Culture method of high-yield anticancer active compound of high-yield Antrodia camphorata

Abstract

The invention belongs to the technical field of production processes of edible and medicinal fungi, and relates to a culture method of a high-yield anticancer active compound of Gaochaigong antrodia camphorata, the preservation name of the Gaochaigong antrodia camphorata is Taiwanofungus gaoligongensis YAFTG001, the preservation number is CCTCC M 20232425, a culture medium used in the fermentation process is composed of 400 parts of an MM culture medium, 200 parts of rice flour and 200 parts of cinnamomum camphora sawdust, and the culture medium is used for culturing the high-yield anticancer active compound of the Gaochaigong antrodia camphorata with the high-yield anticancer active compound of the Gaochaigong antrodia camphorata. The MM culture medium is prepared from 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 1.52 g / L of monopotassium phosphate, 0.52 g / L of magnesium sulfate and the balance of water. According to the method, the content of anticancer active compounds Antcin B, Antcin C and Dankasterone A in the high-yield Antrodia camphorata mycelia is increased through fungus bag culture with camphor sawdust as a matrix, the anticancer drug activity of the high-yield Antrodia camphorata mycelia is greatly improved, and the yield of the high-yield Antrodia camphorata mycelia is increased. The large-scale production of the high-Li tribute antrodia cinnamomea anticancer active compound is promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of the production process of edible and medicinal fungi, and particularly relates to a cultivation method and formula for improving the production efficiency of anticancer active compounds Antcin B, Antcin C and Dankasterone A in the mycelia of Antrodia cinnamomea var. kanoi fermented and cultured, improving the medicinal activity of the mycelia of Antrodia cinnamomea var. kanoi fermented and cultured, and promoting the large-scale production of anticancer active compounds in Antrodia cinnamomea var. kanoi. Background Art

[0002] Antrodia cinnamomea is a precious edible and medicinal fungus; it belongs to the Basidiomycota ( Basidiomycota ), Polyporaceae ( Polyporaceae ), and Antrodia ( Taiwannofungus ). Antrodia cinnamomea exhibits various physiological and pharmacological properties, such as anticancer, antitumor, antioxidant, anti-inflammatory, hypoglycemic, hepatoprotective, immunomodulatory, and intestinal microbiota regulatory activities. Due to its therapeutic effects, especially its prospective application as a chemopreventive agent, it has also attracted increasing research attention for the development of new drugs. Antrodia cinnamomea var. kanoi ( Taiwanofungus gaoligongensis ) was discovered as a new species of Antrodia in 2018, and its distribution area is located in the Gaoligong Mountains in the northwest of Yunnan Province. Through whole-genome analysis, it was found that Antrodia cinnamomea var. kanoi has extremely high similarity with Antrodia cinnamomea in terms of active compound biosynthetic gene clusters. At present, there have been many reports on the active products of Antrodia cinnamomea fermented and cultured, but the research on the production of a large amount of active compounds by Antrodia cinnamomea var. kanoi fermented and cultured is still very limited.

[0003] With further in-depth research, more and more bioactive metabolites have been isolated from the fruiting bodies and cultured mycelia of Antrodia cinnamomea, and their structures and functions have been determined. More than 200 compounds have been isolated and identified so far, including polysaccharides, triterpenoids, ubiquinone derivatives, maleic acid and succinic acid derivatives, benzene ring derivatives and glycoproteins. Studies have shown that triterpenoids are the main anti-tumor active components of Antrodia cinnamomea, and their skeletal structures are mainly ergostane and lanosterol. Among them, ergostane has a unique side chain structure of 24(28)-ene, indicating that its triterpenoid metabolic pathway is very different from that of Ganoderma lucidum and can be used as a characteristic component of Antrodia cinnamomea. Antcin B and Antcin C are ergostane-type triterpenoids isolated from the fruiting bodies of Antrodia cinnamomea, both of which have good anti-cancer and antioxidant activities. Dankasterone A is a sesquiterpenoid compound, and it has been reported that it can be produced from the solid-cultured mycelia of Antrodia cinnamomea and has good anti-cancer potential. Due to the scarcity of the host and slow growth, the wild fruiting bodies of Antrodia cinnamomea are very expensive and in short supply. Therefore, artificial cultivation techniques have been developed to meet the growing demand for the active components of Antrodia cinnamomea. Different cultivation conditions have a great impact on the biological activities of Antrodia cinnamomea mycelia. Therefore, it is of great research significance to study and determine a cultivation method that can produce a large amount of bioactive substances in Antrodia cinnamomea, improve the medicinal activity of the artificially cultured mycelia of Antrodia cinnamomea and is suitable for large-scale production. Summary of the Invention

