A cultivation method for increasing the content of antroquinonol in Antrodia cinnamomea mycelium

Abstract

The present invention provides a cultivation method for increasing the content of androquinol in Antrodia cinnamomea mycelia, which relates to the technical field of medicinal mushroom cultivation. The cultivation method is to inoculate Antrodia cinnamomea mycelia into a liquid medium added with the homogenate of fresh leaves of Toona sinensis (Meliaceae), and the liquid medium is PDA medium, and the addition amount of the homogenate of fresh leaves of Toona sinensis (Meliaceae) is 0.5 - 2 g / 100 mL. The present invention overcomes the deficiencies of the prior art, improves the content of androquinol in Antrodia cinnamomea mycelia and enhances the pharmacological activity of Antrodia cinnamomea mycelia by optimizing the fermentation medium formula and improving the fermentation conditions.

Description

Technical Field

[0001] The invention relates to the technical field of medicinal fungus cultivation, and in particular to a cultivation method for increasing the content of antroquinol in Antrodia cinnamomea mycelium. Background Art

[0002] Antrodia cinnamomea is a precious edible and medicinal fungus; it belongs to the phylum Basidiomycota, family Polyporaceae, genus Taiwannofungus.

[0003] 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 such as cut wood culture, solid-state fermentation, submerged fermentation and dish culture have been developed to supplement the expanding demand for Antrodia cinnamomea. At the same time, due to the significant differences in bioactive metabolites between the fruiting bodies and cultured mycelium of Antrodia cinnamomea, it has been very challenging to obtain sufficient amounts of high-quality Antrodia cinnamomea through artificial cultivation. Therefore, a large number of studies have tested various methods to try to reduce this difference in bioactive metabolites between the fruiting bodies and cultured mycelium. Different cultivation techniques and conditions may lead to very different chemical characteristics. Cut wood culture grows on the wood of Antrodia cinnamomea. The chemical composition is most similar to that of wild Antrodia cinnamomea, but cultivation may take 2-3 years and the Antrodia cinnamomea resources required for basswood culture are scarce and the cost is also high. Liquid submerged fermentation uses mixed nutrients and natural extracts as the culture medium, solid support culture uses grains or agricultural by-products as the matrix, and dish culture usually grows on agar medium containing different nutrients in a culture 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 composition of the culture medium, culture conditions, etc.

[0004] With further in-depth research, more and more bioactive metabolites have been isolated from the fruiting bodies and cultured mycelium of Antrodia cinnamomea, and their structures and functions have been determined. At present, more than 200 compounds have been isolated and identified, including polysaccharides, triterpenoids, ubiquinone derivatives, maleic acid and succinic acid derivatives, benzene ring derivatives and glycoproteins. Antroquinol is a ubiquinone derivative and one of the characteristic metabolites of Antrodia cinnamomea. It is considered to be one of the most bioactive components of Antrodia cinnamomea, showing various biological activities such as anti-cancer, immunosuppression and diet-induced obesity improvement. However, Antrodia cinnamomea grows slowly in nature and has a weak ability to produce antroquinol. Therefore, it is of great research significance to study and determine a culture method that can produce a large amount of antroquinol and is suitable for large-scale production. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a cultivation method for increasing the content of antroquinonol in Antrodia cinnamomea mycelium. By optimizing the fermentation medium formula and improving the fermentation conditions, the content of antroquinonol in Antrodia cinnamomea mycelium is increased, and the pharmacological activity of Antrodia cinnamomea mycelium is enhanced.

[0006] To achieve the above object, the technical solution of the present invention is realized through the following technical solutions:

[0007] A cultivation method for increasing the content of antroquinonol in Antrodia cinnamomea mycelium, wherein the cultivation method is to inoculate Antrodia cinnamomea mycelium into a liquid medium added with the homogenate of fresh leaves of Toona sinensis (Meliaceae) for cultivation.

[0008] Preferably, the liquid medium is a PDA medium, and the formula is: 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 the balance is water. The addition amount of the homogenate of fresh leaves of Toona sinensis (Meliaceae) in the liquid medium is 0.5 - 2 g / 100 mL.

[0009] Preferably, the cultivation conditions are in a constant temperature shaker at 28 ± 2 °C and 150 ± 50 rpm for 15 - 20 d.

[0010] Preferably, the preparation method of the homogenate of fresh leaves of Toona sinensis (Meliaceae) is to soak and wash the fresh leaves of Toona sinensis with tap water, remove impurities, dry them, and then place them in a crusher to make powder, and store them at room temperature for later use.

