Method for promoting hispidin production of phellinus igniarius by using macadamia nut waste as substrate

Abstract

The application relates to the technical field of microbial fermentation, in particular to a culture method for promoting Phellinus baumii to increase Hispidin by using macadamia nut waste as a substrate. The method uses the waste of macadamia nuts as a culture medium, improves the Hispidin content in Phellinus baumii through steps of mycelium culture, liquid strain preparation, solid culture medium preparation and fermentation culture, the method is simple and easy to implement, raw materials are environment-friendly, easy to obtain and low in cost, and the method provides a new and effective way for large-scale production of hispidin.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, and in particular to a method for cultivating *Sanghuang* (a type of medicinal mushroom) using macadamia nut waste as a substrate to promote the increase of Hispidin. Background Technology

[0002] Macadamia nuts, as a widely cultivated cash crop, generate a large amount of waste such as pruning branches, peels, and shells during planting and harvesting. Currently, these wastes are mainly used to make fertilizer or biomass fuel, which is inefficient, not only wasting resources but also potentially causing environmental problems.

[0003] Hispidin is a styrylpyranone compound with broad biological activity found in fungi such as *Phellinus linteus*. It exhibits cytotoxic, hypoglycemic, antibacterial, anti-inflammatory, anti-obesity, antiviral, and neuroprotective properties, showing promising applications, particularly in the field of antitumor therapy. The synthetic pathway of hispidin has been partially elucidated. Its precursor is caffeic acid, and lignin-degrading enzymes in *Phellinus linteus* (such as laccase and polyphenol oxidase) can break down lignin into caffeic acid, thereby promoting hispidin synthesis.

[0004] Meanwhile, current hispidin production mainly relies on traditional cultivation methods of Phellinus linteus, which suffers from high culture medium costs and low hispidin yield, limiting its large-scale application. Therefore, researching a low-cost, high-efficiency hispidin production method, while simultaneously realizing the resource utilization of macadamia nut waste, is of significant practical importance. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of existing methods by proposing a cultivation method that uses macadamia nut waste as a substrate to promote the increase of Hispidin in Phellinus linteus. By using macadamia nut waste as a culture medium, the utilization of waste is promoted, the content of Hispidin in Phellinus linteus is increased, and the method is low in cost and easy to implement.

[0006] To address the aforementioned technical problems, this invention proposes a method for cultivating *Sanghuang* (a type of medicinal mushroom) using macadamia nut waste as a substrate to promote the increase of Hispidin. The technical solution is as follows:

[0007] This method utilizes macadamia nut waste as a substrate to promote the increase of Hispidin in Phellinus linteus. The method involves preparing a solid culture medium from the macadamia nut waste to culture Phellinus linteus. Specific steps include:

[0008] (1) Mycelial culture: When the MY solid medium cools to 45-60℃, add 10-30μL of a mixed solution containing 60-120ppm kanamycin and 60-120ppm ampicillin to the medium at once. Mix thoroughly and pour into plates for solidification. Under sterile conditions, spray 10-20ml of 75% alcohol onto the Sanghuang fungus package and let stand for 30-40min for disinfection. Then, use sterilized tools to pick 0.2-0.5cm mycelia and inoculate them into 60-90mm MY solid medium. Incubate in the dark at 25-30℃ for 8-12 days until yellow mycelia grow.

[0009] (2) Preparation of liquid culture: Take the mycelium cultured in step (1) and crush it for 30-60 seconds using a pulverizer with a speed of 10000-15000 r / min. Inoculate the crushed mycelium into MYGMK liquid culture medium at a volume ratio of mycelium to liquid culture medium of 1:25. Incubate in the dark on a shaker at 25-30℃ for 8-12 days. Add 100 μL of azacytidine stock solution with a concentration of 10000 ppm (final concentration of 10 ppm) at one time and continue to incubate at 25-30℃ for 3-6 days to obtain the liquid culture. During this period, the shaker is continuously rotating at a frequency of 100-180 rpm.