[0004] The present invention provides a fermentation method for increasing the contents of anti-cancer active compounds Antcin B, Antcin C and Dankasterone A in the mycelia of Antrodia cinnamomea var. Kao-Li-Kungensis. By trying different cultivation methods, optimizing the cultivation formula, and improving the fermentation conditions, the biomass of Antrodia cinnamomea var. Kao-Li-Kungensis cultured by fermentation and the contents of Antcin B, Antcin C and Dankasterone A in the mycelia are increased, and the anti-cancer activity of the mycelia of Antrodia cinnamomea var. Kao-Li-Kungensis is improved, which helps to realize the large-scale production of the anti-cancer active compounds Antcin B, Antcin C and Dankasterone A.

[0005] The technical solution of the present invention is as follows: The present invention provides a cultivation method for high-yield anti-cancer active compounds of Antrodia cinnamomea var. Kao-Li-Kungensis, and the Antrodia cinnamomea var. Kao-Li-Kungensis has a deposit name of Taiwanofungus gaoligongensis YAFTG001, deposit number: CCTCC M 20232425. The composition of the culture medium used in the fermentation process consists of 400 parts of MM medium, 200 parts of rice flour and 200 parts of camphor sawdust. The MM medium is composed of 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 1.52 g / L of potassium dihydrogen phosphate and 0.52 g / L of magnesium sulfate, and the balance is water. The cultivation method is: constant temperature dark cultivation at 28°C.

[0006] The present invention also provides a cultivation method for the industrialized large-scale cultivation of high-yield anticancer active compounds of *Antrodia cinnamomea* var. *kawakamii*, which is characterized in that the preserved name of the *Antrodia cinnamomea* var. *kawakamii* is Taiwanofungus gaoligongensis YAFTG001, preservation number: CCTCC M 20232425, and the cultivation method includes the following steps: S1: Activation of mother culture. The mother culture is activated using PDA solid medium and placed in a constant temperature incubator at 26°C and 60% humidity for dark cultivation. S2: Cultivation of original seed. The activated mother culture is used with a modified malt extract broth liquid medium: 5 g / L of animal tissue pepsin digest, 5 g / L of yeast extract, 5 g / L of malt extract, 3 g / L of maltose, and 7 g / L of glucose, with the balance being water; the cultivation method is: dark cultivation in a constant temperature shaker at 28°C and 150 rpm to obtain the original seed. S3: High-yield cultivation. The original seed is further inoculated into a medium, and the medium composition consists of 400 parts of MM medium, 200 parts of rice flour, and 200 parts of camphor sawdust. The MM medium is composed of 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 1.52 g / L of potassium dihydrogen phosphate, and 0.52 g / L of magnesium sulfate, with the balance being water. The cultivation method is: constant temperature dark cultivation at 28°C.

[0007] Furthermore, the anticancer active compound is one or more of Antcin B, Antcin C, or Dankasterone A.

