Fungal polysaccharides, methods for their production and uses thereof

Abstract

The present application relates to the technical field of biological polysaccharide, and particularly relates to a fungal polysaccharide and a preparation method and application thereof. The fungal polysaccharide is extracted from Trametes trogii mycelium, is Trametes trogii mycelium polysaccharide (TGM), is extracted by a composite enzyme high-pressure hot water extraction method, has high purity, small difference between average molecular weights, is at a molecular weight level, has good biological activity, is safe to use, can improve thymus and spleen indexes, increase the proliferation capacity of spleen T and B lymphocytes, improve spleen NK cell activity, increase the number of peripheral white blood cells and lymphocytes, improve the survival rate of spleen lymphocytes, promote the release of a large number of spleen lymphocytes cell inflammatory factors-TNF-alpha, enhance the phagocytosis capacity of mouse peritoneal macrophages, and is used for preparing an immune function regulating product and / or a medicine.

Description

Technical Field

[0001] This invention relates to the field of biopolysaccharide technology, specifically to a fungal polysaccharide, its preparation method, and its uses. Background Technology

[0002] Fungal polysaccharides are active substances isolated from fungal fruiting bodies, mycelia, and fermentation broths. They are natural high-molecular polymers composed of more than 10 monosaccharides mainly linked by β-1,3 and β-1,6 glycosidic bonds. Fungal polysaccharides have numerous applications in the medical and health fields, and are widely believed to possess antioxidant activity and immunomodulatory functions. Numerous studies both domestically and internationally have confirmed that many fungal polysaccharides can enhance human immune function, and some fungal polysaccharides are already used clinically. Research has found that fungal polysaccharides act on lymphocytes, macrophages, and natural killer cells (NK cells), increasing white blood cell count, enhancing the activity of T and B lymphocytes and the killing power of natural killer cells, while also improving the phagocytic capacity of macrophages.

[0003] Chinese patent document CN106916232A discloses a polysaccharide from the mycelium of *Synaps spp.*, its preparation method, and its applications. The polysaccharide obtained has antioxidant and immunomodulatory effects, exhibiting significant activity in scavenging DPPH and hydroxyl radicals. It also protects against DNA damage caused by H2O2 and SH-SY5Y damage to human bone marrow neuroblasts. Furthermore, it promotes the proliferation of spleen lymphocytes without mitogen stimulation and has a synergistic effect on both ConA-induced and LPS-stimulated lymphocyte proliferation in mice, significantly enhancing their biological activity. It can also be used as an antioxidant and immune adjuvant. However, the yield of the prepared polysaccharide is low, its composition is unclear, and its purity is low. Although it has some activity, the activity is not strong.

[0004] The inventors discovered a fungus that parasitizes willow trees, scientifically named *Trametes trogii*, in animal and cell experiments. *Trametes trogii* mycelial polysaccharide (TGM) has significant biological activity. However, due to the deterioration of the natural environment, the scarcity of fungal resources, and the immaturity of fungal polysaccharide extraction methods, fungal polysaccharides are difficult to obtain, with low yields, low purity, or large differences in average molecular weight, making it difficult to detect and apply the activity of fungal polysaccharides. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of low yield, low purity and low activity of fungal polysaccharides in the prior art, thereby providing a fungal polysaccharide and its preparation method, and discovering the technical effect of the fungal polysaccharide in regulating immunity, and providing its use in the preparation of products and / or drugs that enhance immunity.

[0006] This invention discloses a strain of Trametes trogii, TR1, classified as Trametes trogii, with accession number CGMCC No. 19026. The depositary institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101, and the deposit date is November 25, 2019.

[0007] The present invention also discloses a method for isolating and purifying the *Trametes trogii* strain TR1, comprising the following steps: dissecting the fruiting body of *Trametes trogii* strain TR1, taking at least one piece of tissue from the middle of the body, transferring it to a solid culture medium, and after the tissue grows hyphae, picking out colonies for amplification culture.

[0008] Optional, the following steps may be included:

[0009] (1) Preparation of liquid culture medium: Weigh potato glucose broth powder and prepare a liquid culture medium with a mass fraction of 2.5-3.5%. Heat and boil until completely dissolved, dispense into containers, and autoclave at 121℃ for later use.

[0010] (2) Preparation of solid culture medium: Take agar powder and add it to the prepared liquid culture medium. The mass fraction of agar powder in the liquid culture medium containing agar powder is 1.8%-2.0%. After sealing, autoclave at 121℃ for 15-20 minutes. When the temperature drops to 38-42℃ after sterilization, pour it into sterile petri dishes while it is still hot under ultra-clean conditions. The culture medium occupies 1 / 4-1 / 3 of the volume of the petri dish. After cooling and solidification, seal the plate to obtain plate culture medium. Store at 4℃ for later use.

[0011] (3) Isolation and purification of strains: Disinfect the surface of the fruiting body with 75% alcohol under ultra-clean conditions, longitudinally cut open the bacterial body, take one or several pieces of tissue from the middle of the bacterial body, transfer them to a plate culture medium, and incubate at 23-25℃. After the tissue grows uncontaminated hyphae, pick the colonies under aseptic conditions and transfer them to fresh plate culture medium for amplification to obtain strain TR1.

[0012] (4) Preservation of strain: Select strain TR1 and place it in liquid culture medium to obtain mycelial culture medium. Add 1 ml of culture medium containing mycelium to a sterile strain preservation tube containing 0.5 ml of glycerol and store at -80℃.

[0013] The present invention also discloses a fungal polysaccharide extracted from a Trametes trogii strain TR1, wherein the Trametes trogii strain TR1 includes the strain TR1 or the mycelium or a culture medium containing the mycelium.

[0014] Optionally, the fungal polysaccharide is *Terroger's truncatum* mycelial polysaccharide with a molecular weight of 20,000-80,000 Da.

[0015] Optionally, the fungal polysaccharide includes mannose, glucose, and galactose.

[0016] Optional ingredients include idole, inositol, ribose, and sorbitol.

[0017] This invention also discloses a method for preparing the fungal polysaccharide, comprising the following steps:

[0018] (1) Ferment the TR1 strain of Trogectum as described in claim 1, and make the resulting fermentation broth into mycelial dry powder.

[0019] (2) Enzymatic hydrolysis of mycelium: The mycelium dry powder is mixed with water to make a suspension, and 0.8%-1.2% of a compound enzyme is added to the dry powder to make an enzymatic hydrolysate.

[0020] (3) Extraction steps of polysaccharide from mycelium of Trogectella: The enzymatic hydrolysate was extracted at 90-110 kPa pressure and 110-120℃ hot water for 60-120 min, the supernatant was collected by centrifugation, the filter residue was extracted 1-3 times, the supernatants were combined, and the supernatants were concentrated at 60-70℃ under reduced pressure to 20-30% of the original volume and purified.

