare Se-POGP-2b or Se-POGP solutions of different concentrations (0, 100, 200, 300, 400, 500, 600 μg / mL), and then sterilize by filtration through a 0.22 μm filter membrane.

[0166] Cell viability = (OD) 实验组 -OD 阴性对照组 ) / (OD 空白组 -OD 阴性对照组 )

[0167] To investigate the effects of Se-POGP-2b and Se-POGP on the growth status of cancer cells and normal cells, cancer cells and normal cells were prepared at a ratio of 1×10⁶ cells / mL in each confocal glass dish. 5 Cells were seeded at a density of 1000 μL and incubated at 37°C. After 24 hours, the old culture medium was discarded, and the cell surface was washed with PBS. 1 mL of fresh culture medium containing different concentrations of GDPs-1 was added to each dish. After another 24 hours of incubation, following the AO / EB kit instructions, 120 μL of a 10-fold diluted solution C, 15 μL of AO, and 15 μL of EB were added to each dish. The dishes were incubated at room temperature in the dark for 1-5 minutes, the dye was removed, the cells were washed once with PBS, and then soaked in 500 μL of PBS. Observation was performed using a confocal microscope (AO excitation 488 nm, emission 515 nm; EB excitation 518 nm, emission 605 nm).

[0168] To further demonstrate whether Se-POGP-2b and Se-POGP can induce cancer cell apoptosis, cells were collected and seeded into six-well plates at a density of 200,000 cells per well, and cultured at 37°C. After 24 hours, the cell surface was washed with PBS, and fresh medium containing different concentrations of Se-POGP-2b and Se-POGP was added to each well for further culture. After 24 hours, the old medium was discarded, and the cells were washed again with PBS. 500 μL of EDTA-free trypsin was added to each well for 1-5 min until the cells rounded. The cells were centrifuged, and the supernatant was discarded. 500 μL of prepared staining solution (500 μL 1×BB, 5 μL Annexin V-FITC, and 10 μL PI) was added to each tube, and the cells were resuspended. The cells were incubated at room temperature in the dark for 10 min, centrifuged, and the supernatant was discarded. 1 mL of PBS was added for resuspending. The cells were analyzed by flow cytometry within one hour using Flowjo software.

[0169] With increasing concentrations of Se-POGP-2b and Se-POGP, cancer cell growth was significantly inhibited in a concentration-dependent manner. For Skov3, MDA-231, and HeLa, the cell inhibition rates at a concentration of 600 μg / mL were 72% and 60%, 57% and 63%, and 62% and 27%, respectively. For IOSE, MCF-10A, and VK2, cell growth was not affected with increasing concentrations.

[0170] AO / EB staining results showed that when the Se-POGP-2b concentration was 0 μg / mL, cancer cells emitted uniform green fluorescence, and normal cell morphology with intact cell membranes could be observed in Blank. With increasing Se-POGP-2b concentration, the AO / EB fluorescence of cancer cells significantly increased, and cell membrane shrinkage and rounding were observed in Blank, indicating an increase in the number of apoptotic or necrotic cells, consistent with the results obtained from the previous CCK-8 experiment. The AO / EB staining results suggest that Se-POGP-2b and Se-POGP may mediate apoptosis in cancer cells, while having little effect on the growth of normal cells.

[0171] Apoptosis experiments showed that for cancer cells, with increasing Se-POGP-2b concentration, a significant increase in the proportion of cells in quadrants Q2 and Q3 of the apoptosis quadrant diagram was observed, while the proportion of cells in quadrant Q4 decreased, reaching 13.1% (Skov3), 50.9% (MDA-231), and 27.4% (HeLa) at the highest concentration, indicating a gradual increase in the number of apoptotic cells. Annexin V-FITC / PI and AO / EB staining results both confirmed that Se-POGP-2b can induce apoptosis in cancer cells without affecting the growth of normal cells.

[0172] Example 9

[0173] In vivo mouse animal experiments were conducted using either crude selenium glycoprotein Se-POGP or selenium glycoprotein Se-POGP-2b from Pleurotus pulmonaryis mycelium.