[0008] Furthermore, the present invention also provides a production method for an anticancer active compound. The anticancer active compound is one or more of Antcin B, Antcin C, or Dankasterone A. The method is to cultivate *Antrodia cinnamomea* var. *kawakamii* YAFTG001 with the preservation number: CCTCC M 20232425, and after cultivating the product, separate and purify it to obtain the anticancer active compound; the medium composition used in the cultivation process consists of 400 parts of MM medium, 200 parts of rice flour, and 200 parts of camphor sawdust. The MM medium is composed of 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 1.52 g / L of potassium dihydrogen phosphate, and 0.52 g / L of magnesium sulfate, with the balance being water. The cultivation method is: constant temperature dark cultivation at 28°C.

[0009] The present invention has the following beneficial effects: The contents of Antcin B, Antcin C, and Dankasterone A in the mycelia of Antrodia camphorata from Gaoligong produced by the technical solution provided by the present invention are the highest, with their relative peak areas being 316.24, 4825.45, and 3174.24 respectively; and they are all extremely significantly higher than the contents of anti-cancer active ingredients in the mycelia cultured by other methods. Using camphor as the substrate for the fungus bag to culture Antrodia camphorata from Gaoligong shows stronger application potential, providing a way for the efficient development of the medicinal value of Antrodia camphorata from Gaoligong and the efficient production of anti-cancer active compounds Antcin B, Antcin C, and Dankasterone A. At the same time, the fungus bag culture can produce more mycelia, and the process is simple, controllable, and low-cost, which helps to achieve large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is a diagram showing the mycelial growth of Antrodia camphorata from Gaoligong described in the present invention in a PDA liquid medium; Figure 2 It is a diagram showing the mycelial growth of Antrodia camphorata from Gaoligong described in the present invention by solid-state fermentation with added sawdust of Cinnamomum kanehirae branches; Figure 3 It is a diagram showing the mycelial growth of Antrodia camphorata from Gaoligong described in the present invention by solid-state fermentation with added sawdust of Cinnamomum camphora branches; Figure 4 It is a diagram showing the mycelial growth in the fungus bag with Cinnamomum kanehirae substrate described in the present invention; Figure 5 It is a diagram showing the mycelial growth in the fungus bag with Cinnamomum camphora substrate described in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0011] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. For purchased commodities in the test methods, if the specific conditions are not indicated, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0012] The preservation name of Antrodia camphorata from Gaoligong is Taiwanofungus gaoligongensis YAFTG001, preservation number: CCTCC M 20232425, preserved in the China Center for Type Culture Collection, and the preservation address is Wuhan University, China; preservation date: December 1, 2023. This strain has been disclosed in the patent CN202410173373.2, a culture method for inducing Antrodia camphorata from Gaoligong to produce compounds that inhibit plant pathogenic bacteria.

[0013] Antcin B (CAS No.: 163597-25-9), Antcin C (CAS No.: 163565-76-2) and Dankasterone A (CAS No.: 82408-25-3).

[0014] The camphor wood sawdust and kanahimai ... sawdust used in the present invention are both sieved through a 40-mesh sieve for later use.

[0015] The invention provides a fungus bag culture formula for improving the content of anticancer active compounds Antcin B, Antcin C and Dankasterone A in Gaoligong Antrodia cinnamomea mycelium. The fungus bag culture formula comprises: a culture medium is composed of 400 parts of MM culture medium, 200 parts of rice flour and 200 parts of camphor sawdust, the MM culture medium comprises 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 1.52 g / L of potassium dihydrogen phosphate and 0.52 g / L of magnesium sulfate, and the remainder is water. A fungus bag preparation method comprises: firstly preparing the MM culture medium, weighing 6 g of sodium nitrate, 0.52 g of potassium chloride, 1.52 g of potassium dihydrogen phosphate and 0.52 g of magnesium sulfate, adding water to make the volume to one liter, stirring and dissolving, taking 400 parts of MM culture medium, 200 parts of rice flour and 200 parts of camphor sawdust, fully mixing, putting into a polyethylene cultivation bag, sterilizing and cooling, and then using. The culture method comprises: culturing at a constant temperature of 28°C and avoiding light for 50 days.