[0021] Optionally, the complex enzyme includes at least two of cellulase, papain, and pectinase, with an enzymatic hydrolysis temperature of 45-55℃ and a hydrolysis time of 3-5 hours.

[0022] Optionally, the step of extracting polysaccharides from the *Terrogastrocytogenes* mycelium is characterized in that the volume after vacuum concentration is 20%-30% of the volume before concentration.

[0023] Optionally, the purification step includes protein removal and alcohol precipitation;

[0024] The protein removal process includes: removing protein 3-5 times using the Sevage method;

[0025] The alcohol precipitation process includes: adding ethanol to a volume fraction of 70%, allowing it to stand at 4°C, retaining the precipitate, and freeze-drying to obtain TGM dry powder.

[0026] Optionally, in the fermentation step, the inoculum amount of Terogen thrombocytopenia strain TR1 is 5%, and the Terogen thrombocytopenia strain is a suspension containing 20-25g of wet weight of Terogen thrombocytopenia strain TR1 per 200ml of suspension.

[0027] Optionally, in the fermentation step, the culture medium is: glucose broth medium, with an initial pH of 4.5-6.5, a stirring speed of 150-200 rpm in the fermenter, an air flow rate of 3-5 L / min, and a temperature of 23-28℃.

[0028] Optionally, before preparing the mycelium dry powder, a mycelium separation step is also included, in which the fermentation broth is centrifuged at a speed of 7000-9000 rpm for 5-15 min, and the freeze-drying temperature is -50 to -80℃ for 60-80 h.

[0029] Optionally, in the alcohol precipitation step, the volume concentration of ethanol is 92-96%.

[0030] The present invention also discloses the use of the fungal polysaccharide described above or the fungal polysaccharide prepared by the method described above for the preparation of immune function modulation products.

[0031] The present invention also discloses the use of the fungal polysaccharide described above or the fungal polysaccharide prepared by the method described above in the preparation of immune-enhancing products and / or drugs.

[0032] The technical solution of this invention has the following advantages:

[0033] 1. The fungal polysaccharide provided by this invention is extracted from the *Trametes trogii* strain TR1. This strain TR1 has a higher fungal polysaccharide content, with fungal polysaccharides accounting for 75% of the crude polysaccharide mass. The extracted mycelial polysaccharide is *Trametes trogii* mycelial polysaccharide (TGM), with a molecular weight of 20,000-80,000 Da. It has high purity, small differences between average molecular weights, and is within a certain molecular weight range. It has good biological activity and is safe to use.

[0034] 2. The TGM preparation method provided by the present invention adopts a compound enzyme high-pressure hot water extraction method, which yields TGM with high purity and small differences in average molecular weight, all within a uniform molecular weight level, thereby enhancing its biological activity. TGM can be used to prepare immune function modulation products or anti-tumor products and / or drugs.

[0035] 3. The fungal polysaccharide provided by this invention has good safety profile and can improve thymus and spleen indices, increase the proliferation capacity of spleen T and B lymphocytes, enhance spleen NK cell activity, increase the number of peripheral leukocytes and lymphocytes, improve the survival rate of spleen lymphocytes, promote the release of large amounts of cellular inflammatory factor TNF-α from spleen lymphocytes, and enhance the phagocytic capacity of mouse peritoneal macrophages. It can be used to prepare immune function modulation products or anti-tumor products and / or drugs.

[0036] This invention provides a strain TR1 of *Trametes trogii*, classified as *Trametes trogii*, with accession number CGMCC No. 19026. The depository is located at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, on November 25, 2019. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This is a bar chart comparing the purity of crude TGM polysaccharides obtained by the compound enzyme method and the ultrasonic method in Experiment Example 2 of this invention;

[0039] Figure 2 This is a bar chart comparing the average molecular weight of crude TGM polysaccharides obtained by the compound enzyme method and the ultrasonic method in Experiment Example 2 of this invention.

[0040] Figure 3 This is a bar chart comparing the polysaccharide yields of TGM obtained by the compound enzyme method and the ultrasonic method in Experimental Example 2 of this invention.

[0041] Figure 4 This is the GC-MS total ion chromatogram of the acetylated derivatized monosaccharide standard in Experiment Example 3 of this invention;

[0042] Figure 5 This is the total ion chromatogram of TGM monosaccharide acetylation derivatization in Experiment Example 3 of this invention;

[0043] Figure 6 This is a bar chart showing the effect of TGM on the thymus index and spleen index of mice in Experimental Example 5 (5.1) of this invention;

[0044] Figure 7This is a bar chart showing the effect of TGM on the proliferation of mouse spleen lymphocytes in Experimental Example 5 (5.2) of this invention;

[0045] Figure 8 This is a bar chart showing the effect of TGM on the activity of mouse natural killer cells in Experimental Example 5 (5.3) of this invention;

[0046] Figure 9 This is a bar chart showing the effect of TGM on the number of leukocytes and lymphocytes in the peripheral blood of mice in Experimental Example 5 (5.4) of this invention;

[0047] Figure 10 This is a bar chart showing the effect of TGM on the survival rate of mouse spleen lymphocytes in Experimental Example 5 (5.5) of this invention;

[0048] Figure 11 This is a bar chart showing the effect of TGM on the release of TNF-α from mouse splenic lymphocytes in Experimental Example 5 (5.6) of this invention;

[0049] Figure 12 This is a bar chart showing the effect of TGM on the phagocytic capacity of mouse peritoneal macrophages in Experimental Example 5 (5.7) of this invention. Detailed Implementation

[0050] 1.1 Materials and Reagents

[0051] The fruiting bodies of *Trogopterus xanthipes* were collected from willow trees;

[0052] Glucose broth culture medium, Qingdao Rishui Biotechnology Co., Ltd.

[0053] Monosaccharide standards: L(+)-rhamnose, D(+)-xylose, L(+)-arabinose, D(-)-fructose, D-glucuronic acid, D-mannitol, D-(+) glucose, D(+)-galactose, Dr. Ehrenstorfer GmbH, Germany;

[0054] Anhydrous ethanol, n-butanol, chloroform, concentrated sulfuric acid, phenol, 3,5-dinitrosalicylic acid, sodium hydroxide, sodium tartrate tetrahydrate, anhydrous sodium sulfite, Sinopharm Chemical Reagent Co., Ltd.

[0055] 1.2 Instruments

[0056] RE-52AA rotary evaporator;

[0057] SHZ-III circulating vacuum pump;

[0058] FD-1C-50 freeze dryer;

[0059] 752PC UV-Vis spectrophotometer;

[0060] BILON-WSN4B high-precision Ubbelohde viscometer with Ubbelohde viscometer tube;

[0061] Centrifuge;

[0062] JS-3JS-3000 fermentation tank;

[0063] Electronic balance;

[0064] beaker;

[0065] Autoclave;

[0066] Incubator.

