A strain of grifola frondosa gtr2, its culture method and application

Abstract

The application discloses a strain of Wolfiporia hoelen GTR2, a culture method and application thereof, and belongs to the technical field of microorganisms. The strain of Wolfiporia hoelen GTR2 is preserved in the China General Microbiological Culture Collection Center on July 2, 2024, and the preservation number is CGMCC NO.41354. The culture medium and culture conditions of the strain are also disclosed. The strain has high polyphenol and flavonoid production and strong antioxidant capacity in a liquid fermentation process, and lays an important foundation for related pharmacological research and antioxidant product development.

Description

Technical Field

[0001] This invention relates to a strain of Poria cocos GTR2 (Wolfiporia hoelen), its culture method and application, belonging to the field of microbial technology. Background Technology

[0002] Poria cocos (Wolfiporia hoelen (Fr.) YCDai & V.Papp) belongs to the genus Polyporaceae of the order Polyporaceae. The Pharmacopoeia of the People's Republic of China stipulates that Poria cocos is used medicinally as its sclerotium. It is sweet and bland in taste, and neutral in nature, primarily used for edema with scanty urine, phlegm retention with dizziness and palpitations, spleen deficiency with poor appetite, loose stools and diarrhea, restlessness, palpitations, and insomnia. Poria cocos was included in the National List of Food and Medicine Homologous in 2002 and is also an important food source. The secondary metabolites of Poria cocos mainly include polysaccharides, flavonoids, sterols, polyphenols, and triterpenoids, which have anti-tumor, antioxidant, lipid-lowering, blood sugar-lowering, intestinal flora-regulating, and immune-enhancing effects, making it highly valuable for both food and medicine.

[0003] Currently, the regions with large-scale artificial cultivation production include Hunan, Hubei, Guizhou, Yunnan, Fujian, and Anhui. Among them, Jingzhou Miao and Dong Autonomous County in Hunan Province is the largest Poria cocos trading center in China. Jingzhou has a long history of Poria cocos cultivation; the first cultivable Poria cocos strain, 5.78, was isolated from wild Poria cocos collected in Jingzhou. Poria cocos mostly grows on the roots of pine plants and is suitable for cultivation at mid-altitudes, in cool, dry climates, with sandy soil and slopes of 10-35°. The main substrates for artificial cultivation of Poria cocos are Pinus massoniana and Pinus slashani, among other Pinaceae plants. In recent years, due to the impact of pine wilt disease, many artificially cultivated Pinus massoniana forests have been infected with pine wilt nematodes. According to the "Management Measures for Pine Wilt Disease Epidemic Areas and Infected Trees" issued by the National Forestry and Grassland Administration, Pinaceae plants in pine wilt disease epidemic areas can only be harvested for eradication purposes, and the harvested infected trees must be crushed (chipped) or burned on-site. This has, to some extent, affected the source of substrates for artificially cultivated Poria cocos. The area and yield of artificially cultivated Poria cocos have been affected, resulting in significant market fluctuations and a supply shortage.

[0004] Flavonoids are a broad category of compounds consisting of two benzene rings (A- and B-rings) with phenolic hydroxyl groups linked by a central three-carbon atom. Their basic parent nucleus is 2-phenylchromone. Based on the degree of oxidation of the central three-carbon chain, the position of the B-ring (2- or 3-position), and whether the three-carbon chain forms a ring, the main natural flavonoids can be classified into fifteen types: flavones, flavonols, flavonones, flavonols, anthocyanidins, flavan-3,4-diols, xanthones, chalcones, and biflavonoids. Flavonoids and polyphenols are important secondary metabolites of Poria cocos, which have strong antioxidant effects. They can effectively remove free radicals in the body, prevent cell degeneration and aging, improve blood circulation, lower cholesterol, and lower blood sugar, and have high medicinal value. Summary of the Invention

