A culture method for increasing the content of antrodin c in the mycelium of taiwanofungus camphoratus

Abstract

The application provides a culture method for improving the content of Antrodin C in the mycelium of Antrodia cinnamomea, and relates to the technical field of edible and medicinal fungus production processes.The culture method is that the mycelium of Antrodia cinnamomea is inoculated into PDA liquid culture medium added with uniform homogenate of tender leaves of Melia azedarach, and is cultured in a constant-temperature shaking table at 28±2 DEG C and 150±50 rpm for 15-20 days.The application overcomes the defects of the prior art, improves the content of Antrodin C in the mycelium of Antrodia cinnamomea by optimizing the formula of the fermentation medium and improving the fermentation conditions, and strengthens the pharmacological activity of the mycelium of Antrodia cinnamomea.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of edible and medicinal fungus production, and in particular to a culture method for increasing the content of Antrodin C in Antrodia cinnamomea mycelium. BACKGROUND

[0002] Antrodia cinnamomea is a precious edible and medicinal fungus; it belongs to Basidiomycota, Polyporaceae and Taiwannofungus. Antrodia cinnamomea shows various physiological and pharmacological properties, such as anticancer, antitumor, antioxidant, anti-inflammatory, hypoglycemic, liver protection, immune regulation and intestinal microbiota regulation activities. Due to its therapeutic effect, especially its prospective application as a chemopreventive agent, it has also attracted increasing research attention for the development of new drugs. The present application discloses Gaoligong Antrodia cinnamomea, which was discovered as a new species of Taiwannofungus in 2018, and its distribution is located in Gaoligong Mountain in the northwest of Yunnan Province. Through whole genome analysis, it is found that Gaoligong Antrodia cinnamomea has high similarity with Antrodia cinnamomea in active compound biosynthesis gene clusters. At present, there have been more reports on the fermentation culture of active products of Antrodia cinnamomea, but the research on the fermentation culture of Gaoligong Antrodia cinnamomea to produce a large amount of active compounds is still very limited.

[0003] Due to the scarcity of host and slow growth, the wild fruiting bodies of Antrodia cinnamomea are very expensive and in short supply. Therefore, artificial cultivation techniques such as cut wood cultivation, solid-state fermentation, submerged fermentation and petri dish cultivation have been developed to supplement the growing demand for Antrodia cinnamomea. At the same time, due to the significant difference in bioactive metabolites between the fruiting bodies and the cultivated mycelium of Antrodia cinnamomea, it has always been very challenging to obtain sufficient high-quality Antrodia cinnamomea through artificial cultivation. Therefore, a large number of researches have tried various methods to reduce this difference in bioactive metabolites between the fruiting bodies and the cultivated mycelium. Different cultivation techniques and conditions can lead to completely different chemical characteristics. Cut wood cultivation grows on the wood of Antrodia cinnamomea. The chemical composition is most similar to that of wild Antrodia cinnamomea, but it may take 2-3 years to grow and the resources of linden wood cultivation are scarce, which is also costly. Liquid submerged fermentation uses mixed nutrients and natural extracts as culture medium, solid support cultivation uses grains or agricultural by-products as substrate, and petri dish cultivation usually grows on agar medium containing different nutrients in petri dishes. In comparison, liquid fermentation not only has short cycle, low cost, and is easy to mass culture, but also has the potential to obtain various active ingredients by changing the composition of the culture medium, culture conditions, etc.