[0067] Example 1

[0068] This embodiment discloses a specific implementation method for the collection and isolation of Trametes trogii strain TR1, including the following steps:

[0069] Preparation of liquid culture medium: Weigh 30g of potato glucose broth powder into 1L of deionized water, heat to boiling until completely dissolved, dispense into containers, and autoclave at 121℃ for later use.

[0070] Preparation of solid culture medium: Add 19g of agar powder to a 1L Erlenmeyer flask, pour in the prepared liquid culture medium, seal the flask, and autoclave at 121℃ for 18min; when the temperature of the sterilized culture medium drops to 40℃, pour it into a sterile petri dish while it is still hot in a laminar flow hood, with the liquid filling 1 / 4 of the petri dish volume. After cooling and solidification, seal the flask with plastic wrap and store it at 4℃ for later use.

[0071] Strain isolation: Wipe the surface of the fruiting body with 75% alcohol in a clean bench, cut the bacterial body longitudinally with a scalpel, take one or several pieces of tissue from the middle of the bacterial body, transfer them to a plate culture medium, and incubate at 24°C. After the tissue grows uncontaminated hyphae, pick the colonies under aseptic conditions and transfer them to fresh plate culture medium for amplification to obtain the desired strain TR1.

[0072] Strain preservation: Select strains and place them in liquid culture medium to obtain mycelial culture medium. Add 1 ml of mycelial culture medium to a sterile strain preservation tube containing 0.5 ml of glycerol and store at -80℃. The above-preserved strain was deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, on December 25, 2019.

[0073] Example 2

[0074] This embodiment discloses a specific implementation method for the collection and isolation of Trametes trogii strain TR1, including the following steps:

[0075] Preparation of liquid culture medium: Weigh 25g of potato glucose broth powder into 1L of deionized water, heat to boiling until completely dissolved, dispense into containers, and autoclave at 121℃ for later use.

[0076] Preparation of solid culture medium: Add 19g of agar powder to a 1L Erlenmeyer flask, pour in the prepared liquid culture medium, seal the flask, and autoclave at 121℃ for 15min; when the temperature of the sterilized culture medium drops to 38℃, pour it into a sterile petri dish while it is still hot in a laminar flow hood, with the liquid filling 1 / 3 of the petri dish volume. After cooling and solidification, seal the flask with plastic wrap and store it at 4℃ for later use.

[0077] Strain isolation: Wipe the surface of the fruiting body with 75% alcohol in a clean bench, cut the fungal body longitudinally with a scalpel, take one or several pieces of tissue from the middle of the fungal body, transfer them to a plate culture medium, and incubate at 23°C. After the tissue grows uncontaminated hyphae, pick the colonies under aseptic conditions and transfer them to fresh plate culture medium for amplification to obtain the desired strain.

[0078] Strain preservation: Select strain TR1 and place it in liquid culture medium to obtain mycelial culture medium. Add 1 ml of culture medium containing mycelium to a sterile strain preservation tube containing 0.5 ml of glycerol and store at -80℃.

[0079] Example 3

[0080] This embodiment discloses a specific implementation method for the collection and isolation of Trametes trogii strain TR1, including the following steps:

[0081] Preparation of liquid culture medium: Weigh 35g of potato glucose broth powder into 1L of deionized water, heat to boiling until completely dissolved, dispense into containers, and autoclave at 121℃ for later use.

[0082] Preparation of solid culture medium: Add 19g of agar powder to a 1L Erlenmeyer flask, pour in the prepared liquid culture medium, seal the flask, and autoclave at 121℃ for 20min; when the temperature of the sterilized culture medium drops to 42℃, pour it into a sterile petri dish while it is still hot in a laminar flow hood, with the liquid filling 1 / 3 of the petri dish volume. After cooling and solidification, seal the flask with plastic wrap and store it at 4℃ for later use.

[0083] Strain isolation: Wipe the surface of the fruiting body with 75% alcohol in a clean bench, cut the fungal body longitudinally with a scalpel, take one or several pieces of tissue from the middle of the fungal body, transfer them to a plate culture medium, and incubate at 25°C. After the tissue grows uncontaminated hyphae, pick the colonies under aseptic conditions and transfer them to fresh plate culture medium for amplification to obtain the desired strain TR1.

[0084] Strain preservation: Select strain TR1 and place it in liquid culture medium to obtain mycelial culture medium. Add 1 ml of culture medium containing mycelium to a sterile strain preservation tube containing 0.5 ml of glycerol and store at -80℃.

[0085] Example 4

[0086] This embodiment discloses a method for extracting Troglobacter mycelial polysaccharide (TGM), which is carried out according to the following steps:

[0087] (1) TGM fermentation: The inoculum size of Terogen trefoil strain TR1 (preservation number: CGMCC No.19026) was 5%, the concentration of the strain was 20g / 200ml, the initial pH of the culture medium was 5, the stirring speed of the fermenter was 180r / min, the air flow rate was 4L / min, and the temperature was 25℃. During the fermentation process, the growth status of the mycelium was observed, and the wet mycelium was collected and weighed at regular intervals. When the weight no longer increased, the fermentation was terminated, and the Terogen trefoil mycelium fermentation suspension was obtained.

[0088] (2) Centrifuge the fermentation suspension of Trogectella mycelium at 8000 rpm for 10 min to separate solids and liquids, discard the supernatant, freeze dry at -50℃ for 72 h to obtain mycelium dry powder, and weigh it.

[0089] (3) Mix mycelial dry powder and distilled water at a ratio of 1:4 (g / ml), stir evenly, prepare a suspension, add 1% (by mass of dry powder) of compound enzyme, the compound enzyme including cellulase and papain at a mass ratio of 1:1, and enzymatically hydrolyze for 4 hours under a 50℃ water bath.

[0090] (4) Place the sample treated with the compound enzyme into a 100 kPa sterilizer and extract it under high pressure at 115°C for 80 min; centrifuge again to separate the solid and liquid, retain the supernatant, and repeat the extraction twice on the filter residue, and combine the supernatants; concentrate the extract under reduced pressure at 65°C to 25% of the volume before concentration.

[0091] (5) Remove protein three times using the Sevage method and retain the uppermost solution. The Sevage reagent is prepared by n-butanol and hydrochloric acid in a volume ratio of 4:1. Mix the Sevage reagent with the concentrated extract in (4) in a volume ratio of 1:1, shake vigorously for 30 minutes, and let stand to separate the layers. If emulsification occurs, centrifuge at 3000 rpm for 1 minute to separate the layers. There are three layers in total. Take the uppermost layer.

[0092] (6) Add 3 times the volume of 94% ethanol solution for alcohol precipitation, and the final ethanol volume fraction is 70%. Place in a refrigerator at 4°C overnight; filter, retain the precipitate, and freeze dry to obtain crude fungal polysaccharide dry powder sample.