[0016] The present invention increases the content of anticancer active compounds Antcin B, Antcin C and Dankasterone A in Gaoligong Antrodia cinnamomea mycelium by culturing the fungus bag with camphor sawdust as a matrix, greatly increases the anticancer drug activity of Gaoligong Antrodia cinnamomea mycelium, and promotes the large-scale production of Gaoligong Antrodia cinnamomea anticancer active compounds. Example 1

[0017] Gaoligong Antrodia cinnamomea fungus culture 1. Activation of Gaoligong Antrodia cinnamomea strain (mother strain) The fruiting bodies of Ganoderma tsugae from Gaoligong Mountain and the Ganoderma tsugae strain YAFTG001 are stored in the laboratory. The activation preparation of the Ganoderma tsugae strain is as follows: Disinfect the laminar flow hood and turn on the ultraviolet sterilization 15 minutes in advance. Prepare the Ganoderma tsugae strain and PDA solid medium (0.5 g of potato powder / 100 mL, 0.1 g of potassium dihydrogen phosphate / 100 mL, 0.05 g of magnesium sulfate / 100 mL, 0.5 g of yeast powder / 100 mL, 0.01 g of VB1 / 100 mL, 1.1 g of agar / 100 mL, and the balance is water). First, fully burn the forceps or inoculation knife with an alcohol lamp. After cooling, pick up a small piece of the original Ganoderma tsugae strain and inoculate it into the PDA solid medium. After making marks (strain number, date), seal the film and place it in a constant temperature incubator at 26 °C and 60% humidity for 30 days. Observe and record every 5 days during this period. When the Ganoderma tsugae mycelium covers the entire medium, place it at 4 o °C in the refrigerator for storage and standby.

[0018] 2. Liquid culture of Ganoderma tsugae from Gaoligong Mountain (stock culture) Preparation of the liquid culture medium for Ganoderma tsugae from Gaoligong Mountain: Scrape one-fourth of the mycelium in a 60-mm culture dish of Ganoderma tsugae from Gaoligong Mountain and put it into a 2-mL sterilized centrifuge tube. Then add 1 mL of sterile water and break it for 2 minutes. Then take 500 μL of the broken sample solution and inoculate it into 250-mL conical flasks containing 100 mL of modified malt extract broth liquid medium (5 g / L of animal tissue pepsin digest, 5 g / L of yeast extract, 5 g / L of malt extract, 3 g / L of maltose, 7 g / L of glucose, and the balance is water). Place it in a constant temperature shaker at 28 °C and 150 rpm for 10 days. Example 2

[0019] Cultivation of the camphor substrate spawn bag Formulation composition of the camphor substrate spawn bag: 400 parts of MM medium + 200 parts of rice flour + 200 parts of camphor sawdust. MM medium: 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 1.52 g / L of potassium dihydrogen phosphate, 0.52 g / L of magnesium sulfate, and the balance is water; First, break the camphor tree trunk into sawdust (pass through a 40-mesh sieve) and then put it into an autoclave for sterilization (121 o °C, 120 minutes); After cooling, take out 200 g, add 200 g of rice flour, and 400 mL of MM liquid medium. After bagging (16.5 cm × 37 cm polyethylene cultivation bag), put it into an autoclave for sterilization (121 o °C, 120 minutes). After cooling, transfer it to the laminar flow hood. Take the stock culture of Ganoderma tsugae from Gaoligong Mountain in liquid culture and inoculate it into the spawn bag, and culture it at a constant temperature of 28 °C in the dark for 50 days. Comparative Example 1

[0020] Liquid Culture of Antrodia camphorata in PDA Medium The composition of the PDA liquid fermentation medium is as follows: PDA (0.5 g of potato powder / 100 mL, 0.1 g of potassium dihydrogen phosphate / 100 mL, 0.05 g of magnesium sulfate / 100 mL, 0.5 g of yeast powder / 100 mL, 0.01 g of VB1 / 100 mL, and the balance is water). It is placed in an autoclave for sterilization (121 o °C, 20 min). After cooling, it is transferred to a laminar flow hood. The original strain of Antrodia camphorata in liquid culture is inoculated into the PDA liquid medium, and then placed in a constant temperature shaking incubator for cultivation (temperature 28 °C, rotation speed 150 r / min, cultivation time 18 d). Comparative Example 2