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

[0012] S1. Activate the Antrodia cinnamomea strain;

[0013] S2. Perform liquid culture on the activated Antrodia cinnamomea strain to obtain Antrodia cinnamomea mycelium.

[0014] Preferably, the method for activating the Antrodia cinnamomea strain in step S1 is to inoculate the Antrodia cinnamomea strain into an activation medium and cultivate it in a constant temperature incubator at 26 ± 2 °C and 55% - 65% humidity for 28 - 35 d.

[0015] Preferably, the formula of the activation medium is: 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, agar 1.1 g / 100 mL, and the balance is water.

[0016] Preferably, in step S2, the liquid culture method is to inoculate the strain into an improved malt extract broth liquid medium and culture it in a constant temperature shaker at 28±2°C and 150±50 rpm for 8-12 days.

[0017] Preferably, the formula of the improved malt extract broth liquid medium 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.

[0018] The present invention provides a culture method for increasing the content of androquinol in Antrodia cinnamomea mycelium. Compared with the prior art, the advantages are as follows:

[0019] In the present invention, the addition of the homogenate of young leaves of Toona sinensis in the liquid fermentation medium promotes the production of androquinol by Antrodia cinnamomea mycelium. Among them, the addition of 2 g / 100 mL of the homogenate of young leaves of Toona sinensis is the optimal medium for the production of androquinol by liquid fermentation of Antrodia cinnamomea mycelium. The androquinol content in the Antrodia cinnamomea mycelium produced in this way is the highest, reaching 9236.14 mg / kg, which is 1176.97 times that of the Antrodia cinnamomea fruit body, 84.39 times that of solid fermentation culture, and 13.08 times that of PDA liquid fermentation culture, providing a way for the efficient development of the medicinal value of Antrodia cinnamomea and the efficient production of the bioactive ingredient androquinol; and the process is simple and controllable, the culture period is short and the cost is low, and large-scale batch production can be realized. Description of the Drawings

[0020] Figure 1 It is a diagram showing the growth of Antrodia cinnamomea solid fermentation mycelium according to the present invention;

[0021] Figure 2 It is a diagram showing the growth of Antrodia cinnamomea mycelium in PDA liquid medium according to the present invention;

[0022] Figure 3 It is a diagram showing the growth of mycelium in PDA liquid medium with the addition of 0.5 g / 100 mL of the homogenate of young leaves of Toona sinensis according to the present invention;

[0023] Figure 4 It is a diagram showing the growth of mycelium in PDA liquid medium with the addition of 1 g / 100 mL of the homogenate of young leaves of Toona sinensis according to the present invention;

[0024] Figure 5 It is a diagram showing the growth of mycelium in PDA liquid medium with the addition of 2 g / 100 mL of the homogenate of young leaves of Toona sinensis according to the present invention. Detailed Embodiments

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following describes the technical solutions in the embodiments of the present invention clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0026] The Antrodia cinnamomea raw material used in the following embodiments is Antrodia cinnamomea from Gaoligong Mountain, and the preservation name of Antrodia cinnamomea from Gaoligong Mountain is Taiwanofungus gaoligongensis YAFTG001, preservation number: CCTCC M 20232425. Example 1:

[0027] I. Fermentation culture of Antrodia cinnamomea:

[0028] 1. Activation of Antrodia cinnamomea strains:

[0029] The preparation for activating the Antrodia cinnamomea strains is as follows: Disinfect the ultra-clean workbench and turn on the ultraviolet sterilization 15 minutes in advance. Prepare the Antrodia cinnamomea strains 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). First, fully burn the forceps or inoculation knife with an alcohol lamp. After cooling, pick up small pieces of the original Antrodia cinnamomea strains and inoculate them into the PDA solid medium. After making marks (strain number, date), seal the film and place it in a constant temperature incubator at 26°C and 60% humidity for 30 days. Observe and record every 5 days during this period. After the Antrodia cinnamomea mycelia cover the entire medium, store it in a 4°C refrigerator for standby.

[0030] 2. Liquid culture of Antrodia cinnamomea:

[0031] Preparation of the Antrodia cinnamomea liquid culture medium: Scrape one-fourth of the mycelia in a 60 mm culture dish of the Antrodia cinnamomea mycelia into a 2 mL sterilized centrifuge tube, then add 1 mL of sterile water, break it for 2 minutes, and then take 500 µL of the broken sample solution and inoculate it into 250 mL conical flasks filled with 100 ml of improved 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.