[0005] One objective of this invention is to provide a high-yield strain of Poria cocos GTR2 (Wolfiporia hoelen) that produces flavonoids and polyphenols, aiming to efficiently obtain flavonoids and polyphenols through liquid fermentation, laying an important foundation for related pharmacological research and health product development. The scientific name of the Poria cocos GTR2 strain is *Wolfiporia hoelen* (Fr.) YCDai & V. Papp, with accession number CGMCC NO.41354. This Poria cocos GTR2 strain was deposited on July 2, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The second objective of this invention is to provide a liquid culture method for a poria cocos strain GTR2 (Wolfiporiahoelen) that produces high levels of flavonoids and polyphenols. The method includes inoculating the poria cocos strain GTR2 into a solid culture medium for activation, and then inoculating the activated strain into a liquid culture medium for constant temperature culture until the fermentation endpoint.

[0007] Preferably, it includes the following steps:

[0008] Step (1) Inoculate the Poria cocos strain GTR2 (Wolfiporia hoelen) onto a solid culture medium, activate it, and incubate it in a constant temperature incubator at 23℃ for 6 days; the solid culture medium formula is: peeled potato 100-300g / L (for example, it can be 120g / L, 140g / L, 160g / L, 180g / L, 200g / L, 220g / L, 240g / L, 260g / L, 280g / L, etc.), glucose 10.0-30.0g / L (for example, it can be 12g / L, 14g / L, 16g / L, 18g / L, 20g / L). 22g / L, 24g / L, 26g / L, 28g / L, etc.), agar 15.0-25.0g / L (e.g., 17g / L, 20g / L, 22g / L, 24g / L, etc.), potassium dihydrogen phosphate 0.1-4.0g / L (e.g., 0.2g / L, 0.5g / L, 1g / L, 1.5g / L, 2g / L, 2.5g / L, 2.8g / L, 3.0g / L, 2.8g / L, 3.0g / L, 3.5g / L, 3.8g / L, etc.), pH 6.0-6.8 (e.g., 6.2, 6.4, 6.6, 6.7, etc.).

[0009] Preferably, the solid culture medium comprises 200.0 g / L peeled potato, 20.0 g / L glucose, 20.0 g / L agar, 1.0 g / L potassium dihydrogen phosphate, and pH 6.5.

[0010] Step (2) Inoculate the activated strain onto the basic liquid culture medium and culture it in a constant temperature shaker at 20-28℃ (e.g., 22℃, 24℃, 26℃, etc.) and 100-200r / min (e.g., 120r / min, 140r / min, 160r / min, 180r / min) for 14 days until the fermentation is complete. The liquid culture medium includes glucose at 10.0-40.0 g / L (e.g., 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L, 34 g / L, 36 g / L, 38 g / L, etc.), peptone at 1.0-10.0 g / L (e.g., 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, etc.), and yeast extract at 0.5-5.0 g / L (e.g., 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, etc.). Potassium dihydrogen phosphate (0.1-5.0 g / L, e.g., 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, etc.), magnesium sulfate heptahydrate (0.1-5.0 g / L, e.g., 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, etc.), vitamin B1 (0.1-10) × 10 -3 g / L (for example, it can be 0.2×10 g / L) -3 g / L, 0.5×10 -3 g / L, 1 g / L, 2×10 -3 g / L, 3×10 -3 g / L, 4×10 - 3 g / L, 5×10 -3 g / L, 6×10 -3 g / L, 7×10 -3 g / L, 8×10 -3 g / L, 9×10 -3 (g / L, etc.), pH 6.0-6.8 (e.g., 6.2, 6.4, 6.6, 6.7, etc.).

[0011] Preferably, the liquid culture medium comprises: glucose 10-20 g / L, peptone 2.0-6.0 g / L, yeast extract 1.0-3.0 g / L, potassium dihydrogen phosphate 1.0-2.0 g / L, magnesium sulfate heptahydrate 0.5-2.0 g / L, and vitamin B1 (0.5-2.0) × 10⁻⁶. -3g / L, pH 6.3-6.6; liquid culture constant temperature shaker conditions are 22-25℃, 150-180r / min.

