Culture method for increasing content of bioactive substance Emodic acid in talaromyces purpurascens

Abstract

The invention provides a culture method for increasing the content of a bioactive substance Emodic acid in talaromyces purpurascens, and relates to the technical field of medicinal fungus culture. The culture method of the homopurple talaromyces spp. Comprises the following steps: carrying out liquid culture on hyphae of the homopurple talaromyces spp., and then inoculating the hyphae into a liquid culture medium containing black bean powder for culture. The defects in the prior art are overcome, and the pharmacological activity of the talaromyces purpurascens is enhanced by optimizing the formula of the fermentation culture medium and improving the fermentation conditions, so that the content of Emodic acid in the talaromyces purpurascens is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal mushroom cultivation, and particularly relates to a cultivation method for increasing the content of the bioactive substance Emodic acid in Talaromyces purpurogenus. Background Art

[0002] Emodic acid (also known as dihydroxyanthraquinone) is an anthraquinone bioactive compound, and its specific structure is as follows:

[0003] It mainly exists in certain plants, such as Rheum species and Phellodendron amurense, etc. Emodic acid is contained in the roots and rhizomes of Rheum and the bark of Phellodendron amurense. In addition, Emodic acid has also been found in some other plants such as Siraitia grosvenorii and Coptis chinensis. Emodic acid has various biological activities, including antioxidant, anti-inflammatory, anti-tumor, antibacterial, etc. It can reduce the inflammatory response by inhibiting the generation of inflammatory mediators or reducing the activation of inflammatory signaling pathways. It plays an anti-cancer role in inducing apoptosis of cancer cells and inhibiting the proliferation of tumor cells. Some studies have shown that it may exert anti-cancer effects by regulating multiple signaling pathways, such as p53, MAPK, etc. Emodic acid can help improve liver health by inhibiting oxidative damage and inflammatory responses in hepatocytes, and shows potential anti-diabetic activity by improving blood glucose metabolism or reducing oxidative damage caused by diabetes.

[0004] Emodic acid was detected in the metabolome data of the fungus Talaromyces purpurogenus. Talaromyces purpurogenus is a fungus belonging to the family Trichocomaceae, which has significant biological activities and can produce a variety of potential natural metabolites. These products include antibacterial, anti-tumor, antioxidant and other active ingredients, and the enzymes produced by it also have application prospects in the bio-industry. With the further study of its metabolic mechanism, Talaromyces purpurogenus is expected to become an important source of natural products, providing new development opportunities for the pharmaceutical and other industries.

[0005] Different fermentation media and fermentation methods have significant effects on the growth status, nutritional value and biological activities of fungi. Therefore, studying the content change of Emodic acid in Talaromyces purpurogenus under different culture conditions has important value for the efficient utilization of Talaromyces purpurogenus, and provides a new way for fungi to efficiently produce emodic acid. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a cultivation method for increasing the content of the bioactive substance Emodicacid in Talaromyces purpureogenus, by optimizing the fermentation medium formula, improving the fermentation conditions, increasing the content of Emodicacid in Talaromyces purpureogenus, and enhancing the pharmacological activity of Talaromyces purpureogenus.

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions: A cultivation method for increasing the content of the bioactive substance Emodic acid in Talaromyces purpureogenus, wherein the cultivation method is to perform liquid cultivation on the mycelium of Talaromyces purpureogenus and then inoculate it into a liquid medium containing black bean powder for cultivation.

[0008] Preferably, the specific formula of the liquid medium containing black bean powder is 2 - 6 g / L of black bean powder + 15 - 25 g / L of glucose, and the rest is water.

[0009] Preferably, the conditions for cultivating in the liquid medium containing black bean powder are cultivation in a constant temperature shaker at 28 ± 2°C and 150 ± 50 rpm.

[0010] Preferably, the method for obtaining the mycelium of Talaromyces purpureogenus is to inoculate the Talaromyces purpureogenus strain into an activation medium and perform activation cultivation in a constant temperature incubator at 26 ± 2°C and 60 ± 10% humidity for 4 - 8 d until the mycelium covers the entire medium to obtain the mycelium of Talaromyces purpureogenus.

[0011] Preferably, the activation medium is a PDA solid medium, and the formula is: 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 1.1 g / 100 mL of agar.

[0012] Preferably, the method for liquid cultivation of the mycelium of Talaromyces purpureogenus is to scrape and crush the mycelium of Talaromyces purpureogenus and then inoculate it into a modified malt extract broth liquid medium in a constant temperature shaker at 28 ± 2°C and 150 ± 50 rpm for 8 - 12 d.

[0013] Preferably, the formula of the modified 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.