[0021] Solid Fermentation Culture with Added Dry Sawdust of Cinnamomum kanehirae The composition of the medium for solid fermentation culture with added dry sawdust of Cinnamomum kanehirae is as follows: 20 g of Cinnamomum kanehirae sawdust + 20 g of yellow wheat + 50 ml of MM medium (MM medium: 6 g / L sodium nitrate, 0.52 g / potassium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, and the balance is water). The prepared medium is placed in an autoclave for sterilization (121 o °C, 20 min). After cooling, it is transferred to a laminar flow hood. The original strain of Antrodia camphorata in liquid culture is inoculated into the culture flask and cultured at 28 °C in the dark for 20 days. Comparative Example 3

[0022] Solid Fermentation Culture with Added Dry Sawdust of Cinnamomum camphora The composition of the medium for solid fermentation culture with added dry sawdust of Cinnamomum camphora is as follows: 20 g of Cinnamomum camphora sawdust + 20 g of yellow wheat + 50 ml of MM (MM medium: 6 g / L sodium nitrate, 0.52 g / potassium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate, and the balance is water). The prepared medium is placed in an autoclave for sterilization (121 o °C, 20 min). After cooling, it is transferred to a laminar flow hood. The original strain of Antrodia camphorata in liquid culture is inoculated into the culture flask and cultured at 28 °C in the dark for 20 days. Comparative Example 4

[0023] Culture in Cinnamomum kanehirae Substrate Bacterial Bags The formula composition of the Cinnamomum kanehirae substrate bacterial bag is as follows: 400 parts of MM medium + 200 parts of rice flour + 200 parts of Cinnamomum kanehirae sawdust. MM medium: 6 g / L sodium nitrate, 0.52 g / L potassium chloride, 1.52 g / L potassium dihydrogen phosphate, 0.52 g / L magnesium sulfate, and the balance is water. After crushing the Cinnamomum kanehirae trunk into sawdust, it is placed in an autoclave for sterilization (121 oC, 120 min). After cooling, take out 200 g, add 200 g of rice flour, and 400 ml of MM liquid medium. After bagging (16.5 cm × 37 cm polyethylene cultivation bag), put it into the autoclave for sterilization (121 o C, 120 min). After cooling, transfer it to the laminar flow hood. Take the original strain of Antrodia camphorata cultured in liquid and inoculate it into the fungus bag, and culture it for 50 days at a constant temperature of 28°C in the dark. Example 3

[0024] Harvest the culture product of Antrodia camphorata 1. For the mycelia cultured by PDA liquid fermentation, use a funnel to separate the mycelia from the broth. Blot the obtained mycelia dry with absorbent paper, transfer them to a sterilized centrifuge tube with forceps, immediately put them into liquid nitrogen for quick freezing, and then store them in a -80 o °C refrigerator.

[0025] 2. For the mycelia cultured by solid fermentation and fungus bag, carefully pick and separate them with forceps, transfer them to a sterilized centrifuge tube, immediately put them into liquid nitrogen for quick freezing, and then store them in a -80 o °C refrigerator.

[0026] 3. For the fruiting bodies of Antrodia camphorata, carefully pick and separate the fruiting bodies from the basswood with forceps, transfer them to a sterilized centrifuge tube, immediately put them into liquid nitrogen for quick freezing, and then store them in a -80 o °C refrigerator.

[0027] For each sample of the fruiting bodies of Antrodia camphorata and the mycelia of Antrodia camphorata cultured in different formula media, take 3 replicates.