[0012] More preferably, the liquid culture medium comprises 15.0 g / L glucose, 5.0 g / L peptone, 2.0 g / L yeast extract, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and 1.0 × 10⁻⁶ vitamin B1. -3 g / L, pH 6.5, liquid culture constant temperature shaker conditions: 23℃, 170r / min.

[0013] All other specific point values ​​within the above numerical ranges can be selected.

[0014] A third objective of this invention is to provide a fermentation broth obtained by culturing and isolating the *Wolfiporia hoelen* strain GTR2. The fermentation broth is obtained by filtering out the *Wolfiporia hoelen* mycelium after fermentation and collecting the clear liquid. Conventional methods in the art, such as using double-layered sterile filter paper and sterile test tubes, can be employed to collect the fermentation broth.

[0015] Preferably, the fermentation broth does not contain mycelium of the Poria cocos strain GTR2 (Wolfiporia hoelen).

[0016] A fourth object of the present invention is to provide a biological agent comprising the fermentation broth of the *Wolfiporia hoelen* strain GTR2. The biological agent contains flavonoids and polyphenols.

[0017] Preferably, the fermentation broth contains more than 0.08 g / L of flavonoids and more than 0.01 g / L of polyphenols.

[0018] Preferably, the fermentation broth contains flavonoids greater than 0.1 g / L and polyphenols greater than 0.05 g / L.

[0019] A fourth objective of this invention is to provide the application of the biological agent in food, health products, and pharmaceuticals.

[0020] A fourth objective of this invention is to provide the application of the aforementioned Poria cocos strain GTR2 (Wolfiporia hoelen) in the preparation of high-yield flavonoids and polyphenols through fermentation.

[0021] The beneficial effects of this invention are as follows: The extracellular flavonoid and polyphenol yields of the Poria cocos strain GTR2 are significantly higher than those of other strains, and it exhibits strong antioxidant capacity. This invention provides a new Poria cocos strain, a culture medium, and a culture method. After liquid fermentation, the cultured Poria cocos strain has high levels of bioactive components such as flavonoids and polyphenols, resulting in high medicinal value. It can be used to prepare antioxidant foods or health products.

[0022] This invention verifies that using liquid fermentation to obtain extracellular flavonoids and polyphenols from Poria cocos is reasonable and feasible. It can overcome the limitations of long growth cycle and susceptibility to environmental factors such as temperature, moisture, and pests in artificial cultivation of Poria cocos. Furthermore, extracellular secondary metabolites may be effectively secreted into the fermentation broth. Poria cocos flavonoids and polyphenols can be obtained through methods such as filtration, centrifugation, precipitation, extraction, separation, and purification, thereby meeting the market demand for the development of Poria cocos secondary metabolite products. Attached Figure Description

[0023] Figure 1 This is the phylogenetic NJ tree of the *Poria cocos* GTR2 bacterium from Example 1.

[0024] Figure 2 This is a diagram of the hyphae morphology of Poria cocos GTR2 in Example 1. Detailed Implementation

[0025] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the invention and should not be considered as specific limitations thereof. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0026] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not limited to those elements and may also include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0027] The indefinite articles “a” and “an” preceding an element or component of this invention do not impose any limitation on the quantity (i.e., number of times) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0028] The terms "one embodiment," "some embodiments," "exemplary," "specific example," or "some examples," etc., used in this invention refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this document, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example.

[0029] Example 1: Acquisition and identification of strains

[0030] 1.1 Strains Isolation

[0031] Strain isolation: Fresh, intact Poria cocos sclerotia were collected as the material for strain isolation. On a clean bench, the sclerotia were cut open with a scalpel, and the tissue blocks were carefully removed and inoculated into PDA plates. The plates were incubated at 23°C in the dark for 5 days. When white hyphae appeared around the tissue blocks, the pure strain was inoculated onto PDA slant agar plates using aseptic techniques. The plates were incubated at 23°C for 7-10 days. Tubes with well-developed hyphae and no contamination were then stored at 4°C.

