A cultivation method for high-yielding anti-cancer active compounds of Antrodia cinnamomea var. kanoi

Abstract

The present invention belongs to the technical field of the production process of edible and medicinal fungi. It is a cultivation method for producing high-yield anti-cancer active compounds from Antrodia cinnamomea of Gaoligong Mountain. The Antrodia cinnamomea of Gaoligong Mountain 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, with the balance being water. Through the cultivation of mushroom bags with camphor sawdust as the substrate, the present invention increases the contents of anti-cancer active compounds Antcin B, Antcin C, and Dankasterone A in the mycelium of Antrodia cinnamomea of Gaoligong Mountain, greatly improves the anti-cancer drug activity of the mycelium of Antrodia cinnamomea of Gaoligong Mountain, and promotes the large-scale production of anti-cancer active compounds of Antrodia cinnamomea of Gaoligong Mountain.

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 anti-cancer active compounds Antcin B, Antcin C and Dankasterone A in the mycelium of Antrodia cinnamomea var. kanoi fermented in culture, improving the medicinal activity of the mycelium of Antrodia cinnamomea var. kanoi fermented in culture, and promoting the large-scale production of anti-cancer 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 anti-cancer, anti-tumor, antioxidant, anti-inflammatory, hypoglycemic, hepatoprotective, immunomodulatory, and intestinal microbiota-regulating 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 northwestern part 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 the active compound biosynthesis gene cluster. At present, there are many reports on the active products of Antrodia cinnamomea fermented in culture, but the research on the production of a large amount of active compounds by Antrodia cinnamomea var. kanoi fermented in culture 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. So far, more than 200 compounds have been isolated and identified from it, including polysaccharides, triterpenoids, ubiquinone derivatives, maleic acid and succinic acid derivatives, benzene ring derivatives and glycoproteins. Research shows that triterpenoids are the main anti-tumor active ingredients of Antrodia cinnamomea, and their skeletal structures are mainly ergostane and lanostane. 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 it 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 need to be developed to meet the growing demand for the active ingredients of Antrodia cinnamomea. Different cultivation conditions have a great impact on the biological activities of the mycelia of Antrodia cinnamomea. 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 cultivated 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. highligongensis. By trying different cultivation methods, optimizing the cultivation formula, and improving the fermentation conditions, the biomass of Antrodia cinnamomea var. highligongensis 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. highligongensis 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:

[0006] The present invention provides a cultivation method for high-yielding anti-cancer active compounds of Antrodia cinnamomea var. highligongensis, and the preservation name of the Antrodia cinnamomea var. highligongensis is Taiwanofungus gaoligongensisYAFTG001, 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, with the balance being water. The cultivation method is: constant temperature cultivation at 28°C in the dark.

[0007] The present invention also provides a cultivation method for industrialized large-scale cultivation of Ganoderma lucidum (Leyss. ex Fr.) Karst. with high yield of anti-cancer active compounds, characterized in that the name of the deposited Ganoderma lucidum (Leyss. ex Fr.) Karst. is Taiwanofungus gaoligongensis YAFTG001, Deposit Number: CCTCC M 20232425. The cultivation method includes the following steps:

[0008] S1: Mother spawn activation

[0009] The mother spawn is activated using PDA solid medium and placed in a constant temperature incubator at 26°C and 60% humidity for cultivation in the dark.

[0010] S2: Primary spawn cultivation

[0011] The activated mother spawn 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: cultivation in a constant temperature shaker at 28°C and 150 rpm in the dark to obtain the primary spawn.

[0012] S3: High-yield cultivation

[0013] The primary spawn is further inoculated into a culture medium composed 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 cultivation at 28°C in the dark.

[0014] Furthermore, the anti-cancer active compound is one or more of Antcin B, Antcin C, or Dankasterone A.

[0015] Furthermore, the present invention also provides a method for producing an anti-cancer active compound, which is one or more of Antcin B, Antcin C or Dankasterone A. The method is to culture Ganoderma lingzhi YAFTG001 with the preservation number: CCTCC M 20232425, and separate and purify the cultured product to obtain the anti-cancer active compound. The composition of the culture medium used in the culture 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 culture method is: constant temperature dark culture at 28°C.