[0010] (3) Preparation of solid culture medium: After cleaning macadamia nut waste free from mold and pests, dry it at 50-80℃ for 1-4 hours or under natural conditions for 4-6 days and then pulverize it to 20-40 mesh to obtain macadamia nut waste powder.

[0011] Take macadamia nut waste powder, rice flour powder, and NMK liquid in a ratio of 2:2:3, mix them evenly, and sterilize them at 100-140 ℃ for 15-30 min to obtain a solid culture medium.

[0012] (4) Fermentation culture: The liquid culture from step (2) is inoculated into the solid culture medium from step (3). The ratio of liquid culture to solid culture medium is 1:25. The culture is carried out in the dark at 25-30℃ for 23-28 days. The mycelium is collected to extract the crude extract. The Hispidin content is detected according to conventional detection methods.

[0013] Hispidin can be detected using chromatographic, spectroscopic, and bioassay methods, such as high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UHPLC), thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), and determination of physical and chemical constants. In this method, the fermentation product extraction process, compound detection method, and compound identification method are conventional; optimization of the eluting machine is only based on the compound characteristics.

[0014] The MY solid culture medium is a conventional nutrient culture medium in the field, and its main components include malt extract, yeast extract, agar and distilled water.

[0015] The MYGMK liquid culture medium is prepared as follows: Malt extract broth, mannitol, ammonium sulfate, magnesium sulfate, and potassium dihydrogen phosphate are prepared in a ratio of 6-9:10-25:0.1-1:1-2:1-2. The mixture is then diluted with purified water to a final volume of 1L, thoroughly mixed, and sterilized at 100-140℃ for 15-30 minutes. This liquid culture medium is named according to the first letter of its main name.

[0016] The preparation method of the NMK solvent is as follows: Take ammonium sulfate, magnesium sulfate, and potassium dihydrogen phosphate in a ratio of 0.1-1:1-2:1-2, add purified water to a final volume of 1L, mix thoroughly, and sterilize at 100-140℃ for 15-30 min. This liquid culture medium is named according to the first letter of its main name.

[0017] The macadamia nut waste can be one or a combination of two or three of the following: pruned branches, peels or shells from macadamia nut trees that are free from mold and pests.

[0018] When macadamia nut waste is a mixture of pruned branches and husks, the ratio is 3-4:1-2; when it is a mixture of pruned branches and shells, the ratio is 3-4:1-2; when it is a mixture of husks and shells, the ratio is 1:1; when it is a mixture of pruned branches, husks, and shells, the ratio is 6-8:1-2:1-2.

[0019] The weight ratios of the various substances used in the cultivation process of this invention and the various process parameters can be specifically determined according to actual needs.

[0020] This invention is the first to use waste materials such as pruning branches, peels, and shells from macadamia nut cultivation as a substrate for the cultivation of Phellinus linteus, solving the problem of low utilization efficiency of traditional waste materials (such as fertilizer or fuel) and realizing the high-value transformation of agricultural waste. Furthermore, this cultivation method is simple and easy to implement, using environmentally friendly and readily available raw materials at a low cost. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or related technologies, the drawings used in the description of the embodiments of this application or related technologies will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram showing the relative content of hispidin in macadamia nut waste culture according to the present invention.

[0023] Figure 2 This is a schematic diagram of HPLC detection of Phellinus linteus mycelium grown on macadamia nut pruning branch culture medium according to the present invention. By comparing with database data, the compound with a retention time of 9.011 minutes was identified as hispidin, which is the peak indicated by the red arrow.

[0024] Figure 3 This is a schematic diagram of the HPLC detection results of Phellinus linteus mycelium grown on macadamia nut shell culture medium according to the present invention. By comparing with the database data, the compound with a retention time of 9.011 minutes was identified as hispidin, which is the peak indicated by the red arrow.

[0025] Figure 4 This is a schematic diagram of the HPLC detection results of Phellinus linteus mycelium grown on macadamia nut pericarp culture medium according to the present invention. By comparing with database data, the compound with a retention time of 9.011 minutes was identified as hispidin, which is the peak indicated by the red arrow. Detailed Implementation

[0026] The specific embodiments of the present invention will be described in further detail below. Technologies or products not specified in the embodiments are all prior art or conventional products that can be obtained by purchase.