[0032] Characteristics of this strain: On general PDA medium, the mycelium of this Poria cocos strain is white, robust, radial, grows rapidly, has strong vigor, and possesses a distinctive aroma. Figure 2 As shown.

[0033] The culture characteristics of this strain are as follows: After about 6 days of cultivation on PDA medium (potato dextrose agar), the mycelium completely covers the entire petri dish (90 mm in diameter). White hyphae are visible on the surface of the petri dish, radiating outwards. The hyphae are thick, dense, and highly vigorous, with a distinctive aroma. In the later stages of cultivation, the hyphae age and turn light brown.

[0034] 1.2 Strain Identification

[0035] Poria cocos test tube strains were selected, and fungal genomic DNA extraction kits (catalog number: D2300) produced by Beijing Solarbio Biotechnology Co., Ltd. were used to extract DNA from Poria cocos.

[0036] The specific extraction method is as follows:

[0037] Step 1: Take about 100mg of Poria cocos mycelium, pour in an appropriate amount of liquid nitrogen, grind immediately and repeat 3 times to grind the mycelium into powder, add 200μL of solution A, add 20μL of RNase A, and then add 100mg of glass beads, and shake on a high-speed shaker for 5 minutes.

[0038] Step 2: Add 20 μL of proteinase K (10 mg / mL), mix thoroughly, and digest in a 55°C water bath for 30 min. During digestion, the centrifuge tube can be inverted several times to mix. Centrifuge at 12000 rpm for 2 min. Transfer the supernatant to a new centrifuge tube.

[0039] Step 3: Add 200 μL of solution B to the supernatant and mix thoroughly. Incubate in a 55°C water bath for 5 minutes until the precipitate disappears and the solution becomes clear.

[0040] Step 4: Add 200 μL of anhydrous ethanol, mix thoroughly, and add both the solution and flocculent precipitate into the adsorption column. Let stand for 2 minutes.

[0041] Step 5: Centrifuge at 12000 r / min for 1.0 min, discard the waste liquid, and place the adsorption column into the collection tube.

[0042] Step 6: Add 600 μL of washing solution (anhydrous ethanol should have been added before use) to the adsorption column, centrifuge at 12000 r / min for 1.0 min, discard the waste liquid, and place the adsorption column into the collection tube.

[0043] Step 7: Add 600 μL of washing solution to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube. Centrifuge at 12000 rpm for 2 min, and place the adsorption column in a 50℃ incubator for 5 min.

[0044] Step 8: Place the adsorption column into a clean centrifuge tube, add 100 μL of preheated elution solution (preheated in a 65°C water bath) to the center of the adsorption membrane, let it stand at room temperature for 5 min, and centrifuge at 12000 r / min for 1 min.

[0045] Step 9: Add the elution buffer obtained by centrifugation back into the adsorption column, let it stand at room temperature for 2 min, centrifuge at 12000 r / min for 2 min, collect the DNA solution, and store the DNA at -20℃.

[0046] The ITS sequence was extracted from the genomic DNA of Poria cocos using universal fungal primers and amplified by PCR. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific sequences are as follows:

[0047] ITS1(5'-TCCGTAGGTGAACCTGCGG-3')

[0048] ITS4(5'-TCCTCCGCTTATTGATATGC-3')

[0049] PCR amplification reaction system:

[0050] Reaction components Reaction volume (50 μL) <![CDATA[ddH20(μL)]]> 15.00 <![CDATA[10×Buffer (without Mg 2+ )]]> 5.00 <![CDATA[MgCl2(mmoI / L)]]> 2.50 dNTPs (mmol / L) 1.00 Tag enzyme U / μL 2.00 Primer (μmol / L) 10.00 DNA (ng / L) 15.00

[0051] The PCR amplification procedure is as follows:

[0052] Preheat at 95℃ for 5 minutes;

[0053] 94℃ denaturation for 30s; 55℃ annealing for 30s; 72℃ extension for 30s; 30 cycles;

[0054] Extend at 72℃ for 5 minutes;