[0004] With further in-depth study, more and more bioactive metabolites are isolated from the fruiting bodies and cultured mycelium of T. album, and their structures and functions are determined. At present, more than 200 compounds have been isolated and identified in T. album, which include polysaccharides, triterpenoids, ubiquinone derivatives, maleic and succinic acid derivatives, benzene ring derivatives and glycoproteins, and show a wide range of biological activities. Among these bioactive components, antrodins (A-E) are important active components unique to T. album. Antrodin compounds are composed of maleic and succinic acid derivatives, and Antrodin C is the most widely studied antrodin compound, and the research mainly focuses on its biological activity and pharmacology. Antrodin C has been shown to significantly inhibit hepatitis C virus and Lewis lung cancer tumor cells, and shows better inhibition on TGF-beta 1-induced epithelial-mesenchymal transition and breast cancer cell metastasis. However, Gaoligong T. album grows slowly in nature, and has weak ability to produce Antrodin C, therefore, it is of great research significance to research and determine a culture method which can produce a large amount of Antrodin C and is suitable for large-scale production. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a culture method for improving the content of Antrodin C in T. album mycelium, which improves the content of Antrodin C in T. album mycelium of Gaoligong by optimizing the formula of the fermentation medium and improving the fermentation conditions, and strengthens the pharmacological activity of T. album mycelium of Gaoligong.

[0006] To achieve the above object, the technical scheme of the present application is implemented as follows:

[0007] A culture method for improving the content of Antrodin C in T. album mycelium, wherein the culture is inoculating T. album mycelium into a liquid medium added with tender leaf homogenate of Melia azedarach to culture.

[0008] Preferably, the liquid medium is PDA liquid medium, and the formula is: potato powder 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB1 0.01g / 100mL, and the balance is water; wherein the addition amount of tender leaf homogenate of Melia azedarach is 1-3g / 100mL.

[0009] Preferably, the culture conditions are 28±2℃ in a constant temperature shaker at 150±50rpm, and the culture time is 15-20d.

[0010] Preferably, the preparation method of the Melia azedarach tender leaf homogenate is as follows: the Melia azedarach tender leaves are cleaned by soaking with tap water, impurities are removed, and then the leaves are dried and crushed into powder in a crusher, and the powder is stored at room temperature for use.

[0011] Preferably, the method for obtaining the Ganoderma boninense mycelium comprises the following steps:

[0012] S1, inoculating Ganoderma boninense spores into an activated culture medium, and culturing in a constant temperature incubator at 26 DEG C and 60% humidity for 30 days;

[0013] S2, inoculating the activated Ganoderma boninense spore liquid medium in a constant temperature shaker at 28 DEG C and 150 rpm, and culturing for 10 days to obtain Ganoderma boninense mycelium.

[0014] Preferably, the activated culture medium formula in step S1 is as follows: potato powder 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 agar 1.1 g / 100 mL.

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

[0016] The application provides a culture method for increasing the content of Antrodin C in Ganoderma boninense mycelium.

[0017] The application compares the solid culture and liquid culture and the content of Antrodin C in Ganoderma boninense fruiting bodies, and selects the addition of Melia azedarach tender leaf homogenate (KL) as the most suitable culture medium for the production of Antrodin C by Ganoderma boninense, so that the content of Antrodin C in the produced Ganoderma boninense is the highest, which is 3.9 times of that of liquid culture PDA, 14.4 times of that of Ganoderma boninense fruiting bodies (GLG), and 23.7 times of that of solid culture XMF. The application provides a way for the efficient production of the bioactive component Antrodin C and the development of the high-efficiency medicinal value of Ganoderma boninense. The process is simple and controllable, the culture period is short, and the cost is low, so that large-scale batch production can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 FIG. 1 is a Ganoderma boninense solid culture XMF mycelium growth condition diagram according to the application;

[0019] Figure 2 FIG. 3 is a Ganoderma boninense mycelium growth condition diagram in a PDA liquid culture medium according to the application;

[0020] Figure 3 Figure for mycelium growth of the Antrodia cinnamomea in KL liquid medium;

[0021] Figure 4 Column chart for the content of Antrodin C in different samples. DETAILED DESCRIPTION

[0022] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0023] The Antrodia cinnamomea used in the following embodiments is Gaoligong Antrodia cinnamomea with the preservation number CCTCC M 20232425. EMBODIMENT

[0024] Culture of Antrodia cinnamomea:

[0025] (1) Activation of Antrodia cinnamomea strain:

[0026] The ultraclean workbench was sterilized by ultraviolet sterilization 15 min in advance, and the Antrodia cinnamomea strain and PDA solid medium (potato powder 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, VB10.01 g / 100 mL, agar 1.1 g / 100 mL) were prepared. First, the tweezers or inoculation knife was fully burned with an alcohol lamp, and then a small piece of Antrodia cinnamomea original strain was inoculated into the PDA solid medium after cooling. The medium was labeled (strain number, date) and sealed with a film, and then placed in a constant temperature incubator at 26°C and 60% humidity for 30 days. During the period, the culture was observed and recorded every 5 days. When the mycelium of Antrodia cinnamomea covered the entire medium, it was stored in a 4°C refrigerator for standby use.