[0093] Example 5

[0094] This embodiment discloses a method for extracting Troglobacter mycelial polysaccharide (TGM), which is carried out according to the following steps:

[0095] (1) TGM fermentation: The inoculum size of *Terroglossum* strain (preservation number: CGMCC No. 19026) was 5%, the strain concentration was 25g / 200ml, the initial pH of the culture medium was 4.5, the stirring speed of the fermenter was 150r / min, the air flow rate was 3L / min, and the temperature was 28℃. During the fermentation process, the mycelial growth status was observed, and the wet mycelial volume was collected and weighed at regular intervals. When the weight no longer increased, the fermentation was terminated, and *Terroglossum* mycelial fermentation suspension was obtained.

[0096] (2) Polysaccharide extraction by compound enzyme synergistic high pressure hot water extraction: The fermentation suspension of Terrog's mycelium was centrifuged at 7000 rpm for 15 min to separate solid and liquid, the supernatant was discarded, and the mycelium was freeze-dried at -80℃ for 60 h to obtain mycelium dry powder, which was weighed.

[0097] (3) Mix mycelial dry powder and distilled water at a mass ratio of 1:4, stir evenly, prepare a suspension, add 0.8% (by mass of dry powder) of compound enzyme, the compound enzyme including cellulase and pectinase at a mass ratio of 1:1, and enzymatically hydrolyze for 5 hours under a water bath at 45℃.

[0098] (4) Place the sample treated with the compound enzyme into a 90 kPa sterilizer and extract it with high-pressure hot water at 110°C for 120 min; centrifuge again to separate the solid and liquid, retain the supernatant, and repeat the extraction of the filter residue 3 times, and combine the supernatants; concentrate the extract at 60°C under reduced pressure to 30% of the volume before concentration.

[0099] (5) Remove protein three times using the Sevage method and retain the uppermost solution. The Sevage reagent is prepared by n-butanol and hydrochloric acid in a volume ratio of 4:1. Mix the Sevage reagent with the concentrated extract in (4) in a volume ratio of 1:1, shake vigorously for 30 minutes, and let stand to separate the layers. If emulsification occurs, centrifuge at 3000 rpm for 1 minute to separate the layers. There are three layers in total. Take the uppermost layer.

[0100] (6) Add 3 times the volume of 92% ethanol solution for alcohol precipitation, the final ethanol volume fraction is 69%, place in a refrigerator at 4°C overnight; filter, retain the precipitate, freeze dry to obtain crude fungal polysaccharide dry powder sample.

[0101] Example 6

[0102] This embodiment discloses a method for extracting Troglobacter mycelial polysaccharide (TGM), which is carried out according to the following steps:

[0103] (1) TGM fermentation: The inoculum size of Terogen truncatella strain TR1 (preservation number: CGMCC No.19026) was 5%, the concentration of the strain was 22g / 200ml, the initial pH of the culture medium was 6.5, the stirring speed of the fermenter was 200r / min, the air flow rate was 5L / min, and the temperature was 23℃. During the fermentation process, the growth status of the mycelium was observed, and the wet mycelium was collected and weighed at regular intervals. When the weight no longer increased, the fermentation was terminated, and the Terogen truncatella mycelium fermentation suspension was obtained.

[0104] (2) Polysaccharide extraction by compound enzyme synergistic high pressure hot water extraction: The fermentation suspension of Terrog's mycelium was centrifuged at 9000 rpm for 5 min to separate solid and liquid, the supernatant was discarded, and the mycelium powder was freeze-dried at -80℃ for 60 h and weighed.

[0105] (3) Mix mycelial dry powder and distilled water at a ratio of 1:4 (g / ml), stir evenly, prepare a suspension, add 1.2% (by mass of dry powder) of compound enzyme, the compound enzyme including cellulase and papain at a mass ratio of 1:1, and enzymatically hydrolyze for 3 hours under a water bath at 45℃.

[0106] (4) Place the sample treated with the compound enzyme into a 110 kPa sterilizer and extract it under high pressure at 120°C for 60 min; centrifuge again to separate the solid and liquid, retain the supernatant, extract the filter residue once more, and combine the supernatants; concentrate the extract under reduced pressure at 70°C to 20% of the volume before concentration.

[0107] (5) Remove protein three times using the Sevage method and retain the uppermost solution. The Sevage reagent is prepared by n-butanol and hydrochloric acid in a volume ratio of 4:1. Mix the Sevage reagent with the concentrated extract in (4) in a volume ratio of 1:1, shake vigorously for 30 minutes, and let stand to separate the layers. If emulsification occurs, centrifuge at 3000 rpm for 1 minute to separate the layers. There are three layers in total. Take the uppermost layer.

[0108] (6) Add 3 times the volume of 95% ethanol solution for alcohol precipitation, the final ethanol volume fraction is 71%, place in a refrigerator at 4°C overnight; filter, retain the precipitate, freeze dry to obtain crude fungal polysaccharide dry powder sample.

[0109] Example 7

[0110] This embodiment discloses a method for extracting Trogella coarse plug mycelial polysaccharide (TGM), which is carried out according to the following steps:

[0111] (1) TGM fermentation: The inoculum size of the Terogen trefoil strain TR1 (preservation number: CGMCC No.19026) was 5%, the concentration of the strain was 21g / 200ml, the initial pH of the culture medium was 5, the stirring speed of the fermenter was 170r / min, the air flow rate was 3.5L / min, and the temperature was 27℃. During the fermentation process, the growth status of the mycelium was observed, and the wet mycelium was collected and weighed at regular intervals. When the weight no longer increased, the fermentation was terminated, and the Terogen trefoil mycelium fermentation suspension was obtained.

[0112] (2) Polysaccharide extraction by compound enzyme synergistic high pressure hot water extraction: The fermentation suspension of Terrog's mycelium was centrifuged at 8500 rpm for 12 min to separate solid and liquid, the supernatant was discarded, and the mycelium was freeze-dried at -60℃ for 70 h to obtain mycelium dry powder, which was weighed.

[0113] (3) Mix mycelial dry powder and distilled water in a ratio of 1:4 (g / ml), stir evenly, prepare a suspension, add 0.9% (by mass of dry powder) of compound enzyme, the compound enzyme including pectinase, papain and cellulase in a mass ratio of 1:1:1, and enzymatically hydrolyze for 4.5 h in a water bath at 48℃.

[0114] (4) Place the sample treated with the compound enzyme into a 110 kPa sterilizer and extract it with high pressure hot water at 115℃ for 100 min; centrifuge again to separate solid and liquid, retain the supernatant, and repeat the extraction twice with the filter residue, and combine the supernatants; concentrate the extract at 70℃ under reduced pressure to 25% of the volume before concentration.

[0115] (5) Remove protein three times using the Sevage method and retain the uppermost solution. The Sevage reagent is prepared by n-butanol and hydrochloric acid in a volume ratio of 4:1. Mix the Sevage reagent with the concentrated extract in (4) in a volume ratio of 1:1, shake vigorously for 30 minutes, and let stand to separate the layers. If emulsification occurs, centrifuge at 3000 rpm for 1 minute to separate the layers. There are three layers in total. Take the uppermost layer.