[0055] After PCR amplification, the sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were then aligned using BLAST on the National Center for Biotechnology Information website. Sequences with high similarity after alignment were selected, and phylogenetic trees were constructed using the Neighbor-Joining (NJ) method in MEGA 7.0 software. The results are attached. Figure 1 .from Figure 1 It is known that the *Poria cocos* strain GTR2 and *Poria cocos* Wolfiporia hoelen OQ613233.1 belong to the same phylogenetic branch, have a short genetic distance, and are closely related in evolution. The isolated strain GTR2 is identified as *Poria cocos* Wolfiporia hoelen. This strain is named *Poria cocos* strain GTR2 and was deposited on July 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNO.41354; the address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0056] Example 2: Strain culture and fermentation broth preparation

[0057] The Poria cocos GTR2 (Wolfiporia hoelen) strain was picked from the test tube, inoculated onto a solid culture medium, activated, and cultured in a constant temperature incubator at 23°C for 6 days.

[0058] The solid culture medium consisted of: 200.0 g / L peeled potato, 20.0 g / L glucose, 20.0 g / L agar, 1.0 g / L potassium dihydrogen phosphate, and pH 6.5.

[0059] The activated strain was inoculated onto a basic liquid culture medium and cultured in a constant temperature shaker at 23℃ and 170 rpm for 14 days. After fermentation, the Poria cocos mycelium was filtered out using double-layer sterile filter paper, and the fermentation broth was collected in sterile test tubes for direct use in the determination of Poria cocos metabolites.

[0060] The liquid culture medium formula is as follows:

[0061] Element Formula A Formula B Formula C Formula D Formula E Formula F glucose 10 40 15 20 8 50 peptone 10 1 5 8 12 12 Yeast extract 0.5 5 2 4 6 0.2 Potassium dihydrogen phosphate 5 0.1 1 2 6 0.01 Magnesium sulfate heptahydrate 0.1 5 0.5 1 0.01 0.01 Vitamin B1 <![CDATA[10.0×10 -3 ]]> <![CDATA[0.1×10 -3 ]]> <![CDATA[1.0×10 -3 ]]> <![CDATA[5.0×10 -3 ]]> <![CDATA[20×10 -3 ]]> <![CDATA[0.01×10 -3 ]]> pH 6.0 6.8 6.5 6.2 5.8 5.6

[0062] Example 3: Determination of Flavonoid Content

[0063] The study used *Poria cocos* strain GTR2 and six control strains (numbered FLA, FGD, FGZ, FHB, XJ28, and F5.78) as research subjects. Strain XJ28 was purchased from the *Poria cocos* Professional Cooperative in Jingzhou Miao and Dong Autonomous County, Hunan Province; strain F5.78 was purchased from the Shanghai Center for Tibetan Biotechnology Preservation; and strains FLA, FGD, FGZ, and FHB were preserved at the Hunan Provincial Center for the Conservation and Utilization of Mycorrhizal Edible Fungi Germplasm Resources. Using the culture method and formulation described in Example 2, the extracellular metabolic test solutions of 12 *Poria cocos* strains were obtained after culturing.

[0064] The flavonoid content was determined using the aluminum trichloride method, a conventional method in this field. 1.0 mL of the test solution was accurately pipetted into a 10 mL volumetric flask, and 2.0 mL of aluminum trichloride solution and 3.0 mL of potassium acetate solution were added. The mixture was then diluted to the mark with methanol solution, shaken well, and incubated at room temperature for 30.0 min. The solution was then centrifuged at 4000 r / min for 10 min, and the absorbance was measured at 420 nm. Using rutin as a standard, the rutin contents in the standard curve were 0.0125 mg / L, 0.0250 mg / L, 0.0500 mg / L, 0.100 mg / L, 0.150 mg / L, and 0.200 mg / L, respectively. The rutin content was plotted on the x-axis, and the absorbance on the y-axis. The regression equation was calculated as: y = 3.9005x - 0.0058, with a correlation coefficient R0. 2 =0.9995. Calculate the flavonoid content of Poria cocos based on the standard curve.

