A culture method for increasing the content of Antrocinnamomin H in Antrodia cinnamomea mycelium

Abstract

The present invention provides a cultivation method for increasing the antrocinnamomin H content in Antrodia cinnamomea mycelium, relating to the technical field of edible and medicinal fungi production processes. The cultivation method comprises inoculating Antrodia cinnamomea mycelium into a liquid fermentation medium supplemented with Polygonum multiflorum powder for cultivation; the liquid fermentation medium is a WDF liquid fermentation medium, and the formula is 0.5g / 100mL pea powder, 0.1g / 100mL potassium dihydrogen phosphate, 0.05g / 100mL magnesium sulfate, 0.5g / 100mL yeast powder, 0.01g / 100mL VB1, and the balance is water. The present invention overcomes the shortcomings of the prior art by optimizing the fermentation medium formula and improving the fermentation conditions, thereby increasing the antrocinnamomin H content in Gaoligong Antrodia cinnamomea mycelium and enhancing the pharmacological activity of Gaoligong Antrodia cinnamomea mycelium.

Description

Technical Field

[0001] The present invention relates to the technical field of edible and medicinal fungi production processes, and in particular to a cultivation method for increasing the content of antrocinnamomin H in Antrodia cinnamomea mycelium. Background Art

[0002] Antrodia cinnamomea is a valuable edible and medicinal fungus belonging to the phylum Basidiomycota, family Polyporaceae, and genus Taiwannofungus. Antrodia cinnamomea exhibits diverse physiological and pharmacological properties, including anticancer, antitumor, antioxidant, anti-inflammatory, hypoglycemic, hepatoprotective, immunomodulatory, and gut microbiota-modulating activities. Due to its therapeutic effects, particularly its prospective application as a chemopreventive agent, it has attracted increasing research attention for the development of new drugs.

[0003] Due to the scarcity of host plants and slow growth, wild Antrodia cinnamomea fruiting bodies are very expensive and in short supply. Therefore, artificial cultivation techniques, such as cut wood culture, solid-state fermentation, submerged fermentation, and plate culture, have been developed to supplement the growing demand for Antrodia cinnamomea. At the same time, obtaining sufficient quantities of high-quality Antrodia cinnamomea through artificial cultivation has been extremely challenging due to significant differences in bioactive metabolites between the fruiting bodies and cultured mycelium. Consequently, numerous studies have tested various methods to mitigate these differences in bioactive metabolites between the fruiting bodies and cultured mycelium. Different cultivation techniques and conditions can result in distinct chemical profiles. Cut wood culture, grown on Cinnamomum camphora wood, has the most similar chemical composition to wild Antrodia cinnamomea, but cultivation can take 2-3 years, and the Cinnamomum camphora trees required for basswood culture are scarce and expensive. Liquid submerged fermentation uses a mixture of nutrients and natural extracts as the culture medium, solid-support culture uses grains or agricultural byproducts as the substrate, and plate culture typically grows on agar medium containing various nutrients in a Petri dish. In comparison, liquid fermentation not only has a short cycle and low cost, but also facilitates large-scale cultivation. In addition, it is possible to obtain a variety of active ingredients by changing the culture medium composition and culture conditions.

[0004] The addition of Chinese herbal powder to the cultivation of medicinal fungi can significantly stimulate the production of bioactive metabolites in the fungi. Chinese herbal powder can provide additional carbon sources, nitrogen sources, and trace elements to promote fungal growth and metabolism. Secondary metabolites in Chinese herbal medicines may act as signal molecules to regulate key metabolic enzymes in fungi, affect metabolic flux, induce the activation of specific biosynthetic pathways in fungi, and increase the production of specific active ingredients. In addition, certain Chinese herbal ingredients may cause mild stress to fungi, prompting them to produce more secondary metabolites to adapt to environmental changes. The addition of Chinese herbal powder can be used as an effective regulatory strategy to increase the production of active metabolites in medicinal fungi and enhance their medicinal value.