[0032] 3. Solid fermentation culture and PDA liquid fermentation culture of Antrodia cinnamomea:

[0033] Solid state fermentation culture: The solid state fermentation medium is 20 g of Cinnamomum kanehirai stem powder + 50 ml of nutrient solution (the nutrient solution composition is: 6 g / L sodium nitrate, 0.52 g / L potassium chloride, 0.52 g / L magnesium sulfate, 1.52 g / L potassium dihydrogen phosphate).

[0034] PDA liquid fermentation culture: 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 it is placed in a constant temperature shaking incubator for culture (temperature 28 °C, rotation speed 150 r / min, culture time 18 d).

[0035] 4. Addition culture with the homogenate matrix of fresh leaves of Toona sinensis (Meliaceae):

[0036] Homogenate of fresh leaves of Toona sinensis (Meliaceae): The fresh leaves of Toona sinensis are soaked and washed clean with tap water, removing impurities such as cobwebs and dust on the leaves. After drying, they are placed in a crusher to be ground into powder and stored at room temperature for later use.

[0037] Addition culture with the homogenate of fresh leaves of Toona sinensis (Meliaceae): In the 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) liquid fermentation medium of Antrodia cinnamomea, the homogenate of fresh leaves of Toona sinensis (Meliaceae) with concentration gradients of 0.5 g / 100 mL, 1 g / 100 mL, and 2 g / 100 mL is added respectively, and then it is placed in an autoclave for sterilization (121 °C, 20 min).

[0038] After cooling, transfer it to a laminar flow hood. Take the Antrodia cinnamomea strain cultured in liquid and inoculate it into the liquid medium, and then place it in a constant temperature shaking incubator for culture (temperature 28 °C, rotation speed 150 r / min, culture time 18 d).

[0039] II. Harvest and extract the crude extract of Antrodia cinnamomea cultured by fermentation:

[0040] After the fermentation of Antrodia cinnamomea is completed, harvest the mycelium. The specific operation steps are as follows:

[0041] 1. Use a funnel to separate the mycelium from the bacterial liquid. The obtained bacterial liquid is placed in a 250 mL Erlenmeyer flask, and ethyl acetate with a 1:1 ratio is added for extraction. After ultrasonic oscillation for 40 min, let it stand for 12 h. Then pour the bacterial liquid after extraction into a separating funnel for sufficient extraction. During this period, shake it up and down (3 to 5 times). After standing for 3 h, pour the upper extraction liquid of the separating funnel into a round bottom flask, and use a rotary evaporator (temperature 50 °C, rotation speed 80 r / min) to obtain the crude extract. Rinse the crude extract with acetone into a 2 mL weighing centrifuge tube and dry it.

[0042] 2. The obtained mycelia were blotted dry with absorbent paper, transferred to a sterilized centrifuge tube with forceps, immediately frozen in liquid nitrogen, and then stored in a -80 °C refrigerator.

[0043] 3. At the same time, samples of Antrodia cinnamomea fruiting bodies and solid-fermented mycelia were taken and preserved. Three replicates were taken for each sample of Antrodia cinnamomea fruiting bodies and Antrodia cinnamomea mycelia fermented and cultured in different formulated media.

[0044] III. Antroquinonol Content Detection

[0045] The Antroquinonol content of Antrodia cinnamomea fruiting bodies and Antrodia cinnamomea mycelia of Gaoligongshan fermented and cultured in different formulated media was detected. The specific operation steps are as follows:

[0046] 1. Metabolite Extraction

[0047] Weigh 60 mg of the sample into a 2 mL centrifuge tube;

[0048] 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;

[0049] Place the centrifuge tube containing the sample in a 2 mL adapter provided with the instrument, immerse it in liquid nitrogen for rapid freezing for 5 min, take out the centrifuge tube and thaw it at room temperature, 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;

[0050] Take out the centrifuge tube, centrifuge at 12000 rpm at 4 °C for 10 min, take the supernatant, and centrifuge and concentrate to dryness;

[0051] 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 on-machine detection; perform LC-MS detection with the sample to be tested.

[0052] 2. Preparation of Antroquinonol Standard Curve

[0053] The Antroquinonol reference standard was prepared after extraction and purification. After UV, HPLC-MS, and NMR analysis, its purity was ≥92%. For the preparation of the Antroquinonol reference standard, accurately weigh 25 mg of Antroquinonol, dissolve it with acetonitrile to prepare a 1 mg / mL mother liquor, and then dilute and make up the volume to 5, 10, 20, 40, 60, 80, and 100 mg / L respectively. After microfiltration, perform HPLC analysis to prepare the Antroquinonol standard curve.