[0027] (2) Liquid culture of Antrodia cinnamomea:

[0028] The mycelium of the above Antrodia cinnamomea was scraped, and one-fourth of the mycelium in a 60 mm culture dish was placed in a 2 mL sterilized centrifuge tube. Then 1 mL of sterile water was added, and the sample was broken for 2 min. Then 500 µL of the broken sample liquid was inoculated into a 250 mL conical flask containing 100 ml of modified malt extract broth liquid medium (animal tissue pepsin digest 5 g / L, yeast extract 5 g / L, malt extract 5 g / L, maltose 3 g / L, glucose 7 g / L, and the rest water), and then placed in a constant temperature shaker at 28°C and 150 rpm for 10 days.

[0029] (3) Fermentation culture of Antrodia camphorata in different culture media:

[0030] S1, PDA liquid fermentation medium: potato flour 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, vitamin B1 0.01g / 100mL, and the remainder is water.

[0031] 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, VB1 0.01g / 100mL, Melia azedarach young leaf homogenate 2g / 100mL, the remainder is water;

[0032] The process of homogenizing tender chinaberry leaves is as follows: Soak and wash the tender chinaberry leaves with tap water to remove spider webs, dust and other impurities, dry them and then grind them into powder in a crusher. Store at room temperature for later use.

[0033] S3, Solid Culture Medium (XMF): 20g wheat flour + 50ml MM medium, wherein the MM medium formula is 6g / L sodium nitrate, 0.52g / L potassium chloride, 0.52g / L magnesium sulfate, and 1.52g / L potassium dihydrogen phosphate.

[0034] All the above culture media were sterilized in an autoclave (121℃, 20 min).

[0035] (4) Fermentation culture:

[0036] The liquid cultured Antrodia camphorata strains were inoculated into the above-mentioned PDA, KL, and XMF culture media. The culture conditions for PDA and KL were 28℃, 150 r / min, and 18 days. The culture conditions for XMF were 28℃ and cultured in the dark for 30 days.

[0037] (5) Harvesting mycelium:

[0038] S1. After the fermentation of PDA and KL is completed, the mycelium and bacterial solution are separated using a funnel. The obtained bacterial solution is placed in a 250mL Erlenmeyer flask, and ethyl acetate is added in a 1:1 ratio for extraction. After ultrasonic shaking for 40 min, it is allowed to stand for 12 h. Then, the extracted bacterial solution is poured into a separatory funnel for full extraction, shaking up and down 5 times during the process. After standing for 3 h, the upper extract of the separatory funnel is poured into a round-bottom flask and the crude extract is obtained using a rotary evaporator (temperature 50℃, speed 80 r / min). The crude extract is washed with acetone into a 2mL weighing centrifuge tube and dried.

[0039] The obtained mycelium was dried with water-absorbing paper, transferred to a sterilized centrifuge tube with a pair of tweezers, immediately frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0040] S2, sampling and preservation of XMF fermented mycelium and Ganoderma lingzhi fruiting bodies (GLG)

[0041] (6) Antrodin C content detection:

[0042] S1, metabolite extraction

[0043] 60 mg of the above sample was weighed into a 2 mL centrifuge tube; 500 µL of methanol (-20°C) and 500 µL of H2O (4°C) were added, vortexed for 30 s, and 100 mg of glass beads were added;

[0044] The centrifuge tube containing the sample was placed in the 2 mL adapter provided with the instrument, immersed in liquid nitrogen for rapid freezing for 5 min, the centrifuge tube was taken out and thawed at room temperature, the centrifuge tube was again placed in the 2 mL adapter, and installed into the grinding instrument, oscillated at 55 Hz for 2 min;