[0027] Example 1: This method utilizes macadamia nut waste as a substrate to promote the increase of Hispidin in Phellinus linteus. The macadamia nut waste is prepared into a solid culture medium for culturing Phellinus linteus. Specific methods include:

[0028] (1) Mycelial culture: When the temperature of the MY solid medium reaches 50℃, add 20μL of a mixture of 100ppm kanamycin and 100ppm ampicillin solution to the medium at one time. Under sterile conditions, spray 10ml of 75% alcohol onto the Sanghuang fungus bag and let it stand for 40min for disinfection. Then, use sterilized tools to pick 0.4cm mycelia and inoculate them into 75mm MY solid medium. Incubate in the dark at 28℃ for 10 days to grow yellow mycelia, which are then ready for use.

[0029] (2) Preparation of liquid culture: Take one 2*2cm mycelium and crush it for 60s using a pulverizer at 10000r / min. Inoculate the crushed mycelium into 100ml of MYGMK liquid culture medium. Incubate in the dark at 28℃ using a shaker at 180rpm for 10 days. Add 100μL of 10000ppm azacytidine stock solution (final concentration 10ppm) and continue incubation at 28℃ for 5 days to obtain the liquid culture. Preparation method of MYGMK liquid culture medium: Take malt extract broth, mannitol, ammonium sulfate, magnesium sulfate and potassium dihydrogen phosphate in a ratio of 7.5:20:0.5:1:1, add purified water to make up to 1L, mix well, and sterilize at 130℃ for 20min.

[0030] (3) Preparation of solid culture medium: Pruned branches of macadamia nuts grown in Xishuangbanna were selected. After washing the pruned branches free from mold and pests, they were dried at 60℃ for 3 hours and then pulverized to 30 mesh to obtain macadamia nut waste powder. The macadamia nut pruning branch powder, rice flour powder, and NMK liquid were taken in a ratio of 2:2:3, mixed evenly, and sterilized at 121℃ for 20 minutes to obtain solid culture medium. Preparation method of NMK solvent: Ammonium sulfate, magnesium sulfate, and potassium dihydrogen phosphate were taken in a ratio of 1:2:2, and purified water was added to make up to 1L. The mixture was mixed evenly and sterilized at 121℃ for 20 minutes.

[0031] (4) Fermentation culture: The liquid culture from step (2) is inoculated into the solid culture medium from step (3). The ratio of liquid culture to solid culture medium is 1:25. The culture is carried out in the dark at 28°C for 25 days. The mycelium is collected and crude extract is extracted. The Hispidin content is detected according to conventional detection methods.

[0032] Example 2: This method utilizes macadamia nut waste as a substrate to promote the increase of Hispidin in Phellinus linteus. The macadamia nut waste is used to prepare a solid culture medium for culturing Phellinus linteus. Specific methods include:

[0033] (1) Mycelial culture: When the temperature of the MY solid medium reaches 45℃, add 10μL of a mixture of 60ppm kanamycin and 60ppm ampicillin solution to the medium at once. Under sterile conditions, spray 15ml of 75% alcohol onto the Sanghuang fungus package and let it stand for 30min for disinfection. Then, use sterilized tools to pick 0.2cm mycelia and inoculate them into 60mm MY solid medium. Incubate in the dark at 25℃ for 12 days to grow yellow mycelia, which are then ready for use.

[0034] (2) Preparation of liquid culture: Take one 2*2cm mycelium and crush it for 45s using a pulverizer at 13000r / min. Inoculate the crushed mycelium into 100ml of MYGMK liquid culture medium. Incubate in the dark at 25℃ using a shaker at 100rpm for 12 days. Add 100μL of 10000ppm azacytidine stock solution (final concentration 10ppm) and continue incubation at 25℃ for 6 days to obtain the liquid culture. Preparation method of MYGMK liquid culture medium: Take malt extract broth, mannitol, ammonium sulfate, magnesium sulfate and potassium dihydrogen phosphate in a ratio of 6:10:0.1:1.5:1.5, add purified water to make up to 1L, mix well and sterilize at 100℃ for 30min.