[0065] Table 1 shows that the flavonoid production capacity of different Poria cocos strains varied (P = 0.05). The Poria cocos GTR2 strain had the highest flavonoid production, reaching 0.113 g / L, significantly higher than the other six strains. The extracellular flavonoid contents of Poria cocos FLA, FHB, FGZ, and XJ28 strains were next, ranging from 0.043 to 0.049 g / L, with no significant differences among them. The Poria cocos FGD strain had the lowest flavonoid content, at 0.019 g / L.

[0066] Table 1 Comparison of flavonoid production capacity of different Poria cocos strains

[0067]

[0068]

[0069] Note: Different letters indicate significant differences, while the same letter indicates no significant differences. The same applies to the following table.

[0070] This example demonstrates that, after liquid culture, the Poria cocos strain GTR2 has the highest flavonoid content in its fermentation broth compared to the other six strains, indicating that this strain has the ability to produce high levels of extracellular flavonoids.

[0071] Example 4: Determination of Polyphenol Content

[0072] The study used *Poria cocos* strain GTR2 and six other *Poria cocos* strains (numbered FLA, FGD, FGZ, FHB, XJ28, and F5.78). Strain XJ28 was purchased from the *Poria cocos* Professional Cooperative in Jingzhou Miao and Dong Autonomous County, Hunan Province; strain F5.78 was purchased from the Shanghai Center for Tibetan Biotechnology; and strains FLA, FGD, FGZ, and FHB were preserved at the Hunan Provincial Center for the Conservation and Utilization of Mycorrhizal Edible Fungi Germplasm Resources. Using the culture method and culture medium formulation described in Example 2, the extracellular metabolites of the 12 *Poria cocos* strains were cultured separately to obtain test solutions.

[0073] The polyphenol content of Poria cocos was determined using the Folin-Ciocalteu colorimetric method, a conventional method in this field. 1.00 mL of the Poria cocos test solution was pipetted, and 1.0 mL of FC colorimetric reagent and 3.0 mL of 7.5% sodium carbonate solution were added. The volume was then diluted to 25.0 mL with distilled water, mixed, and allowed to develop at room temperature for 60.0 min. The absorbance was measured at 765 nm. Gallic acid was used as a standard, and the concentrations of gallic acid in the standard curve were 0.0250 mg / L, 0.0500 mg / L, 0.100 mg / L, 0.200 mg / L, 0.300 mg / L, and 0.400 mg / L, respectively.

[0074] A standard curve was plotted with gallic acid content on the x-axis and absorbance on the y-axis. The regression equation was calculated as: y = 3.8313x + 0.0190, and the correlation coefficient R0 was 1. 2 =0.9985. Calculate the polyphenol content of Poria cocos based on the standard curve.

[0075] Table 2 shows that there were significant differences in polyphenol content among different strains of Poria cocos (P = 0.05). The GTR2 strain had the highest polyphenol content, reaching 0.206 g / L, significantly higher than the other six strains. The FLA, XJ28, F5.78, FGZ, and FHB strains had the next highest total extracellular phenol content, ranging from 0.080 to 0.091 g / L, with no significant differences among them. The FGD strain had the lowest polyphenol content, only 0.064 g / L, significantly lower than the other six strains.

[0076] Table 2 Comparison of polyphenol production capacity of different Poria cocos strains

[0077]

[0078] This example demonstrates that, after liquid culture, the Poria cocos strain GTR2 has the highest polyphenol content in its fermentation broth compared to the other six strains, indicating that this strain has the ability to produce high levels of extracellular polyphenols.