[0045] Step 3 was repeated twice;

[0046] The centrifuge tube was taken out, centrifuged at 12000 rpm at 4°C for 10 min, the supernatant was taken out, and the centrifuge was concentrated and dried;

[0047] The sample was dissolved with 300 µL of 50% 2-chlorophenylalanine (4 ppm) methanol solution (1:1, 4°C), 0.22 µm membrane filtration was performed, and the sample to be tested was obtained, and LC-MS detection was performed;

[0048] LC-MS detection with the sample to be tested

[0049] S2, on-machine detection

[0050] Chromatographic conditions: ACQUITY UPLC® HSS T3 1.8 µm (2.1×150 mm) column was used, the temperature of the automatic sampler was set to 8°C, the flow rate was 0.25 mL / min, the column temperature was 40°C, 2 μL of sample was injected for gradient elution, and the mobile phase was 0.1% formic acid water (C) - 0.1% formic acid acetonitrile (D) for positive ion; 5 mM ammonium formate water (A) - acetonitrile (B) for negative ion. Gradient elution program: 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).

[0051] Mass spectrometry conditions: The instrument uses an electrospray ion source (ESI), positive and negative ionization mode, positive ion spray voltage is 3.50 kV, negative ion spray voltage is 2.50 kV, sheath gas 30 arb, auxiliary gas 10 arb. Capillary temperature 325 ℃, full scan with resolution 70 000, scan range 81~1 000, and HCD is used for secondary fragmentation, collision voltage is 30 eV, and dynamic exclusion is used to remove unnecessary MS / MS information.

[0052] Data analysis: According to the above detection results, the detected samples were subjected to metabolomics data analysis. The specific steps are as follows: data preprocessing, including format conversion, peak identification, filtering, alignment and normalization. Data checking was performed by chromatogram and mass 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, and the content difference of Antrodin C in high-guanbaoshan Antrodia camphorata fruiting body and in high-guanbaoshan Antrodia camphorata mycelium fermented in different formula culture medium was compared. The results are shown in Table 1 and Figure 4 :

[0053] Table 1

[0054]

[0055] As can be seen from Table 1, the content of Antrodin C in KL fermentation medium is greatly improved compared with that in Antrodia camphorata fruiting body and XMF and PDA culture medium, which provides a new and effective way for large-scale production of Antrodin C.

[0056] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can modify the technical solutions described in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A cultivation method for increasing the Antrodin C content in Antrodia camphorata mycelium from Gaoligong Mountain, characterized in that, The cultivation method involves inoculating the mycelium of *Antrodia camphorata* from Gaoligong Mountain into a liquid culture medium containing homogenized young leaves of *Melia azedarach* for cultivation. The liquid culture medium is 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 and VB1 0.01g / 100mL, with the remainder being water; wherein the amount of neem tender leaf homogenate added is 1-3g / 100mL.

2. The cultivation method according to claim 1, characterized in that: The culture conditions were 28±2℃ and 150±50rpm in a constant temperature shaker for 15-20 days.

3. The cultivation method according to claim 1, characterized in that: The preparation method of the neem leaf homogenate is to soak and wash the neem leaves with tap water to remove impurities, dry them, grind them into powder in a crusher, and store them at room temperature for later use.

4. The cultivation method according to claim 1, characterized in that: The method for obtaining the *Antrodia camphorata* mycelium from Gaoligong Mountain includes the following steps: S1. Inoculate the Gaoligong Antrodia camphorata strain into the activation medium and incubate it in a constant temperature incubator at 26℃ and 60% humidity for 30 days; S2. The activated *Antrodia cinnamomea* strain was inoculated into a modified malt extract broth liquid culture medium and cultured for 10 days in a constant temperature shaker at 28°C and 150 rpm to obtain *Antrodia cinnamomea* mycelium. The modified malt extract broth liquid culture medium was formulated as follows: 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 remainder being water.

5. The cultivation method according to claim 4, 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, vitamin B1 0.01g / 100mL and agar 1.1g / 100mL.

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