[0035] (3) Preparation of solid culture medium: Pruned branches of macadamia nuts grown in Xishuangbanna were selected. After washing the pruned branches that were free from mold and pests, they were dried at 50℃ for 4 hours and then pulverized to 20 mesh to obtain macadamia nut waste powder. The macadamia nut pruning branch powder, rice flour powder, and NMK liquid were taken in a ratio of 2:2:3, mixed evenly, and sterilized at 100℃ for 30 minutes to obtain solid culture medium.

[0036] Preparation method of NMK solvent: Take ammonium sulfate, magnesium sulfate and potassium dihydrogen phosphate in a ratio of 0.5:1.5:1.5, add purified water to make up to 1L, mix well, and sterilize at 100℃ for 30min.

[0037] (4) Fermentation culture: The liquid culture from step (2) is inoculated into the solid culture medium from step (3). The ratio of liquid culture to solid culture medium is 1:25. The culture is carried out in the dark at 25°C for 28 days. The mycelium is collected and crude extract is extracted. The Hispidin content is detected according to conventional detection methods.

[0038] Example 3: This method utilizes macadamia nut waste as a substrate to promote the increase of Hispidin in Phellinus linteus. The macadamia nut waste is used to prepare a solid culture medium for culturing Phellinus linteus. Specific methods include:

[0039] (1) Mycelial culture: When the temperature of the MY solid medium reaches 60℃, add 30μL of a mixture of 120ppm kanamycin and 120ppm ampicillin solution to the medium at one time. Under sterile conditions, spray 20ml of 75% alcohol on the Sanghuang fungus bag and let it stand for 35min for disinfection. Then, use sterilized tools to pick 0.5cm mycelia and inoculate them into 90mm MY solid medium. Incubate in the dark at 30℃ for 8 days to grow yellow mycelia, which are then ready for use.

[0040] (2) Preparation of liquid culture: Take one 2*2cm mycelium and crush it for 30s using a pulverizer at 15000r / min. Inoculate the crushed mycelium into 100ml of MYGMK liquid culture medium. Incubate in the dark at 30℃ using a shaker at 145rpm for 8 days. Add 100μL of 10000ppm azacytidine stock solution (final concentration 10ppm) and continue incubation at 30℃ for 3 days to obtain the liquid culture. Preparation method of MYGMK liquid culture medium: Take malt extract broth, mannitol, ammonium sulfate, magnesium sulfate and potassium dihydrogen phosphate in a ratio of 9:25:1:2:2, add purified water to make up to 1L, mix well, and sterilize at 140℃ for 15min.

[0041] (3) Preparation of solid culture medium: Selected macadamia nut pruning branches grown in Xishuangbanna, cleaned the branches free from mold and pests, dried them at 80℃ for 1 hour, and then pulverized them to 40 mesh to obtain macadamia nut waste powder. Take macadamia nut pruning branch powder, rice flour powder, and NMK liquid in a ratio of 2:2:3, mix them evenly, and sterilize them at 140℃ for 15 minutes to obtain solid culture medium.

[0042] Preparation method of NMK solvent: Take ammonium sulfate, magnesium sulfate and potassium dihydrogen phosphate in a ratio of 0.1:1:1, add purified water to make up to 1L, mix well, and sterilize at 140℃ for 15min.

[0043] (4) Fermentation culture: The liquid culture from step (2) is inoculated into the solid culture medium from step (3). The ratio of liquid culture to solid culture medium is 1:25. The culture is carried out in the dark at 30°C for 23 days. The mycelium is collected and crude extract is extracted. The Hispidin content is detected according to conventional detection methods.