[0116] (6) Add 3 times the volume of 96% ethanol solution for alcohol precipitation, the final ethanol volume fraction is 72%, place in a 4°C refrigerator overnight; filter, retain the precipitate, freeze dry to obtain crude fungal polysaccharide dry powder sample.

[0117] Comparative Example 1

[0118] This comparative example provides an ultrasound-assisted hot water extraction method for extracting TGM. The specific operation steps are as follows:

[0119] Trametes trogii strain TR1 (preservation number: CGMCC No. 19026) was prepared according to steps (1)-(2) in Example 4. The mycelial powder was mixed with distilled water at a ratio of 1:10 (g / ml). The mixture was stirred evenly and homogenized to prepare a suspension. The suspension was ultrasonically broken up at 120W for 20 min. Then, an equal volume of distilled water was added to the suspension and the mixture was refluxed in a water bath at 90℃ for 2 h. The solid and liquid components were separated by centrifugation. The precipitate was extracted again once, and the supernatants from both extractions were combined. The extract was concentrated at 65℃ to a volume of 1 / 4 of the original volume. The protein was removed using the Sevage method, as in step (5) of Example 4. The alcohol precipitation was performed as in step (6) of Example 4. Finally, a crude polysaccharide sample was obtained.

[0120] Experimental Example 1

[0121] TGM content determination and molecular weight determination

[0122] (1) TGM content determination: The TGM content is (total sugar content of sample - reducing sugar content). The total sugar content is tested by phenol-concentrated sulfuric acid method, and the reducing sugar is tested by dinitrosalicylic acid method (DNS method).

[0123] (2) Determination of polysaccharide molecular weight by viscosity method: The average molecular weight of crude polysaccharide samples was measured by the Ubbelohde viscometer method, based on the viscosity-average molecular weight.

[0124] The test results are shown in Table 1 below. The crude fungal polysaccharide dry powder sample obtained in Example 4 was divided into two parts and measured separately, as shown in No. 1-1 and 1-2. The crude fungal polysaccharide sample obtained in Example 5 was divided into two parts and measured separately, as shown in No. 2-1 and 2-2. Examples 6 and 7 are shown in No. 3 and No. 4.

[0125] Table 1. Crude polysaccharide content of TGM

[0126] 1-1 900 604.1 67.1 25106.1 1-2 832 587.4 70.6 22191.6 2-1 1102 828.9 75.2 37449.6 2-2 1108 780.8 70.5 42245.2 3 1020 710.6 69.7 42878.3 4 1670 1097.7 65.7 77304.7 Total 6632 4609.6 - -

[0127] Note: *The numbers before the sample number, such as 1- or 2-, indicate the batch of mycelium, while the numbers after the sample number, such as -1 or -2, indicate that the mycelium from the same batch was extracted in multiple samples.

[0128] Experimental Example 2

[0129] A batch of Terrog's mycelial samples was extracted simultaneously using a compound enzyme-assisted high-pressure hot water extraction method and a traditional ultrasound-assisted hot water extraction method for comparison. The mycelial powder was weighed, divided into two equal portions, and extracted using the compound enzyme-assisted high-pressure hot water extraction method as described in Example 4 and the ultrasound-assisted hot water extraction method as described in Comparative Example 1, respectively.

[0130] The purity (percentage of polysaccharides in the crude polysaccharide product) of the crude polysaccharide samples extracted by the two methods was determined by the phenol-concentrated sulfuric acid method, and the molecular weight (viscosity-average molecular weight) was determined by the Ubbelohde viscometer method. The polysaccharide yield of the product (the ratio of net polysaccharide content in the crude polysaccharide product to the mass of mycelial raw material) was calculated. The compound enzyme synergistic high-pressure hot water extraction method and the ultrasonic synergistic hot water extraction method will be referred to as the compound enzyme method and the ultrasonic method, respectively. The experimental results are shown in [Figure number missing]. Figure 1-3 As shown.

[0131] Conclusion: It is evident that the compound enzyme method has advantages over the ultrasonic method in terms of polysaccharide purity and polysaccharide yield. Although the average molecular weight is lower than that of the ultrasonic method, the difference is small and they are at the same molecular weight level. Therefore, the compound enzyme method can improve polysaccharide yield, and the polysaccharide purity in the product is higher. The molecular weight of the product is also comparable to that of the ultrasonic method. Considering all factors, the compound enzyme method is more advantageous for extracting crude polysaccharides.

[0132] Experimental Example 3

[0133] Test for the composition of Trogella truncatella fungal polysaccharide (TGM):

[0134] The monosaccharide composition of TGM was identified using acetylation derivatization-GC-MS. The specific test steps are as follows:

[0135] (1) TGM hydrolysis: Take 10 mg of TGM sample prepared in Example 4 into an ampoule, add 2 mL of 3 mol / L trifluoroacetic acid, seal and hydrolyze at 120℃ for 6 h. Dry the trifluoroacetic acid with nitrogen, add methanol and dry. Add 0.6 mL of 0.05 mol / L NaOH solution to the hydrolysis product, then add 5 mg of NaBH4. React at room temperature for 8-10 h. Add a small amount of acetic acid to decompose the excess NaBH4 until no bubbles are produced. Evaporate the reaction solution to dryness. Wash the reaction product with acidic methanol, evaporate the methanol solution to dryness, repeat 3-4 times to remove borate. Finally, add methanol to dryness and dry in an oven at 105℃ to remove moisture.

[0136] (2) Acetylation derivatization: Take 3 mg of the hydrolyzed monosaccharide, add 0.5 mL of pyridine and 1 mL of acetic anhydride, heat for 2 h, cool, add 2 mL of toluene, dry with N2, add chloroform, wash with distilled water, and dry with N2 to obtain a brownish-yellow product. This acetylated monosaccharide alcohol was diluted with CHCl3 and analyzed by gas chromatography-mass spectrometry.

[0137] (3) Mass spectrometry identification and analysis conditions: HP-5MS quartz capillary column (50m×0.22mm.id, 0.25μm); EI source, electron energy 70eV, mass number scan range 33~550m / z, injection port temperature 260℃, interface temperature 270℃, carrier gas: helium, flow rate 1.2mL / min, split ratio 20:1, temperature program: initial temperature 100℃, increase to 150℃ at 10℃ / min, then increase to 240℃ at 15℃ / min, hold for 40min.

[0138] (4) The seven monosaccharide standards were subjected to acetylation treatment and GC-MS analysis according to the test procedure. The test results are shown in [Figure 1]. Figure 4 As shown, 1 is rhamnose, 2 is xylose, 3 is arabinose, 4 is fructose, 5 is glucuronic acid, 6 is mannose, 7 is glucose, and 8 is galactose. There are also some other impurities in the standard sample spectrum, which are intermediate products and isomers in the derivatization process of the standard sample.