[0016] The present invention has the following beneficial effects:

[0017] The contents of Antcin B, Antcin C and Dankasterone A in the Ganoderma lingzhi mycelium produced by the technical solution provided by the present invention are the highest, and their relative peak areas are 316.24, 4825.45 and 3174.24 respectively; they are all extremely significantly higher than the contents of anti-cancer active ingredients in the mycelium cultured by other methods. Using camphor as the substrate of the fungus bag to culture Ganoderma lingzhi shows stronger application potential, providing a way for the efficient development of the medicinal value of Ganoderma lingzhi 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 mycelium, and the process is simple, controllable and low-cost, which helps to achieve large-scale production. Description of the Drawings

[0018] Figure 1 It is a diagram of the mycelial growth of Ganoderma lingzhi described in the present invention in PDA liquid medium;

[0019] Figure 2 It is a diagram of the mycelial growth of Ganoderma lingzhi described in the present invention with the addition of solid fermentation of Cinnamomum kanehirae branch sawdust;

[0020] Figure 3 It is a diagram of the mycelial growth of Ganoderma lingzhi described in the present invention with the addition of solid fermentation of Cinnamomum camphora branch sawdust;

[0021] Figure 4 It is a diagram of the mycelial growth in the fungus bag of Cinnamomum kanehirae substrate described in the present invention;

[0022] Figure 5 It is a diagram of the mycelial growth in the fungus bag of Cinnamomum camphora substrate described in the present invention. Specific Embodiments

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. The purchased commodities in the test method, if the specific conditions are not indicated, are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer can all be conventional products purchased from the market.

[0024] The preservation name of Gaoligong Antrodia cinnamomea is Taiwanofungus gaoligongensis YAFTG001, deposit number: CCTCC M 20232425, deposited in China Center for Type Culture Collection, deposit address: Wuhan University, China; deposit date: December 1, 2023. This strain has been disclosed by patent CN202410173373.2 A method for inducing Gaoligong Antrodia cinnamomea to produce bacterial compounds that inhibit plant diseases.

[0025] Antcin B (CAS No.: 163597-25-9), Antcin C (CAS No.: 163565-76-2) and DankasteroneA.

[0026] The camphor wood sawdust and kanahimai ... sawdust used in the present invention are both sieved through a 40-mesh sieve for later use.

[0027] 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.

[0028] 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. Embodiment 1:

[0029] Gaoligong Antrodia cinnamomea fungus culture

[0030] 1. Activation of Antrodia camphorata strain from Gaoligong Mountains (mother strain)

[0031] Both the fruiting body of Antrodia camphorata from Gaoligong Mountains and the Antrodia camphorata YAFTG001 strain are stored in the laboratory. The preparation for activating the Antrodia camphorata strain is as follows: Disinfect the laminar flow bench and turn on the ultraviolet sterilization 15 minutes in advance. Prepare the Antrodia camphorata strain and PDA solid medium (0.5 g / 100 mL of potato powder, 0.1 g / 100 mL of potassium dihydrogen phosphate, 0.05 g / 100 mL of magnesium sulfate, 0.5 g / 100 mL of yeast powder, 0.01 g / 100 mL of VB1, 1.1 g / 100 mL of agar, and the balance is water). First, fully burn the forceps or inoculation knife with an alcohol lamp. After cooling, pick a small piece of the original Antrodia camphorata strain and inoculate it into the PDA solid medium. After making marks (strain number, date), seal the film and place it in an incubator at 26 °C and 60% humidity for 30 days. Observe and record every 5 days during this period. When the Antrodia camphorata mycelium covers the entire medium, place it in a refrigerator at 4 o °C for storage for later use.

[0032] 2. Liquid culture of Antrodia camphorata from Gaoligong Mountains (primary culture)

[0033] Preparation of the liquid culture medium for Antrodia camphorata from Gaoligong Mountains: Scrape one-fourth of the mycelium in a 60-mm culture dish of Antrodia camphorata mycelium into a 2-mL sterilized centrifuge tube, 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

[0034] Cultivation of Antrodia camphorata in camphor tree substrate bags

[0035] Formulation composition of the Antrodia camphorata in camphor tree substrate bags: 400 parts of MM medium + 200 parts of rice flour + 200 parts of camphor tree 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 oC, 120 min). After cooling, transfer it to a laminar flow hood. Take the original strain of Antrodia camphorata cultured in liquid and inoculate it into the fungus bag, and culture it at a constant temperature of 28 °C in the dark for 50 days.