[0044] Example 4 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches and pericarps from macadamia trees grown in Xishuangbanna are selected. After washing the pruned branches and pericarps (free from mold, disease, and pests), they are dried naturally for 4 days and pulverized to 22 mesh. The pruned branch powder and pericarp powder are taken in a 3:2 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0045] Example 5 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches and pericarps from macadamia trees grown in Lincang are selected. After washing the pruned branches and pericarps (free from mold, disease, and pests), they are dried naturally for 4 days and pulverized to 35 mesh. The pruned branch powder and pericarp powder are taken in a 4:1 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0046] Example 6 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches and pericarps from macadamia trees grown in Lincang are selected. After washing the pruned branches and pericarps (free from mold, disease, and pests), they are dried naturally for 4 days and pulverized to 25 mesh. The pruned branch powder and pericarp powder are taken separately in a 3.5:1.5 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0047] Example 7 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches and shells from macadamia trees grown in Pu'er are selected. After washing the pruned branches and shells to ensure they are free of mold, pests, and diseases, they are dried naturally for 4 days and pulverized to 32 mesh. The pruned branch powder and shell powder are taken separately in a 3:1.5 ratio and mixed evenly to obtain macadamia nut waste powder. The rest of the process is the same as in Example 1.

[0048] Example 8 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches and shells of macadamia nuts grown in Baoshan are selected. After washing the pruned branches and shells to ensure they are free of mold, disease, and pests, they are dried naturally for 4 days and pulverized to 22 mesh. The pruned branch powder and shell powder are taken in a 4:1 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0049] Example 9 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches and shells from macadamia trees grown in Pu'er are selected. After washing the pruned branches and shells (free from mold, disease, and pests), they are dried naturally for 6 days and pulverized to 30 mesh. The pruned branch powder and shell powder are taken in a 4:1 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0050] Example 10 describes a method for cultivating *Sanghuang* (a type of medicinal mushroom) using macadamia nut waste as a substrate to promote the increase of Hispidin. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial cultivation, liquid inoculum preparation, solid culture medium preparation, and fermentation cultivation. In the solid culture medium preparation, the pericarps and shells of macadamia nuts grown in Pu'er are selected. After washing the pericarps and shells of macadamia nuts free from mold, disease, and pests, they are dried naturally for 5 days and pulverized to 33 mesh. The pericarp powder and shell powder are taken in a 1:1 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0051] Example 11 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches, pericarps, and shells of macadamia nuts grown in Pu'er are selected. After washing the pruned branches, pericarps, and shells (free from mold, pests, and diseases), they are dried at 60°C for 3 hours and then pulverized to 30 mesh. The powdered pruned branches, pericarps, and shells are taken in a 6:2:2 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0052] Example 12 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches, pericarps, and shells of macadamia nuts grown in Pu'er are selected. After washing the pruned branches, pericarps, and shells (free from mold, disease, and pests), they are dried at 65°C for 2.5 hours and then pulverized to 35 mesh. The powdered pruned branches, pericarps, and shells are taken in a 7.5:1.5:1.5 ratio and mixed evenly to obtain macadamia nut waste powder. The rest is the same as in Example 1.

[0053] Example 13 describes a method for promoting Hispidin production in *Sanghuang* using macadamia nut waste as a substrate. The method involves preparing a solid culture medium from the macadamia nut waste for *Sanghuang* cultivation. The specific steps include: mycelial culture, liquid inoculum preparation, solid culture medium preparation, and fermentation culture. In the solid culture medium preparation, pruned branches, pericarps, and shells of macadamia nuts grown in Pu'er are selected. After washing the pruned branches, pericarps, and shells (free from mold, pests, and diseases), they are dried at 70°C for 1.5 hours and then pulverized to 30 mesh. The powdered pruned branches, pericarps, and shells are taken in an 8:1:1 ratio and mixed evenly to obtain macadamia nut waste powder. The rest of the process is the same as in Example 1.