[0174] (1) Laboratory animals

[0175] Strain: Balb / c nude mouse (female, 4-6 weeks old, weight 16-18g);

[0176] Quantity: 56 (7 per group);

[0177] Keeping conditions: SPF-rated environment, 12-hour day-night cycle, free access to food and water;

[0178] (2) Grouping scheme (Table 7);

[0179]

[0180] (3) Establishment of tumor model

[0181] Cell line: Human ovarian cancer SKOV-3 cells (ATCC certified);

[0182] Seeding method: Logarithmic growth phase cells (5 × 10⁻⁶) 6 0.1 mL / mouse was injected subcutaneously into the ovarian tissue of mice.

[0183] Twelve hours after the last administration, all experimental mice were weighed and blood samples were taken. Subsequently, the mice were euthanized by cervical dislocation, and their spleens and ovaries (tumors) were carefully collected and accurately weighed.

[0184] Calculation formula: Organ index (mg / g) = Organ weight (mg) / Body weight (g);

[0185] Sample collection: Mice were sacrificed 12 hours after the last administration, and spleen and tumor tissue were separated under aseptic conditions.

[0186] Experimental results showed that, compared with the model group, the tumor weight in the drug group and the positive control group was smaller, indicating a better inhibitory effect. The spleen index can indicate the body's immune status and reflect the immunotoxicity of antitumor drugs; compared with the blank group, the spleen in the model group was significantly swollen. However, in the drug group, the spleen index was significantly lower than that in the model group, showing a dose-dependent effect. 5-FU treatment achieved a relatively high tumor inhibition rate, but compared with the model group, it also showed strong cytotoxic effects on these immune organs, which limits its further application.

Claims

1. A lung-shaped Pyrorhizium bacillus PpXN08, characterized in that, Its accession number is CCTCC NO: M 20242531.

2. The mycelium of *Pleurotus pulmonaryus* PpXN08 as described in claim 1, characterized in that, It was obtained by culturing Pleurotus pulmonale PpXN08 in selenium-containing liquid medium.

3. The mycelium of *Pleurotus pulmonaryus* PpXN08 as described in claim 2, characterized in that, Pleurotus pulmonale PpXN08 was first cultured in basal medium to obtain the mother culture, and then the mother culture was cultured in selenium-containing basal medium to obtain mycelium.

4. The mycelium producing selenium glycopeptides as described in claim 3, characterized in that, The basic culture medium is an aqueous solution containing 150-250 g / L potato, 15-25 g / L glucose, 1-5 g / L yeast, 0.1-1 g / L magnesium sulfate and 0.5-1.5 g / L potassium dihydrogen phosphate. In the selenium-containing basal culture medium, the selenium is sodium selenite, and its concentration can be 5~50 mg / L, or more specifically 10~20 mg / L; The incubation temperature is 24~25℃, the stirring pressure is controlled at 0.1~0.15 Pa, and the incubation time is more than 5 days.

5. The application of *Pleurotus pulmonarius* PpXN08 and its mycelium, characterized in that... Functional products can be prepared directly from it, and / or functional products can be prepared after extracting active ingredients from it; The *Pleurotus pulmonae* PpXN08 is the *Pleurotus pulmonae* PpXN08 as described in claim 1; the mycelium is the mycelium as described in any one of claims 2 to 4.

6. The application of *Pleurotus pulmonaryus* PpXN08 and its mycelium as described in claim 5, characterized in that... An extract containing at least one of selenoglycopeptides and / or selenoproteins, which are active anticancer components, is extracted from the extract and used to prepare functional products.

7. The application as described in claim 6, characterized in that, The active ingredients for anti-cancer treatment include those that are effective against at least one of the following cancers: stomach cancer, liver cancer, bronchial cancer, ovarian cancer, breast cancer, and cervical cancer.

8. A functional article, characterized in that, It comprises at least one of Pleurotus pulmonaryus PpXN08 and its mycelium and extract; wherein, the Pleurotus pulmonaryus PpXN08 is the Pleurotus pulmonaryus PpXN08 as described in claim 1; and the mycelium is the mycelium as described in any one of claims 2 to 4.

9. The functional article as described in claim 8, characterized in that, A functional article containing the active ingredient described in any one of claims 5 to 7.

10. The functional article as described in claim 8 or 9, characterized in that, It is at least one of the following: medicine, health product, and food.

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