[0139] (5) After hydrolysis and acetylation, TGM was analyzed by GC-MS. The total ion chromatogram of the sample is shown in the figure. Figure 5 As shown in Table 2, the spectra were searched using NIST 11 data and compared with standard spectra. A total of 7 sugar derivatives were identified, of which 3 matched the standard spectra: acetylated derivatives of mannose, glucose, and galactose. The remaining 4 sugar derivatives did not match the standard spectra. The NIST data matching results were for ribose, sorbose, idole, and inositol derivatives. Among these four monosaccharide derivatives, the matching degree for ribose and sorbose was low (38% and 40%, respectively), while the matching degree for idole and inositol derivatives was high (74% and 91%, respectively). These 7 sugar derivatives were relatively quantified based on peak area, with glucose as 100. The monosaccharide composition ratio was ribose: sorbose: idole: mannose: inositol: glucose: galactose = 3:20:12:29:40:100:92.

[0140] Table 2. Determination of TGM Monosaccharide Composition

[0141]

[0142] Note: * indicates qualitative analysis using the NIST database; "**" indicates qualitative analysis using both the NIST 11 database and reference standards.

[0143] Test Example 4 Safety Testing

[0144] Kunming mice (KM mice), male, weighing 30-35g, were randomly divided into a normal group and a TGM group, with 10 mice in each group, and were fed routinely.

[0145] Mice in the TGM group were administered 3.0g of TGM (75.2% fungal polysaccharide content) per kg of body weight by gavage, while mice in the normal group were administered 0.1mL of ultrapure water per 10g of TGM by gavage, once daily for 60 days.

[0146] The weight, mental state, fecal morphology, and mortality of the mice were observed, and the results are shown in Table 3.

[0147] Table 3. Effects of TGM on mouse body weight, mental state, fecal morphology, and mortality.

[0148] normal group 30±3 (starting); 48±5 (ending) good No abnormalities none TGM Group 30±3 (starting); 46±5 (ending) good No abnormalities none

[0149] Experimental Example 5

[0150] 5.1 Effects of TGM on thymus index and spleen index in mice

[0151] Material: TGM, with a TGM content of 75.2%;

[0152] Animals: KM mice, male, weighing 30-35g;

[0153] Instruments and surgical instruments: bench scale, scalpel, scissors, forceps, etc.;

[0154] method:

[0155] (1) Mice were randomly divided into normal group, model group, low-dose TGM group, medium-dose TGM group and high-dose TGM group, with 8 mice in each group. The normal group and model group were given 0.1 mL of ultrapure water / 10 g by gavage once a day for 15 days. The low-dose TGM group was given 150 mg / kg body weight by gavage, the medium-dose TGM group was given 300 mg / kg body weight by gavage, and the high-dose TGM group was given 600 mg / kg body weight by gavage. All were given once a day for 15 days.

[0156] 48 hours and 24 hours before the end of the experiment, mice in the other groups, except for the normal group, were injected intraperitoneally with cyclophosphamide once each at a dose of 40 mg / kg body weight.

[0157] (2) After euthanizing the animal, weigh its body mass, remove the thymus and spleen, weigh them separately, and calculate the thymus and spleen index (thymus / spleen index = thymus or spleen mass / body mass). The results are as follows: Figure 6 As shown;

[0158] Conclusion: The thymus and spleen indices of low, medium and high dose TGM were significantly higher than those of the model group, and showed a certain degree of dose-effect relationship. The p-values ​​marked with * were compared with the normal group, with p < 0.01.

[0159] 5.2 Effects of TGM on the proliferation of mouse splenic lymphocytes

[0160] Materials: TGM (75.2% purity), 96-well culture plate, centrifuge tubes, petri dishes, syringe, scissors, tweezers, 200-mesh sieve;

[0161] Experimental animals: KM mice, male, weighing 30-35g;

[0162] Instruments: benchtop microcentrifuge, DT5-2B low-speed benchtop centrifuge, CO2 constant temperature incubator, SCB-1520 ultra-clean workbench, MF-30 microplate reader, FM40 snowflake ice maker, GR60DA high-pressure steam sterilizer, water bath, inverted microscope.

[0163] Reagents: RPMI-1640 medium, erythrocyte lysis buffer, concanavalin A (Con A), lipopolysaccharides (LPS), fetal bovine serum, PBS buffer, and thiazolyl blue tetrazolium bromide (MTT).

[0164] method

[0165] (1) Preparation of spleen lymphocytes: Animals were euthanized by cervical dislocation, the abdominal villi were removed, and the spleen was immersed in 75% alcohol for 1 min in a clean bench. The spleen was removed by opening the abdomen, washed with PBS, ground on a 200-mesh sieve, and rinsed into a culture dish with pre-cooled medium containing 10% fetal bovine serum. The suspension was centrifuged at 800 rpm for 3 min, the supernatant was discarded, 3 mL of erythrocyte lysis buffer was added, mixed, placed on ice for 3 min, centrifuged at 800 rpm for 3 min, the supernatant was discarded, 3 mL of medium containing 10% fetal bovine serum was added, mixed, centrifuged at 800 rpm for 3 min, the supernatant was discarded, the suspension was resuspended in medium, and stained with trypan blue. The viable cell count was >95%.

[0166] (2) Adjust the mouse spleen cell suspension to 2×10 7 Cells / mL were added to each well of a 96-well flat-bottomed culture plate with 100 μL of cell suspension. Each group had 3 wells. 50 μL of Con A was added to each well, with a final concentration of 10 μg / mL, or 50 μL of LPS was added to each well, with a final concentration of 10 μg / mL. A negative control without Con A and LPS was also included. 50 μL of TGM was added to each well, with final concentrations of 100 μg / mL for the low-dose group, 150 μg / mL for the medium-dose group, and 200 μg / mL for the high-dose group. After culturing for 24 h, 20 μL of 5 mg / mL MTT solution was added, and the cells were cultured for another 4 h. After centrifugation at 5000 r / min for 10 min, the supernatant was discarded, and 150 μL of LDMSO was added. The cells were mixed and dissolved, and the absorbance was measured at 570 nm using a microplate reader.

[0167] T or B lymphocyte proliferation rate = OD of Con A group or LPS group / OD of blank control group × 100%

[0168] The results are as follows Figure 7 As shown;

[0169] Conclusion: The proliferation capacity of spleen T and B lymphocytes in low, medium and high doses of TGM was significantly higher than that in the normal group, showing a certain dose-response relationship. The * indicates p < 0.01 compared with the normal group.

[0170] 5.3 Effects of TGM on mouse NK cell activity

[0171] Reagents and consumables: TGM (75.2% TGM content), RPMI-1640 medium, fetal bovine serum, antibiotics, PBS buffer, Con A, MTT, DMSO, 2-mercaptoethanol (2-ME), 200-mesh sieve, 96-well flat-bottom culture plate, surgical instruments, large syringe core, LDH reagent kit, etc.