[0054] The technical content of the present invention has been described above, but the scope of protection of the present invention is not limited to the described content. Within the scope of knowledge possessed by those skilled in the art, various changes can be made to the technical content of the present invention without departing from the spirit of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cultivating *Sanghuang* (a type of medicinal mushroom) using macadamia nut waste as a substrate to promote the increase of Hispidin, characterized in that: The method for culturing Phellinus linteus using macadamia nut waste as a solid culture medium includes: (1) Mycelial culture: Sterilize the Sanghuang fungus package under sterile conditions. After cooling the MY solid medium to 45-60℃, add 10-30μL of mixed solution containing 60-120ppm kanamycin and 60-120ppm ampicillin at once. After thorough mixing, pour into plates and wait for solidification. Then pick out mycelia and inoculate them into the MY solid medium. Incubate in the dark at 25-30℃ for 8-12 days. Yellow mycelia will grow and be ready for use. (2) Preparation of liquid culture: After the mycelium cultured in step (1) is broken, it is inoculated into MYGMK liquid culture medium at a volume ratio of mycelium to liquid culture medium of 1:

25. It is then cultured in the dark on a shaker at 25-30℃ for 8-12 days with continuous shaking at a frequency of 100-180 rpm. 100 μL of azacytidine stock solution with a concentration of 10000 ppm is added at once, and the culture is continued in the dark at 25-30℃ for 3-6 days to obtain the liquid culture. (3) Preparation of solid culture medium: The macadamia nut waste was dried and crushed to obtain macadamia nut waste powder. The macadamia nut waste powder, rice flour powder and NMK solvent were taken in a ratio of 2:2:3, mixed evenly, and sterilized at high temperature and high pressure to obtain solid culture medium. (4) Fermentation culture: The liquid culture from step (2) is inoculated into the solid culture medium from step (3). The ratio of liquid culture to solid culture medium is 1:

25. The culture is carried out in the dark at 25-30℃ for 23-28 days. The mycelium is collected to extract the crude extract. The Hispidin content is detected according to conventional detection methods.

2. The cultivation method according to claim 1, characterized in that: In step (3), the macadamia nut waste is one or a combination of two or three of the following: macadamia tree pruning branches, peels or shells that are free from mold and pests.

3. The cultivation method according to claim 2, characterized in that: The macadamia nut waste mixture is composed of macadamia tree pruning branches and pericarps in a ratio of 3-4:1-2, or macadamia tree pruning branches and pericarps in a ratio of 3-4:1-2, or pericarps and pericarps in a ratio of 1:1, or macadamia tree pruning branches, pericarps and pericarps in a ratio of 6-8:1-2:1-2.

4. The cultivation method according to any one of claims 1-3, characterized in that: The macadamia nut waste mentioned is dried macadamia nut waste with a moisture content of 10-20%.

5. The cultivation method according to any one of claims 1-3, characterized in that: The macadamia nut waste is dried at 50-80℃ for 1-4 hours or under natural conditions for 4-6 days, and then crushed to 20-40 mesh.

6. The cultivation method according to claim 1, characterized in that: In step (1), spray 10-20 ml of 75% alcohol onto the Sanghuang fungus bag and let it stand for 30-40 minutes. Select 0.2-0.5 cm mycelia and inoculate them into 60-90 mm MY solid culture medium.

7. The cultivation method according to claim 1, characterized in that: In step (2), malt extract broth, mannitol, ammonium sulfate, magnesium sulfate and potassium dihydrogen phosphate are taken in a ratio of 6-9:10-25:0.1-1:1-2:1-2, and purified water is added to make up to 1L. The mixture is then mixed evenly to prepare MYGMK liquid culture medium. The mycelium is pulverized for 30-60s using a pulverizer with a rotation speed of 10000-15000r / min. The concentration of azacytidine is 10ppm.

8. The cultivation method according to claim 1, characterized in that: In step (3), ammonium sulfate, magnesium sulfate, and potassium dihydrogen phosphate are taken in a ratio of 0.1-1:1-2:1-2, and diluted with 1L of purified water to obtain NMK solvent.

9. The cultivation method according to claim 1, characterized in that: The MY solid culture medium, MYGMK liquid culture medium, NMK solvent and solid culture medium were all sterilized at 100-140 ℃ for 15-30 min.

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