[0036] Comparative Example 1:

[0037] Liquid culture of Antrodia camphorata in PDA medium

[0038] The composition of the PDA liquid fermentation medium is: PDA (0.5 g / 100 mL of potato powder, 0.1 g / 100 mL of potassium dihydrogen phosphate, 0.05 g / 100 mL of magnesium sulfate, 0.5 g / 100 mL of yeast powder, 0.01 g / 100 mL of VB1, and the balance is water), put it into an autoclave for sterilization (121 o C, 20 min). After cooling, transfer it to a laminar flow hood. Take the original strain of Antrodia camphorata cultured in liquid and inoculate it into the PDA liquid medium, and then put it into a constant temperature shaking incubator for culture (temperature 28 °C, rotation speed 150 r / min, culture time 18 d).

[0039] Comparative Example 2:

[0040] Solid fermentation culture with added dry sawdust of Cinnamomum kanehirae

[0041] The composition of the medium for solid fermentation culture with dry sawdust of Cinnamomum kanehirae is: 20 g of Cinnamomum kanehirae sawdust + 20 g of yellow wheat + 50 ml of MM medium (MM medium: 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 0.52 g / L of magnesium sulfate, 1.52 g / L of potassium dihydrogen phosphate, and the balance is water). Put the prepared medium into an autoclave for sterilization (121 o C, 20 min). After cooling, transfer it to a laminar flow hood. Take the original strain of Antrodia camphorata cultured in liquid and inoculate it into the culture bottle, and culture it at a constant temperature of 28 °C in the dark for 20 days.

[0042] Comparative Example 3:

[0043] Solid fermentation culture with added dry sawdust of Cinnamomum camphora

[0044] The composition of the medium for solid fermentation culture with dry sawdust of Cinnamomum camphora is: 20 g of Cinnamomum camphora sawdust + 20 g of yellow wheat + 50 ml of MM (MM medium: 6 g / L of sodium nitrate, 0.52 g / L of potassium chloride, 0.52 g / L of magnesium sulfate, 1.52 g / L of potassium dihydrogen phosphate, and the balance is water). Put the prepared medium into an autoclave for sterilization (121 o C, 20 min). After cooling, transfer it to a laminar flow hood. Take the original strain of Antrodia camphorata cultured in liquid and inoculate it into the culture bottle, and culture it at a constant temperature of 28 °C in the dark for 20 days.

[0045] Comparative Example 4:

[0046] Cultivation of Antrodia cinnamomea Matrix Bacterial Bags

[0047] The formula composition of the Antrodia cinnamomea matrix bacterial bag is: 400 parts of MM medium + 200 parts of rice flour + 200 parts of Antrodia cinnamomea sawdust. MM medium: sodium nitrate 6 g / L, potassium chloride 0.52 g / L, potassium dihydrogen phosphate 1.52 g / L, magnesium sulfate 0.52 g / L, and the balance is water. After breaking the Antrodia cinnamomea tree trunk into sawdust, it is put into an autoclave for sterilization (121 o °C, 120 min). After cooling, 200 g is taken out, 200 g of rice flour is added, and 400 ml of MM liquid medium is added. After bagging (16.5 cm × 37 cm polyethylene cultivation bag), it is put into an autoclave for sterilization (121 o °C, 120 min). After cooling, it is transferred to a laminar flow hood, and the original strain of Antrodia cinnamomea cultured in liquid is inoculated into the bacterial bag and cultured at a constant temperature of 28°C in the dark for 50 days.

[0048] Example 3:

[0049] Harvesting the Cultivation Products of Antrodia cinnamomea

[0050] 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.

[0051] 2. For the mycelia of solid fermentation and bacterial bag cultivation, 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.

[0052] 3. For the fruiting bodies of Antrodia cinnamomea, carefully pick and separate the fruiting bodies from the log 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.

[0053] For each sample of the fruiting bodies of Antrodia cinnamomea and the mycelia of Antrodia cinnamomea cultured in different formula media, 3 replicates are taken.