[0028] Detection of the contents of anti-cancer active compounds Antcin B, Antcin C and Dankasterone A Detect the contents of Antcin B, Antcin C and Dankasterone A in the fruiting bodies of Antrodia camphorata and the products of Antrodia camphorata cultured under different treatments. The specific operation steps are as follows: 1. Metabolite extraction Weigh 60 mg of the sample into a 2 mL centrifuge tube; add 500 µL of methanol (-20°C) and 500 µL of H 2O (at 4 °C), vortex for 30 s, add 100 mg of glass beads; place the centrifuge tube containing the sample in a 2 mL adapter provided with the instrument, immerse it in liquid nitrogen for rapid freezing for 5 min, take out the centrifuge tube and thaw it at room temperature, place the centrifuge tube in the 2 mL adapter again, install it in the grinder, and oscillate at 55 Hz for 2 min; repeat twice; take out the centrifuge tube, centrifuge at 12,000 rpm at 4 °C for 10 min, take the supernatant, centrifuge and concentrate to dryness; dissolve the sample with 300 μL of 50% 2-chlorophenylalanine (4 ppm) methanol aqueous solution (1:1, at 4 °C), filter through a 0.22 µm membrane to obtain the sample to be tested, and perform LC-MS analysis on the machine; 2. Machine detection Chromatographic conditions: Use an ACQUITY UPLC® HSS T3 1.8 µm (2.1×150 mm) chromatographic column, set the auto sampler temperature to 8 °C, with a flow rate of 0.25 mL / min, a column temperature of 40 °C, inject 2 μL for gradient elution, and the mobile phase is 0.1% formic acid water (C) - 0.1% formic acid acetonitrile (D) for positive ions; 5 mM ammonium formate water (A) - acetonitrile (B) for negative ions. The gradient elution program is 0 - 1 min, 2% B / D; 1 - 9 min, 2% - 50% B / D; 9 - 12 min, 50% - 98% B / D; 12 - 13.5 min, 98% B / D; 13.5 - 14 min, 98% - 2% B / D; 14 - 20 min, 2% D - positive mode (14 - 17 min, 2% B - negative mode).

[0029] Mass spectrometry conditions: The instrument uses an electrospray ionization source (ESI), positive and negative ion ionization modes, the positive ion spray voltage is 3.50 kV, the negative ion spray voltage is 2.50 kV, the sheath gas is 30 arb, and the auxiliary gas is 10 arb. The capillary temperature is 325 °C, perform a full scan at a resolution of 70,000, the scan range is 81 - 1,000, and use HCD for secondary fragmentation, the collision voltage is 30 eV, and at the same time use dynamic exclusion to remove unnecessary MS / MS information.

[0030] Data analysis: According to the detection results in Step 3, metabolomics data analysis is performed on the detected samples. The specific steps are as follows: Perform data preprocessing, including format conversion, peak identification, filtering, alignment, and normalization. Check the data through chromatograms and quality control. Screen for differential compounds through multivariate statistical analysis, and identify the compounds by comparing with the mass spectrometry data in the database. Obtain the peak areas of compounds Antcin B, Antcin C, and Dankasterone A in different samples, and then compare them with the standard curves respectively. Finally, obtain the content differences of the detected compounds in different samples, so as to determine the content differences of the anti-cancer active compounds Antcin B, Antcin C, and Dankasterone A in the fruiting bodies of Cinnamomum kanehirae Hayata from Gaoligong Mountain and the mycelia of Cinnamomum kanehirae Hayata cultured by different treatments. The results are shown in Table 1.

[0031] Table 1 Contents of Antcin B, Antcin C, and Dankasterone A in Cinnamomum kanehirae Hayata from Different Samples

[0032] As can be seen from Table 1, the contents of Antcin B and Antcin C in the fruiting bodies of Cinnamomum kanehirae Hayata from Gaoligong Mountain are much higher than those of the mycelia cultured by artificial fermentation. However, the growth and development cycle of the fruiting bodies is too long, making it difficult to cultivate and utilize them on a large scale. Therefore, it is urgent to improve the artificial culture conditions to achieve the efficient production and utilization of Cinnamomum kanehirae Hayata from Gaoligong Mountain. And the content of Dankasterone A in the preferred camphor substrate mushroom bag culture of the present invention is 3.73 times that of the fruiting body, providing an effective artificial production method for the production of the anti-cancer active compound Dankasterone A.

