Culture method for increasing content of Antrodin C in antrodia camphorata mycelium

Abstract

The invention provides a culture method for increasing the content of Antrodin C in antrodia camphorata mycelia, and relates to the technical field of edible and medicinal fungus production processes. The culture method comprises the following steps: inoculating antrodia camphorata mycelia into a PDA liquid culture medium added with melia azedarach tender leaf homogenate, and culturing in a constant-temperature shaking table at 28 + / -2 DEG C and 150 + / -50 rpm for 15-20 days. According to the method, the defects in the prior art are overcome, the content of Antrodin C in the Gaochaigong antrodia camphorata mycelia is increased by optimizing the formula of the fermentation culture medium and improving the fermentation conditions, and the pharmacological activity of the Gaochaigong antrodia camphorata mycelia is enhanced.

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 Antrodin C 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. The Gaoligong Antrodia cinnamomea described in this paper was discovered in 2018 as a new species of the genus Taiwannofungus, native to the Gaoligong Mountains in northwestern Yunnan Province. Whole-genome analysis revealed that Gaoligong Antrodia cinnamomea and Antrodia cinnamomea share high similarity in the gene clusters responsible for the biosynthesis of active compounds. While there have been numerous reports on active products produced by fermentation of Antrodia cinnamomea, research on the production of large quantities of active compounds by fermentation of Gaoligong Antrodia cinnamomea is limited.

[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, low cost, and is convenient for large-scale cultivation, but also has the potential to produce a variety of active ingredients by changing the culture medium composition, culture conditions, etc.

[0004] With further research, a growing 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, antrodins (AE) are unique and important active ingredients in Antrodia cinnamomea. The antrodin class of compounds consists of maleic and succinic acid derivatives, with Antrodin C being the most extensively studied antrodin compound, with research primarily focused on its bioactivity and pharmacology. Antrodin C has been shown to significantly inhibit hepatitis C virus and Lewis lung cancer cells, and exhibits enhanced inhibitory effects on TGF-β1-induced epithelial-mesenchymal transition and breast cancer cell metastasis. However, Gaoligong Antrodia cinnamomea grows slowly in nature and has a low capacity to produce antrodin C. Therefore, developing a culture method that can produce large amounts of antrodin C and is suitable for large-scale production is of great research significance. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a cultivation method for increasing the content of Antrodin C in Antrodia cinnamomea mycelium. By optimizing the fermentation medium formula and improving the fermentation conditions, the content of Antrodin C in Gaoligong Antrodia cinnamomea mycelium is increased, and the pharmacological activity of Gaoligong Antrodia cinnamomea mycelium is enhanced.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions: A cultivation method for increasing the content of Antrodin C in Antrodia cinnamomea mycelia, comprising inoculating the Antrodia cinnamomea mycelia into a liquid culture medium to which a homogenate of young leaves of Melia azedarach is added for cultivation.

[0007] Preferably, the liquid culture medium is a PDA liquid culture medium, and the formula is: potato flour 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB10.01g / 100mL, and the balance is water; wherein the addition amount of neem young leaf homogenate is 1-3g / 100mL.

[0008] Preferably, the culture conditions are 28±2° C., 150±50 rpm in a constant temperature shaker, and culture for 15-20 days.

[0009] Preferably, the preparation method of the chinaberry young leaf homogenate is to soak the chinaberry young leaves in tap water, clean them, remove impurities, dry them, grind them into powder in a crusher, and store them at room temperature for later use.

[0010] Preferably, the method for obtaining the Antrodia cinnamomea mycelium comprises the following steps: S1. Inoculate Antrodia cinnamomea strains into activation culture medium and culture 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.

[0011] Preferably, the activation culture medium formula 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.

[0012] 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.