[0172] Instruments: CO2 incubator, clean bench, microplate reader, centrifuge, constant temperature water bath.

[0173] Cells and animals: YAC-1 cells (mouse leukemia cells); KM mice, male, 30-35g.

[0174] Drug: Cyclophosphamide.

[0175] Methods: Grouping and administration methods are the same as in 5.1.

[0176] Spleen was aseptically harvested, and spleen cells were prepared into a suspension, with the cell concentration adjusted to 2 × 10⁻⁶. 7 The target cells were YAC-1 cells per mL, which were used as effector cells. YAC-1 cells were passaged as target cells 24 hours before the experiment, and the cell concentration was adjusted to 4 × 10⁶ cells / mL using RPMI-1640 complete culture medium. 5 The target cells were 100 μl each of target cells and effector cells, with an effector cell to target cell ratio of 50:1, and added to a 96-well culture plate. In the target cell natural release well, 100 μl each of target cells and culture medium were added. In the target cell maximum release well, 100 μl each of target cells and 2.5% Triton were added. All of the above were set up in triplicate. The plates were incubated at 37°C in a 5% CO2 incubator for 4 h, centrifuged at 1500 rpm for 5 min, and the supernatant was used to measure LDH activity.

[0177] NK cell activity % = (OD of reaction well - OD of spontaneous release well / OD of maximum release well - OD of spontaneous release well) × 100%

[0178] The results are as follows Figure 8 As shown, the figure shows that the p-value of this group is less than 0.01 compared to the model group.

[0179] Conclusion: The activity of NK cells in the spleen of mice was significantly higher than that in the model group.

[0180] 5.4 Effects of TGM on the number of leukocytes and lymphocytes in peripheral blood of mice

[0181] Reagents and consumables: TGM, with a TGM content of 75.2%;

[0182] Instrument: Complete blood cell analyzer;

[0183] Animals: KM mice, male, 30-35g;

[0184] Drug: Cyclophosphamide.

[0185] method

[0186] (1) Animal grouping and administration are the same as in 5.1.

[0187] (2) Collect venous blood, add anticoagulant, and measure the number of white blood cells and lymphocytes in the whole blood using a whole blood cell analyzer.

[0188] The results are as follows Figure 9 As shown, "*" indicates that the p-value of this group is less than 0.05 compared to the model group, and "**" indicates that the p-value of this group is less than 0.01 compared to the model group.

[0189] Conclusion: Low, medium, and high doses of TGM all antagonized the effect of cyclophosphamide in reducing the total white blood cell count and lymphocyte count in mouse peripheral blood. The effect of increasing the total white blood cell count was more pronounced, suggesting that TGM also has an effect on increasing other types of white blood cells besides lymphocytes.

[0190] Effect of 5.5TGM on the survival rate of mouse spleen lymphocytes

[0191] Materials: TGM (75.2% TGM content), 96-well culture plate, centrifuge tubes, culture dishes, syringe, scissors, tweezers, 200-mesh sieve;

[0192] Experimental animals: KM mice, male, weighing 30-35g;

[0193] Instruments: benchtop microcentrifuge, DT5-2B low-speed benchtop centrifuge, CO2 constant temperature incubator, SCB-1520 ultra-clean workbench, MF-30 microplate reader, FM40 snowflake ice maker, GR60DA high-pressure steam sterilizer, water bath, inverted microscope.

[0194] Reagents: RPMI-1640 medium, erythrocyte lysis buffer, fetal bovine serum, PBS buffer;

[0195] method

[0196] (1) Preparation of spleen lymphocytes is the same as in 5.2.

[0197] (1) Adjust the lymphocyte suspension to 2×10 6 Cells / mL. First, add 100 μl of diluted cell suspension to each well of a 96-well culture plate; add 100 μl of culture medium to the blank group; add 100 μl of Con A solution to the Con A group, with a final concentration of 10 μg / mL; add 100 μl of LPS to the LPS group, with a final concentration of 5 μg / mL; add 50 μL of TGM to the TGM+Con A group, with a final concentration of 200 μg / mL, and add 50 μL of Con A solution, with a final concentration of 10 μg / mL; add 50 μL of TGM to the TGM+LPS group, with a final concentration of 200 μg / mL, and add 50 μL of LPS, with a final concentration of 5 μg / mL. Each group has 3 replicates. Culture for 24 h and count cells by trypan blue staining.

[0198] The results are as follows Figure 10 As shown, the asterisk (*) indicates that the group is compared with Con A / LPS with p < 0.01.

[0199] Experiments showed that nearly 50% of mouse spleen lymphocytes died 24 hours after administration of Con A or LPS, while the cell death rate was significantly reduced after TGM treatment (p < 0.01).

[0200] 5.6 Effects of TGM on TNF-α release from mouse splenic lymphocytes

[0201] Materials: TGM (75.2% TGM content), 96-well culture plate, centrifuge tubes, culture dishes, syringe, scissors, tweezers, 200-mesh sieve;

[0202] Experimental animals: KM mice, male, weighing 30-35g;

[0203] Instruments: benchtop microcentrifuge, DT5-2B low-speed benchtop centrifuge, CO2 constant temperature incubator, SCB-1520 ultra-clean workbench, MF-30 microplate reader, FM40 snowflake ice maker, GR60DA high-pressure steam sterilizer, water bath, inverted microscope.

[0204] Reagents: RPMI-1640 medium, erythrocyte lysis buffer, fetal bovine serum, PBS buffer, TNF-α ELISA kit;

[0205] method:

[0206] (1) Mouse lymphocyte preparation is the same as in 5.2.

[0207] (2) Adjust the lymphocyte suspension to 2×10 6100 μl of TGM solution was added to a 96-well plate. 100 μl of culture medium was added to the blank control group; 100 μl of TGM solution was added to the TGM groups. The final concentrations were: 100 μg / mL for the low-dose group, 150 μg / mL for the medium-dose group, and 200 μg / mL for the high-dose group. Each group was divided into three replicates. After culturing for 24 h, the plates were centrifuged at 1000 rpm for 10 min, and the supernatant was collected. The assay was performed according to the instructions of the mouse TNF-α ELISA kit.

[0208] The results are as follows Figure 11 As shown, the figure shows that p < 0.01 when compared with the normal group.

[0209] Conclusion: Low, medium and high doses of TGM all induced the release of large amounts of the inflammatory cytokine TNF-α from splenic lymphocytes.

[0210] 5.7 Effects of TGM on the phagocytic capacity of mouse peritoneal macrophages

[0211] Instruments: benchtop microcentrifuge, carbon dioxide incubator, SCB-1520 clean bench, MF-30 microplate reader, FM40 snowflake ice maker, DT5-2B low-speed benchtop centrifuge, GR60DA autoclave, water bath.