[0054] 3. On-machine Detection

[0055] Chromatographic conditions: ACQUITY UPLC was used ® HSS T3 1.8 µm (2.1×150 mm) chromatographic column, the temperature of the autosampler was set at 8 °C, with a flow rate of 0.25 mL / min, a column temperature of 40 °C, and an injection volume of 2 μL for gradient elution. The mobile phase was 0.1% formic acid in water (C) - 0.1% formic acid in acetonitrile (D) for positive ions; 5 mM ammonium formate in water (A) - acetonitrile (B) for negative ions. 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).

[0056] Mass spectrometry conditions: The instrument used an electrospray ionization source (ESI), positive and negative ion ionization modes, the positive ion spray voltage was 3.50 kV, the negative ion spray voltage was 2.50 kV, the sheath gas was 30 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325 °C, full scan was performed at a resolution of 70,000, the scan range was 81 - 1,000, and HCD was used for secondary fragmentation with a collision voltage of 30 eV. At the same time, dynamic exclusion was used to remove unnecessary MS / MS information.

[0057] Data analysis: Based on the above detection results, metabolomics data analysis was performed on the detected samples. The specific steps were as follows: Data preprocessing was carried out, including format conversion, peak identification, filtering, alignment, and normalization. Data inspection was performed through chromatograms and quality control. Differential compounds were screened through multivariate statistical analysis, and compounds were identified by comparing with the mass spectrometry data in the database. The peak areas of androquinol in different samples were obtained, and then compared with the standard curve of androquinol. Finally, the content differences of androquinol in different samples were obtained, so as to determine the content differences of androquinol in the fruiting bodies of Antrodia cinnamomea var. kanoi and the mycelia of Antrodia cinnamomea var. kanoi fermented and cultured in different formula media. The results are shown in Table 1:

[0058] Table 1

[0059]

[0060] As can be seen from Table 1, compared with the fruiting bodies of Antrodia cinnamomea var. kanoi and solid fermentation culture, the content of androquinol was greatly increased by culturing in PDA liquid fermentation medium. And by adding the homogenate of young leaves of Toona sinensis in the Meliaceae family to the PDA liquid fermentation medium, a higher yield of androquinol was obtained, providing a new and effective way for the large-scale production of androquinol.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some 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 various embodiments of the present invention.

Claims

1. A cultivation method for increasing the content of antroquinonol in Antrodia cinnamomea mycelium, characterized in that, The cultivation method is to inoculate Antrodia cinnamomea mycelium into a liquid medium supplemented with a homogenate of fresh leaves of Toona sinensis (Meliaceae), and the liquid medium is a PDA medium with the following formula: 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, with the balance being water, and the addition amount of the homogenate of fresh leaves of Toona sinensis (Meliaceae) in the liquid medium is 0.5 - 2 g / 100 mL.

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

3. The cultivation method according to claim 1, characterized in that: The preparation method of the homogenate of fresh leaves of Toona sinensis (Meliaceae) is to soak and wash the fresh leaves of Toona sinensis with tap water, remove impurities, dry them, and then place them in a crusher to make powder, which is stored at room temperature for later use.

4. The culturing method according to claim 1, wherein: The method for obtaining Antrodia cinnamomea mycelium includes the following steps: S1. Activate the Antrodia cinnamomea strain. S2. Carry out liquid cultivation on the activated Antrodia cinnamomea strain to obtain Antrodia cinnamomea mycelium.

5. The culturing method according to claim 4, wherein: In step S1, the method for activating the Antrodia cinnamomea strain is to inoculate the Antrodia cinnamomea strain into an activation medium and cultivate it in a constant temperature incubator at 26 ± 2 °C and 55% - 65% humidity for 28 - 35 d.

6. The culturing method according to claim 5, characterized in that, The formula of the activation medium is: 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, agar 1.1 g / 100 mL, with the balance being water.

7. The cultivation method according to claim 4, characterized in that: In step S2, the method for liquid cultivation is to inoculate the strain into a modified malt extract broth liquid medium and cultivate it in a constant temperature shaker at 28 ± 2 °C and 150 ± 50 rpm for 8 - 12 d.

8. The cultivation method according to claim 7, wherein The formula of the modified malt extract broth liquid medium is: peptone from animal tissue 5 g / L, yeast extract 5 g / L, malt extract 5 g / L, maltose 3 g / L, glucose 7 g / L, with the balance being water.

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