[0013] The present invention provides a cultivation method for increasing the content of Antrodin C in Antrodia cinnamomea mycelium, which has the following advantages over the prior art: By comparing the antrodin C content in solid and liquid cultures, as well as in Gaoligong Antrodia cinnamomea fruiting bodies, this study identified the optimal medium for antrodin C production in Antrodia cinnamomea using a homogenate of young leaves of Melia azedarach (KL). This method resulted in the highest antrodin C content in Antrodia cinnamomea, reaching 3.9 times that of liquid-cultured PDA, 14.4 times that of Antrodia cinnamomea fruiting bodies (GLG), and 23.7 times that of solid-cultured XMF. This method provides a pathway for the efficient development of the medicinal value of Antrodia cinnamomea and the efficient production of the bioactive ingredient, antrodin C. The process is simple and controllable, with a short culture cycle and low cost, enabling large-scale batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram showing the growth of XMF mycelium in solid culture of Antrodia cinnamomea according to the present invention; Figure 2 This is a diagram showing the mycelial growth of Antrodia cinnamomea described in the present invention in a PDA liquid culture medium; Figure 3 This is a diagram showing the mycelial growth of Antrodia cinnamomea described in the present invention in KL liquid culture medium; Figure 4 This is a bar chart showing the content of Antrodin C in different samples. DETAILED DESCRIPTION

[0015] 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.

[0016] The Antrodia cinnamomea used in the following examples is Gaoligong Antrodia cinnamomea with a deposit number of CCTCC M 20232425. Example

[0017] Cultivation of Antrodia cinnamomea: (1) Activation of Antrodia cinnamomea strains: Disinfect the clean bench and use UV sterilizer 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 a pair of tweezers or an inoculating knife with an alcohol lamp. Once cooled, pick a small piece of the original Antrodia cinnamomea culture and inoculate it onto the PDA solid culture medium. Label the medium (strain number, date), seal the medium, and incubate in a constant temperature incubator at 26°C and 60% humidity for 30 days. Observe and record the culture every 5 days. Once the Antrodia cinnamomea mycelium has spread throughout the medium, store it in a 4°C refrigerator until it is ready for use.

[0018] (2) Liquid culture of Antrodia cinnamomea: Scrape a quarter of the mycelium from a 60 mm culture dish and place it in a 2 mL sterile centrifuge tube. Add 1 mL of sterile water and crush it for 2 minutes. Then, take 500 µL of the crushed sample liquid and inoculate it into a 250 mL conical flask containing 100 ml of 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, 7 g / L glucose, and the balance is water). Place it in a constant temperature shaker at 28 ° C and 150 rpm and culture for 10 days.

[0019] (3) Fermentation culture of Antrodia cinnamomea in different culture media: S1. PDA liquid fermentation medium: potato starch 0.5 g / 100 mL, potassium dihydrogen phosphate 0.1 g / 100 mL, magnesium sulfate 0.05 g / 100 mL, yeast powder 0.5 g / 100 mL, VB1 0.01 g / 100 mL, and the balance is water.

[0020] S2, PDA + Melia azedarach young leaf homogenate liquid fermentation medium (KL): potato flour 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB10.01g / 100mL, Melia azedarach young leaf homogenate 2g / 100mL, the balance is water; The neem young leaf slurry is prepared by soaking the neem young leaves in tap water and cleaning them, removing spider webs, dust and other impurities from the leaves, drying them, crushing them into powder in a crusher, and storing them at room temperature for later use.

[0021] S3. Solid culture medium (XMF): 20 g wheat flour + 50 ml MM medium. The MM medium formula is 6 g / L sodium nitrate, 0.52 g / L potassium chloride, 0.52 g / L magnesium sulfate, and 1.52 g / L potassium dihydrogen phosphate.

[0022] All the above culture media were sterilized in an autoclave (121°C, 20 min).

[0023] (4) Fermentation culture: The liquid cultured Antrodia cinnamomea strains were inoculated into the above-mentioned PDA, KL, and XMF culture media respectively. The culture conditions for PDA and KL were as follows: temperature 28°C, rotation speed 150 r / min, and culture time 18 days; the culture conditions for XMF were as follows: culture at 28°C in the dark for 30 days.