[0054] Detection of the Contents of Anticancer Active Compounds Antcin B, Antcin C and Dankasterone A

[0055] Detect the contents of Antcin B, Antcin C and Dankasterone A in the fruiting bodies of Antrodia cinnamomea and the products of Antrodia cinnamomea cultured under different treatments. The specific operation steps are as follows:

[0056] 1. Metabolite Extraction

[0057] Weigh 60 mg of the sample into a 2 mL centrifuge tube; add 500 µL of methanol (-20 °C) and 500 µL of H2O (4 °C), vortex for 30 s, and add 100 mg of glass beads; place the centrifuge tube containing the sample into 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, then place the centrifuge tube into the 2 mL adapter again, install it into the grinder, and oscillate at 55 Hz for 2 min; repeat twice; take out the centrifuge tube, centrifuge at 12000 rpm at 4 °C for 10 min, take the supernatant, and centrifuge and concentrate to dryness; dissolve the sample with 300 µL of 50% 2-chlorophenylalanine (4 ppm) methanol aqueous solution (1:1, 4 °C), filter it through a 0.22 µm membrane to obtain the sample to be tested, and perform LC-MS analysis on the machine;

[0058] 2. Machine detection

[0059] Chromatographic conditions: Use an ACQUITY UPLC® HSS T3 1.8 µm (2.1×150 mm) chromatographic column, set the temperature of the autosampler 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) in positive ion mode; 5 mM ammonium formate water (A) - acetonitrile (B) in negative ion mode. 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).

[0060] 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 scanning range is 81 - 1 000, and perform HCD for secondary fragmentation, the collision voltage is 30 eV, and at the same time use dynamic exclusion to remove unnecessary MS / MS information.

[0061] Data analysis: According to the detection results in step 3, metabolomic data analysis is performed on the detected samples. The specific steps are as follows: Data preprocessing is carried out, including format conversion, peak identification, filtering, alignment, and normalization. Data inspection is performed through chromatograms and quality control. Differential compounds are screened through multivariate statistical analysis, and compounds are identified by comparing with mass spectrometry data in the database. The peak areas of compounds Antcin B, Antcin C, and Dankasterone A in different samples are obtained, and then compared with the standard curves respectively. Finally, the content differences of the detected compounds in different samples are obtained, 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.

[0062] Table 1 Contents of Antcin B, Antcin C, and Dankasterone A in Cinnamomum kanehirae Hayata from Different Samples

[0063]

[0064] 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 be cultured and utilized 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 matrix fungus 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.

[0065] The camphor matrix fungus bag culture of the present invention has increased 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 and the solid fermentation culture with the addition of Lauraceae plant matrix, and both exceed the addition of the camphor tree matrix, confirming that camphor can be used as an excellent substitute tree species for the camphor tree 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 fungus 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.

[0066] 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 cultivation method for high-yielding anti-cancer active compounds of Antrodia cinnamomea var. kanoi, characterized in that, The preservation name of the Ganoderma tsugae from Gaoligong is Taiwanofungus gaoligongensis YAFTG001, preservation 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, with the balance being water. The cultivation method is: constant temperature cultivation at 28°C in the dark. The anti-cancer active compounds are Antcin B, Antcin C, and Dankasterone A.

2. A cultivation method for large-scale industrialized cultivation of Antrodia cinnamomea from Gaoligong Mountain with high yield of anti-cancer active compounds, characterized in that, The preservation name of the Ganoderma tsugae from Gaoligong is Taiwanofungus gaoligongensis YAFTG001, preservation number: CCTCC M 20232425, and the cultivation method includes the following steps: S1: Activation of stock culture. The stock culture is activated using PDA solid medium and cultured in the dark in a constant temperature incubator at 26°C and 60% humidity. S2: Cultivation of spawn. The activated stock 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: culturing in the dark in a constant temperature shaker at 28°C and 150 rpm to obtain the spawn. S3: High-yield cultivation. The spawn is further inoculated into a medium composed of 400 parts of MM medium, 200 parts of rice flour, and 200 parts of camphor sawdust. The MM medium consists 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: culturing in the dark at a constant temperature of 28°C. The anti-cancer active compounds are Antcin B, Antcin C, and Dankasterone A.

3. A method for producing an anti-cancer active compound, characterized in that, The anti-cancer active compounds are Antcin B, Antcin C, and Dankasterone A. The method is to culture Taiwanofungus gaoligongensis YAFTG001 with the deposit number: CCTCC M 20232425, and separate and purify the cultured product to obtain the anti-cancer active compounds. The medium composition used in the cultivation process is composed of 400 parts of MM medium, 200 parts of rice flour, and 200 parts of camphor sawdust. The MM medium consists 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: culturing in the dark at a constant temperature of 28°C.

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