[0005] With further in-depth research, an increasing number of bioactive metabolites have been isolated from the fruiting bodies and cultured mycelium of Antrodia cinnamomea, and their structures and functions have been determined. Over 200 compounds have been isolated and identified, including polysaccharides, triterpenoids, ubiquinone derivatives, maleic and succinic acid derivatives, benzene ring derivatives, and glycoproteins, exhibiting a wide range of biological activities. Among these bioactive components, antrocinnamomin H, a maleimide derivative isolated from Antrodia cinnamomea mycelium, exhibits a significant inhibitory effect on nitric oxide (NO) production by macrophages. The antrocinnamomin H content in Gaoligong Antrodia cinnamomea mycelium varies significantly under different culture conditions. Therefore, it is of great research significance to determine a culture method that can produce a large amount of antrocinnamomin H by varying culture conditions and is suitable for large-scale production. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides a cultivation method for increasing the antrocinnamomin H content in Antrodia cinnamomea mycelium. By optimizing the fermentation medium formula and improving the fermentation conditions, the antrocinnamomin H content in Gaoligong Antrodia cinnamomea mycelium is increased, thereby enhancing the pharmacological activity of Gaoligong Antrodia cinnamomea mycelium.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] A cultivation method for increasing the content of antrocinnamomin H in Antrodia cinnamomea mycelia, comprising inoculating the Antrodia cinnamomea mycelia into a liquid fermentation medium to which Polygonum multiflorum powder has been added for cultivation; the liquid fermentation medium is a WDF liquid fermentation medium having a formula of 0.5 g / 100 mL of pea 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 being water.

[0009] Preferably, the amount of the Polygonum multiflorum powder added to the liquid fermentation medium is 0.3-0.8 g / mL.

[0010] Preferably, the culture is carried out at 28±2° C. and 150±50 rpm in a constant temperature shaker for 15-20 days.

[0011] Preferably, the method for obtaining the Antrodia cinnamomea mycelium comprises the following steps:

[0012] S1. Place the Antrodia cinnamomea strain in an activation culture medium and culture it in a constant temperature incubator at 26°C and 60% humidity for 30 days;

[0013] S2. The activated Antrodia cinnamomea strain was inoculated into a liquid culture medium, and cultured in a constant temperature shaker at 28° C. and 150 rpm for 10 days to obtain Antrodia cinnamomea mycelium.

[0014] Preferably, the formula of the activation culture medium in step S1 is: potato flour 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB1 0.01g / 100mL, and agar 1.1g / 100mL.

[0015] Preferably, the formula of the liquid culture medium in step S2 is: 5 g / L of animal tissue pepsin digest, 5 g / L of yeast extract powder, 5 g / L of malt extract powder, 3 g / L of maltose, 7 g / L of glucose, and the balance is water.

[0016] The present invention provides a cultivation method for increasing the content of antrocinnamomin H in Antrodia cinnamomea mycelium, which has the following advantages over the prior art:

[0017] This method uses WDF liquid fermentation medium supplemented with Polygonum multiflorum powder as the fermentation medium for Antrodia cinnamomea mycelium. Within 20 days of cultivation, the antrocinnamomin H content in the mycelium is significantly increased, reaching 7.5 times the antrocinnamomin H content in WDF liquid fermentation medium and 13.9 times the antrocinnamomin H content in WDF liquid fermentation medium plus Astragalus powder. This provides a pathway for the efficient development of the medicinal value of Antrodia cinnamomea and the efficient production of the bioactive ingredient antrocinnamomin H. The process is simple and controllable, with a short cultivation cycle and low cost, enabling large-scale batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the growth of Antrodia cinnamomea solid culture WDFS mycelium according to the present invention;

[0019] Figure 2 This is a diagram showing the mycelial growth of Antrodia cinnamomea described in the present invention in WDF liquid culture medium;

[0020] Figure 3 This is a graph showing the mycelial growth in a liquid culture medium (HQ) supplemented with 0.5 g of astragalus powder per 100 mL of the present invention;

[0021] Figure 4This is a graph showing mycelial growth in a liquid culture medium (HSW) supplemented with 0.5 g of Polygonum multiflorum powder per 100 mL of the present invention. DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] The Antrodia cinnamomea used in the following examples is Gaoligong Antrodia cinnamomea, and its preservation name is Taiwanofungus gaoligongensis YAFTG001, deposit number: CCTCC M 20232425, deposited in the China Center for Type Culture Collection, Wuhan University, China; deposit date: December 4, 2023; deposit number: CCTCC M 20232425; this strain has been disclosed by patent CN202410173373.2 "A cultivation method for inducing Gaoligong Antrodia cinnamomea to produce bacterial compounds that inhibit plant diseases." Example 1:

[0024] Antrodia cinnamomea fermentation culture:

[0025] 1. Activation of Antrodia cinnamomea fungi

[0026] To activate the Antrodia cinnamomea culture, disinfect the clean bench and perform UV sterilization 15 minutes in advance. Prepare the Antrodia cinnamomea culture and PDA solid culture medium (potato starch 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB1 0.01g / 100mL, agar 1.1g / 100mL). First, thoroughly burn the inoculation knife with an alcohol lamp. After cooling, pick a small piece of the original Antrodia cinnamomea culture and inoculate it into the PDA solid culture medium. Label it (strain number, date), seal it with film, and place it in a constant temperature incubator at 26°C and 60% humidity for 30 days. During this period, observe and record every 5 days. When the Antrodia cinnamomea mycelium has covered the entire culture medium, store it in a 4°C refrigerator until it is ready for use.

[0027] 2. Liquid culture of Antrodia cinnamomea

[0028] Preparation of Antrodia cinnamomea liquid culture medium: scrape a quarter of the mycelium from a 60mm culture dish and place it in a 2mL sterilized centrifuge tube, then add 1mL of sterile water and crush it for 2min. Then take 500µL of the crushed sample liquid and inoculate it into 250mL conical flasks containing 100ml of modified malt extract broth liquid culture medium (5g / L animal tissue pepsin digest, 5g / L yeast extract, 5g / L malt extract, 3g / L maltose, 7g / L glucose, and the rest is water), place it in a constant temperature shaker at 28℃ and 150rpm, and culture for 10d to obtain Antrodia cinnamomea mycelium.

[0029] 3. Preparation of Antrodia cinnamomea solid culture medium and liquid fermentation culture medium:

[0030] The formula of WDFS solid medium is as follows: first add 20g of pea flour into the tissue culture bottle, then add 50ml of MM solution and mix well (MM solution formula: 6g / L sodium nitrate, 0.52g / L potassium chloride, 0.52g / L magnesium sulfate, 1.52g / L potassium dihydrogen phosphate);

[0031] WDF liquid fermentation medium: pea powder 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB1 0.01g / 100mL;

[0032] HQ liquid fermentation medium: 0.5 g / 100 mL of Astragalus powder was exogenously added to WDF liquid fermentation medium;

[0033] HSW liquid fermentation medium: 0.5 g / 100 mL Polygonum multiflorum powder was exogenously added to WDF liquid fermentation medium;

[0034] Each group of culture medium was sterilized in an autoclave (121°C, 20 min) and then used.

[0035] 4. Antrodia cinnamomea fermentation culture:

[0036] The above-mentioned liquid-cultured Gaoligong Antrodia cinnamomea strain (Antrodia cinnamomea mycelium) was inoculated into solid culture medium, WDF liquid fermentation medium, HQ liquid fermentation medium and HSW liquid fermentation medium, respectively, and placed in a constant temperature shaking incubator for culture (temperature 28°C, rotation speed 150 r / min, culture time 18 d).

[0037] 5. Harvest, extract, ferment and culture Gaoligong Antrodia cinnamomea crude extract

[0038] (1) Use a funnel to separate the mycelium from the bacterial solution. The obtained bacterial solution is placed in a 250 mL Erlenmeyer flask and extracted with 1:1 ethyl acetate. Ultrasonic vibration is performed for 40 min and then allowed to stand for 12 h. The extracted bacterial solution is then poured into a separatory funnel for full extraction. During this period, the solution is shaken up and down (3 to 5 times). After standing for 3 h, the upper layer of the extract in the separatory funnel is poured into a round-bottom flask. A rotary evaporator (temperature 50 °C, speed 80 r / min) is used to obtain a crude extract. The crude extract is rinsed with acetone and placed in a 2 mL weighed centrifuge tube and air-dried.

[0039] (2) The obtained mycelium was dried with absorbent paper, transferred to a sterilized centrifuge tube with tweezers, immediately placed in liquid nitrogen for quick freezing, and stored in a -80℃ refrigerator.

[0040] (3) At the same time, the fruiting bodies of Antrodia cinnamomea and solid fermentation mycelium were sampled and preserved; three replicates were taken for each sample of Antrodia cinnamomea fruiting bodies and Gaoligong Antrodia cinnamomea mycelium fermented and cultured in different culture media.