[0212] Reagents: RPMI-1640 medium, LPS, TNF-α ELISA kit, fetal bovine serum.

[0213] Materials: TGM (75.2% TGM content), 96-well culture plate, syringe;

[0214] Animals: KM mice, male, 30-35g;

[0215] method

[0216] (1) The mice were euthanized by pulling their necks, the hair on their abdomen was shaved, the abdomen was disinfected by spraying with 75% alcohol, the peritoneum was opened and exposed, 8 mL of pre-cooled RPMI-1640 culture medium was injected into the peritoneum, the mouse abdomen was gently massaged for 5 min, and the irrigation fluid was aspirated into a pre-cooled centrifuge tube.

[0217] (2) Centrifuge at 800 r / min for 5 min, discard the supernatant, wash with 5 mL of pre-cooled cell culture medium, resuspend the cells, stain with trypan blue to count, and then seed the cell suspension into a 96-well culture plate, 200 μl per well, incubate for 24 h, gently shake the culture plate, discard the supernatant, rinse the culture wells twice with cell culture medium preheated at 37℃, and the remaining adherent cells are macrophages.

[0218] (3) Add TGM to a final concentration of 200 μg / mL. A normal control group without TGM was also included. After culturing for 24 h, the supernatant was discarded, and the cells were washed twice with PBS. 100 μl of neutral red solution was added to each well, and the cells were cultured for another 3 h. The supernatant was discarded, and 100 μl of cell lysis buffer was added to each well. The cells were incubated at room temperature for 2 h until complete lysis. The OD value at 540 nm was measured using a microplate reader. Results Figure 12 As shown, the TGM group was compared with the normal group by p < 0.01.

[0219] Conclusion: TGM significantly enhances the phagocytic capacity of mouse peritoneal macrophages.

[0220] Obviously, the above embodiments and experimental examples are merely illustrative and not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of Troglobacter ( Trametes trogii strain TR1, characterized in that, The accession number is: CGMCCNo.19026.

2. The Trogectatus strain according to claim 1 ( Trametes trogii The method for isolating and purifying strain TR1 includes the following steps: Dissecting the Trog thrombus ( Trametes trogii The fruiting bodies of strain TR1 were obtained by taking at least one piece of tissue from the middle of the body, transferring it to a solid culture medium, and then picking out colonies for amplification culture after mycelium had grown from the tissue.

3. The Trogectatus tuberculosis strain according to claim 2 ( Trametes trogii A method for isolating and purifying strain TR1, characterized in that, Includes the following steps: (1) Preparation of liquid culture medium: Weigh potato glucose broth powder and prepare a liquid culture medium with a mass fraction of 2.5-3.5%. Heat and boil until completely dissolved, dispense into containers, and autoclave at 121℃ for later use. (2) Preparation of solid culture medium: Take agar powder and add it to the prepared liquid culture medium. The mass fraction of agar powder in the liquid culture medium containing agar powder is 1.8%-2.0%. After sealing, autoclave at 121℃ for 15-20 min. When the temperature drops to 38-42℃ after sterilization, pour it into sterile petri dishes while it is still hot under ultra-clean conditions. The culture medium occupies 1 / 4-1 / 3 of the volume of the petri dish. After cooling and solidification, seal the plate to obtain plate culture medium. Store at 4℃ for later use. (3) Isolation and purification of strains: Disinfect the surface of the fruiting body of *Terroger's truncatula* with 75% alcohol under ultra-clean conditions, longitudinally cut open the fungal body, take one or several pieces of tissue from the middle of the fungal body, transfer them to a plate culture medium, and incubate at 23-25℃. After the tissue grows uncontaminated hyphae, pick the colonies under aseptic conditions and transfer them to fresh plate culture medium for amplification to obtain strain TR1. (4) Preservation of strain: Select strain TR1 and place it in liquid culture medium to obtain mycelial culture medium. Add 1 ml of culture medium containing mycelium to a sterile strain preservation tube containing 0.5 ml of glycerol and store at -80℃.

4. A method for preparing fungal polysaccharides, characterized in that, Includes the following steps: (1) Ferment the TR1 strain of Trogectum as described in claim 1, and make the resulting fermentation broth into mycelial dry powder; (2) Enzymatic hydrolysis of mycelium: The mycelium dry powder is mixed with water to make a suspension, and a compound enzyme of 0.8%-1.2% of the dry powder mass is added to make an enzymatic hydrolysate; (3) Extraction steps of Trogella mycelium polysaccharide: The enzymatic hydrolysate is extracted at 90-110 kPa pressure and 110-120℃ hot water for 60-120 min, centrifuged and the supernatant is collected. The filter residue is extracted 1-3 times, the supernatants are combined, and the mixture is concentrated under reduced pressure at 60-70℃ to 20-30% of the original volume and purified. The complex enzyme includes at least two of cellulase, papain and pectinase. The enzymatic hydrolysis temperature is 45-55℃ and the enzymatic hydrolysis time is 3-5 h.

5. The method for preparing fungal polysaccharides according to claim 4, characterized in that, In the process of extracting polysaccharides from the mycelium of Trogectella esculenta, the volume after vacuum concentration is 20%-30% of the volume before concentration.

6. The method for preparing fungal polysaccharides according to any one of claims 4-5, characterized in that, The purification steps include protein removal and alcohol precipitation; The protein removal process includes: removing protein 3-5 times using the Sevage method; The alcohol precipitation process includes: adding ethanol to a volume fraction of 70%, allowing it to stand at 4°C, retaining the precipitate, and freeze-drying to obtain TGM dry powder.

7. The method for preparing fungal polysaccharides according to claim 4, characterized in that, In the fermentation step, the inoculum amount of Trogectatus strain TR1 is 5%, and the Trogectatus strain is a suspension, with each 200ml suspension containing 20-25g of wet weight of Trogectatus strain TR1.

8. The method for preparing fungal polysaccharides according to claim 4 or 5, wherein in the fermentation step, the culture medium is: glucose broth medium, the initial pH of the culture medium is 4.5-6.5, the stirring speed of the fermenter is 150-200 rpm, the air flow rate is 3-5 L / min, and the temperature is 23-28℃.

9. The method for preparing fungal polysaccharides according to any one of claims 4-5, characterized in that, Before the mycelium powder is prepared, a mycelium separation step is also included, in which the fermentation broth is centrifuged at a speed of 7000-9000 rpm for 5-15 min, and the freeze-drying temperature is -50~-80℃ for 60-80 h.

10. The method for preparing fungal polysaccharides according to claim 6, characterized in that, In the alcohol precipitation step, the volume concentration of ethanol is 92-96%.

11. Use of the fungal polysaccharide prepared by the method of any one of claims 4-10 for the preparation of immune function modulatory products.

12. Use of the fungal polysaccharide prepared by the method of any one of claims 4-10 in the preparation of immune-enhancing products and / or pharmaceuticals.

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