[0024] (5) Mycelium harvest: S1. After the fermentation of PDA and KL is completed, use a funnel to separate the mycelium from the bacterial liquid. The obtained bacterial liquid is placed in a 250 mL Erlenmeyer flask and extracted with ethyl acetate in a ratio of 1:1. After ultrasonic vibration for 40 minutes, let it stand for 12 hours. Then, pour the extracted bacterial liquid into a separatory funnel for full extraction. During this period, shake it up and down 5 times. After standing for 3 hours, pour the upper layer of the extract in the separatory funnel into a round-bottom flask and use a rotary evaporator (temperature 50°C, speed 80 r / min) to obtain a crude extract. Rinse the crude extract with acetone and transfer it to a 2 mL weighing centrifuge tube and dry it.

[0025] The obtained mycelium was dried with absorbent paper, transferred to a sterilized centrifuge tube with tweezers, immediately frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0026] S2. Samples of the mycelium and Antrodia cinnamomea fruiting bodies (GLG) after XMF fermentation are collected and preserved.

[0027] (6) Antrodin C content detection: S1. Metabolite Extraction Weigh 60 mg of the above 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 in the 2 mL adapter provided with the instrument, immerse it in liquid nitrogen for rapid freezing for 5 min, remove the centrifuge tube and freeze-thaw it at room temperature, then place the centrifuge tube in the 2 mL adapter again, install it in the grinder, and oscillate at 55 Hz for 2 min; Repeat step 3 twice; Remove the centrifuge tube, centrifuge at 12000 rpm at 4℃ for 10 min, take the supernatant, and centrifuge and concentrate to dryness; Dissolve the sample in 300 µL of a 50% 2-chlorophenylalanine (4 ppm) methanol-water solution (1:1, 4°C), filter through a 0.22 µm membrane, and perform LC-MS analysis. LC-MS detection with the sample to be tested S2. On-machine testing 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 phase 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).

[0028] 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.

[0029] Data analysis: Based on the above test results, metabolomics data analysis was performed on the tested samples. The specific steps are as follows: data preprocessing, including format conversion, peak identification, filtering, alignment and normalization. Data inspection was performed through chromatograms and quality control. Differential compounds were screened by multivariate statistical analysis, and the compounds were identified by comparing the mass spectrometry data in the database. The peak area of Antrodin C in different samples was obtained to compare the content differences of Antrodin C in Gaoligong Antrodia cinnamomea fruiting bodies and Gaoligong Antrodia cinnamomea mycelium fermented in different formula culture media. The results are shown in Tables 1 and Figure 4 : Table 1

[0030] As can be seen from Table 1, KL fermentation medium greatly increased the content of Antrodin C compared with Antrodia cinnamomea fruiting body and XMF and PDA medium, providing a new and effective way for the large-scale production of Antrodin C.

[0031] 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 Antrodin C in Antrodia cinnamomea mycelium, characterized in that: The culture method comprises the steps of inoculating Antrodia cinnamomea mycelium into a liquid culture medium to which a homogenate of young leaves of Melia azedarach is added for culture.

2. The culture method according to claim 1, wherein: The liquid culture medium is a PDA liquid culture medium, and the formula is: 0.5g / 100mL of potato flour, 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; wherein the addition amount of the chinaberry young leaf homogenate is 1-3g / 100mL.

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

4. The culture method according to claim 1, wherein: The preparation method of the chinaberry young leaf homogenate is as follows: soaking the chinaberry young leaves in tap water, cleaning them, removing impurities, drying them, crushing them into powder in a crusher, and storing them at room temperature for later use.

5. The culture method according to claim 1, wherein: The method for obtaining the Antrodia cinnamomea mycelium comprises the following steps: S1. Inoculate Antrodia cinnamomea strains into activation culture medium and culture 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.

6. The culture method according to claim 5, characterized in that The activation culture medium formula 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.

7. The culture method according to claim 5, characterized in that 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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