[0041] 6. Antrocinnamomin H content detection:

[0042] (1) Metabolite extraction

[0043] Weigh 60 mg of sample into a 2 mL centrifuge tube; add 500 µL of methanol (-20°C) and 500 µL of H2O (4°C), vortex for 30 seconds, and add 100 mg of glass beads; place the centrifuge tube containing the sample in the 2 mL adapter provided with the instrument and quickly freeze it in liquid nitrogen for 5 minutes. Remove the centrifuge tube and freeze and thaw it at room temperature. Place the centrifuge tube again in the 2 mL adapter, install it in the grinder, and oscillate at 55 Hz for 2 minutes. Repeat step 3 twice;

[0044] Remove the centrifuge tube, centrifuge at 12,000 rpm and 4°C for 10 min, collect the supernatant, concentrate and dry by centrifugation; dissolve the sample in 300 µL of 50% 2-chlorophenylalanine (4 ppm) methanol aqueous solution (1:1, 4°C), filter through a 0.22 µm membrane to obtain the sample to be tested, and perform LC-MS detection; perform LC-MS detection on the sample to be tested.

[0045] 2. On-machine testing

[0046] Chromatographic conditions: An ACQUITY UPLC® HSS T3 1.8 µm (2.1 × 150 mm) column was used. The autosampler temperature was set at 8°C. Gradient elution was performed with a 2 µL injection at a flow rate of 0.25 mL / min and a column temperature of 40°C. The mobile phases consisted of positive ion 0.1% formic acid in water (C) - 0.1% formic acid in acetonitrile (D); negative ion 5 mM ammonium formate in water (A) - acetonitrile (B). The gradient elution program was: 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).

[0047] Mass spectrometry conditions: The instrument used an electrospray ionization (ESI) source in positive and negative ionization modes, with a positive spray voltage of 3.50 kV and a negative spray voltage of 2.50 kV, a sheath gas of 30 arb, and an auxiliary gas of 10 arb. The capillary temperature was 325°C. Full scans were performed at a resolution of 70,000, with a scan range of 81 to 1,000. HCD was used for secondary fragmentation, with a collision voltage of 30 eV. Dynamic exclusion was used to remove unnecessary MS / MS information.

[0048] Data Analysis: Based on the above test results, metabolomics data analysis was performed on the samples tested. The specific steps are as follows: Data preprocessing, including format conversion, peak identification, filtering, alignment, and normalization. Data verification was performed through chromatograms and quality control. Differential compounds were screened through multivariate statistical analysis and identified by comparing mass spectrometry data in a database. The peak areas of antrocinnamomin H in different samples were determined to compare the content of antrocinnamomin H in Gaoligong Antrodia cinnamomea fruiting bodies and mycelia cultured in different culture media. The results are shown in the table below:

[0049]

[0050] As can be seen from the table above, HSW liquid fermentation medium greatly increases the content of antrocinnamomin H compared to other culture methods, providing a new and effective way for the large-scale production of antrocinnamomin H.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for increasing the content of Antrocinnamomin H in Antrodia cinnamomea mycelium, characterized in that: The culture method comprises inoculating Antrodia cinnamomea mycelium into a liquid fermentation medium to which Polygonum multiflorum powder is added for culture; the liquid fermentation medium is a WDF liquid fermentation medium, and the formula is 0.5g / 100mL of pea powder, 0.1g / 100mL of potassium dihydrogen phosphate, 0.05g / 100mL of magnesium sulfate, 0.5g / 100mL of yeast powder, 0.01g / 100mL of VB1, and the balance is water; The amount of the Polygonum multiflorum powder added to the liquid fermentation medium is 0.3-0.8 g / mL.

2. The culture method according to claim 1, wherein: The culture conditions are 28±2°C, 150±50 rpm in a constant temperature shaker for 15-20 days.

3. The culture method according to claim 1, wherein The method for obtaining the Antrodia cinnamomea mycelium comprises the following steps: S1. Place the Antrodia cinnamomea strain in an activation culture medium and culture it in a constant temperature incubator at 26°C and 60% humidity for 30 days; S2. The activated Antrodia cinnamomea strain was inoculated into a liquid culture medium, and cultured in a constant temperature shaker at 28° C. and 150 rpm for 10 days to obtain Antrodia cinnamomea mycelium.

4. The culture method according to claim 3, wherein: The formula of the activation culture medium in step S1 is: potato flour 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB1 0.01g / 100mL, and agar 1.1g / 100mL.

5. The culture method according to claim 3, wherein: The formula of the liquid culture medium in step S2 is: 5 g / L of animal tissue pepsin digest, 5 g / L of yeast extract powder, 5 g / L of malt extract powder, 3 g / L of maltose, 7 g / L of glucose, and the balance is water.

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