[0033] The camphor substrate mushroom bag culture of the present invention improves the contents of the anti-cancer active compounds Antcin B, Antcin C, and Dankasterone A compared with the PDA liquid culture of Cinnamomum kanehirae Hayata from Gaoligong Mountain and the solid fermentation culture with the addition of Lauraceae plant substrates, and both exceed the addition of camphor substrates. It is confirmed that camphor can be used as an excellent substitute tree species for Cinnamomum kanehirae in promoting the growth of Cinnamomum kanehirae Hayata from Gaoligong Mountain and the production of bioactive substances, thus greatly reducing the production cost. At the same time, the mushroom bag culture can produce a large amount of mycelia, greatly improving the production efficiency of the anti-cancer active compounds Antcin B, Antcin C, and Dankasterone A, and the process is simple, controllable, and low-cost, providing a new and effective way for the large-scale production of the anti-cancer active compounds Antcin B, Antcin C, and Dankasterone A.

[0034] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for cultivating Gaoligong Antrodia cinnamomea with high yield of anticancer active compounds, characterized in that: The Gaoligong Antrodia cinnamomea preservation name is Taiwanofungus gaoligongensis YAFTG001, deposit number: CCTCC M 20232425, the culture medium used in the fermentation process consists of 400 parts of MM culture medium, 200 parts of rice flour and 200 parts of camphor sawdust, the MM culture medium is composed of 6 g / L sodium nitrate, 0.52 g / L potassium chloride, 1.52 g / L potassium dihydrogen phosphate and 0.52 g / L magnesium sulfate, and the balance is water, and the culture method is: constant temperature and light-proof culture at 28°C.

2. A method for factory-scale expansion of Gaoligong Antrodia cinnamomea to produce high-yield anticancer active compounds, characterized in that: The Gaoligong Antrodia cinnamomea preservation name is Taiwanofungus gaoligongensis YAFTG001, deposit number: CCTCC M 20232425, the culture method comprises the following steps: S1: Activation of mother culture: PDA solid medium is used for mother culture activation, and the culture is placed in a constant temperature incubator at 26°C and 60% humidity in the dark; S2: stock culture, the activated mother strain is cultured in a modified malt extract broth liquid culture medium: 5 g / L animal tissue pepsin digest, 5 g / L yeast extract, 5 g / L malt extract, 3 g / L maltose and 7 g / L glucose, the remainder is water; the culture method is: 28°C, 150 rpm constant temperature shaker in the dark to obtain the stock strain; S3: High-yield culture, the original seed is further inoculated into a culture medium, the culture medium composition consists of 400 parts of MM culture medium, 200 parts of rice flour and 200 parts of camphor sawdust, the MM culture medium is composed of 6 g / L sodium nitrate, 0.52 g / L potassium chloride, 1.52 g / L potassium dihydrogen phosphate and 0.52 g / L magnesium sulfate, and the balance is water, and the culture method is: constant temperature culture at 28°C in the dark.

3. The culture method according to claim 1 or 2, characterized in that The anticancer active compound is one or more of Antcin B, Antcin C or Dankasterone A.

4. A method for producing an anticancer active compound, characterized in that: The anticancer active compound is one or more of Antcin B, Antcin C or Dankasterone A. The method comprises the following steps: culturing Gaoligong Antrodia cinnamomea YAFTG001 with a preservation number of CCTCC M 20232425, separating and purifying the cultured product to obtain the anticancer active compound; the culture medium used in the culture process comprises 400 parts of MM culture medium, 200 parts of rice flour and 200 parts of camphor sawdust, wherein the MM culture medium comprises 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 1.52 g / L of potassium dihydrogen phosphate and 0.52 g / L of magnesium sulfate, and the remainder is water; and the culture method comprises: culturing at a constant temperature of 28°C in the dark.

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