[0079] Example 5 Antioxidant Capacity Determination

[0080] The study used *Poria cocos* strain GTR2 and six other *Poria cocos* strains (numbered FLA, FGD, FGZ, FHB, XJ28, and F5.78). Strain XJ28 was purchased from the *Poria cocos* Professional Cooperative in Jingzhou Miao and Dong Autonomous County, Hunan Province; strain F5.78 was purchased from the Shanghai Center for Tibetan Biotechnology Preservation; and strains FLA, FGD, FGZ, and FHB were preserved at the Hunan Provincial Center for the Conservation and Utilization of Mycorrhizal Edible Fungi Germplasm Resources. Using the culture method and culture medium formulation described in Example 2, the extracellular metabolites of the 12 *Poria cocos* strains were cultured and analyzed.

[0081] The antioxidant activity of seven Poria cocos strains was evaluated by the DPPH· free radical scavenging rate of the fermentation broth. The specific method is as follows: accurately measure 1.00 mL of the Poria cocos test solution, and then add 4.00 mL of 0.24 mmol·L⁻¹. -1 After mixing the DPPH solution, react for 20.0 min and then measure the absorbance at 516 nm. Anhydrous ethanol was used instead of DPPH solution as the sample background, and dimethyl sulfoxide was used as a blank control. The scavenging rate was calculated according to formula (1).

[0082]

[0083] In the formula: C is the free radical scavenging rate; A0 is the absorbance of the blank control; A i A represents the absorbance of the experimental group; j The absorbance is the background absorbance of the sample.

[0084] Table 3 Comparison of DPPH· free radical scavenging ability of different Poria cocos strains

[0085]

[0086] Table 3 shows that the extracellular metabolites of different Poria cocos strains exhibited significant differences in their DPPH· free radical scavenging ability (P = 0.05). The Poria cocos GTR2 strain showed the highest DPPH· free radical scavenging rate, reaching 44.60%, significantly higher than the other seven strains. The Poria cocos F5.78, FLA, FHB, XJ28, and FGZ strains showed good DPPH· free radical scavenging rates, ranging from 19.11% to 22.68%, with no significant differences among them. The Poria cocos FGD strain showed the lowest DPPH· free radical scavenging rate, at 14.17%, significantly lower than the other six strains.

[0087] This embodiment demonstrates that the fermentation broth of Poria cocos strain GTR2 has a strong scavenging effect on DPPH free radicals and exhibits strong antioxidant activity, which can be further developed into a health food or medicine with antioxidant effects.

[0088] The fermentation broth of the *Wolfiporia hoelen* GTR2 strain of this invention has high flavonoid and polyphenol content, indicating that the *Wolfiporia hoelen* GTR2 strain of this invention has high medicinal value and can be used to prepare drugs with antioxidant effects in food or health products, or to prepare drug components for anticancer drugs, with high added value and high economic benefits.

[0089] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Wolfiporia hoelen GTR2, characterized in that, The preservation number of the strain is CGMCC NO. 41354.

2. The culture method of Wolfiporia hoelen GTR2 according to claim 1, wherein, After the strain is activated, liquid medium is used for culture.

3. The culture method of the Wolfiporia hoelen strain GTR2 according to claim 2, characterized by, The liquid culture medium comprises glucose 10-40 g / L, protein peptone 1.0-10.0 g / L, yeast extract 0.5-5.0 g / L, potassium dihydrogen phosphate 0.1-5.0 g / L, magnesium sulfate heptahydrate 0.1-5.0 g / L, vitamin B1 (0.1-10) × 10 -3 g / L, pH 6.0-6.8; the liquid culture constant-temperature shaking table condition is 20-28 ℃, 100-200 r / min.

4. A biological agent, characterized in that, The fermentation liquor of the Wolfiporia hoelen GTR2 strain according to claim 1, wherein the fermentation liquor comprises mycelium of the Wolfiporia hoelen GTR2 strain.

5. The biological preparation of claim 4, wherein the biological preparation is a vaccine. The fermentation liquor contains flavones and polyphenols.

6. The biological preparation of claim 4, wherein, The content of the flavones in the fermentation liquor is greater than 0.08 g / L, and the content of the polyphenols is greater than 0.01 g / L.

7. The Wolfiporia hoelen GTR2 strain according to claim 1 is used for preparing fermentation liquor with high yield of flavones and polyphenols.

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