[0014] The present invention provides a cultivation method for increasing the content of the bioactive substance Emodic acid in Talaromyces purpureogenus, and the advantages compared with the prior art are: The present invention uses black bean powder to prepare a liquid medium for culturing Talaromyces purpureogenus. The content of Emodic acid in the cultured Talaromyces purpureogenus is 2.6 times that of Gause liquid medium, 8.5 times that of PDA liquid medium, and 16.4 times that of ISP liquid medium. It provides a way for the efficient development of the medicinal value of Talaromyces purpureogenus and the high-efficient production of the bioactive ingredient Emodic acid. Description of the Drawings

[0015] Figure 1 It is a growth situation diagram of Talaromyces purpureogenus of the present invention in ISP liquid medium; Figure 2 It is a growth situation diagram of Talaromyces purpureogenus of the present invention in PDA liquid medium; Figure 3 It is a growth situation diagram of Talaromyces purpureogenus of the present invention in Gause liquid medium; Figure 4 It is a growth situation diagram of Talaromyces purpureogenus of the present invention in HD liquid medium; Figure 5 It is a bar chart of the content difference of Emodic acid in Talaromyces purpureogenus cultured in different media. Detailed Embodiments

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below 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 the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. Embodiment

[0017] Select the Talaromyces purpureogenus isolated in the laboratory and preserved in Wuhan University, China on May 19, 2022, with the preservation number of CCTCC M 2022612 as the experimental strain. This strain has been disclosed in the related patent: CN202211368972.7 "Endophytic fungus YAFEF086 strain from the root of Polygonatum kingianum and its application" applied on November 3, 2022.

[0018] 1. Activation of the Talaromyces purpureogenus strain: The activation preparation of the Talaromyces purpureogenus strain is as follows: Disinfect the laminar flow hood and turn on the ultraviolet sterilization 15 minutes in advance. Prepare the strain and PDA solid medium (0.5 g / 100 mL potato powder, 0.1 g / 100 mL potassium dihydrogen phosphate, 0.05 g / 100 mL magnesium sulfate, 0.5 g / 100 mL yeast powder, 0.01 g / 100 mL VB1, 1.1 g / 100 mL agar). First, fully burn the forceps or inoculation knife with an alcohol lamp. After cooling, pick up a small piece of the original Talaromyces purpureogenus strain and inoculate it onto the PDA solid medium. After making marks (strain number, date), seal it with a film and place it in an incubator at 26°C and 60% humidity for 5 days. Wait until the mycelium covers the entire medium and then store it in a 4°C refrigerator for later use.

[0019] 2. Liquid culture of Talaromyces purpureogenus: Preparation of the liquid culture medium of Talaromyces purpureogenus: Scrape one-fourth of the mycelium in a 60 mm petri dish of the above-cultured Talaromyces purpureogenus 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 animal tissue pepsin digest, 5 g / L yeast extract powder, 5 g / L malt extract powder, 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 for 10 days.

[0020] 3. Inoculate Talaromyces purpureogenus in different fermentation media for culture Absorb the Talaromyces purpureogenus fungal balls from the above liquid culture and inoculate them into the following various media: ISP liquid medium: 4 g / L yeast extract powder, 10 g / L malt extract powder, 4 g / L glucose; PDA liquid medium: 4 g / L potato powder, 20 g / L glucose; Gause liquid medium: 20 g / L soluble starch, 0.5 g / L NaCl, 1 g / L KNO3, 0.5 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·7H2O; HD liquid medium: 4 g / L black bean powder, 20 g / L glucose.

[0021] All media have been sterilized by high-temperature high-pressure steam (121°C, 20 minutes). The culture conditions are: culture in a constant temperature shaking incubator (temperature 28°C, rotation speed 150 r / min, culture time 2 days).

[0022] 4. Harvest and extract the crude extract of the fermented Talaromyces purpureogenus: After the liquid fermentation of Talaromyces purpureogenus is completed using the above-mentioned culture media respectively, the mycelia are harvested. The specific operation steps are as follows: (1) Use a funnel to separate the mycelia from the broth. The obtained broth is placed in a 250 mL Erlenmeyer flask, and ethyl acetate in a 1:1 ratio is added for extraction. After ultrasonic oscillation for 40 min, it is left to stand for 12 h. Then, the extracted broth is poured into a separatory funnel for thorough extraction. During this period, it is shaken up and down (3 - 5 times). After standing for 3 h, the upper-layer extract in the separatory funnel is poured into a round-bottom flask, and the crude extract is obtained using a rotary evaporator (temperature 50 °C, rotation speed 80 r / min). The crude extract is rinsed with acetone into a 2 mL weighing centrifuge tube and air-dried.

[0023] (2)The obtained mycelia are 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.

[0024] (3)At the same time, samples of the mycelia of Talaromyces purpureogenus in solid fermentation are taken and preserved. Three replicates are taken for each sample of Talaromyces purpureogenus fermented and cultured in different formulated culture media.

[0025] 5. Detection of Emodic acid content: The Emodic acid content in the mycelia of Talaromyces purpureogenus fermented and cultured in different formulated culture media is detected. The specific operation steps are as follows: (1)Metabolite extraction 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 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 and install it in a grinder, and oscillate at 55 Hz for 2 min; Repeat step 3 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; perform LC-MS detection using the sample to be tested; (2)Analysis on the machine Chromatographic conditions: An ACQUITY UPLC® HSS T3 1.8 µm (2.1×150 mm) chromatographic column was used. The temperature of the autosampler was set at 8°C. Gradient elution was performed at a flow rate of 0.25 mL / min, a column temperature of 40°C, and an injection volume of 2 μL. 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 as follows: 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).

[0026] Mass spectrometry conditions: The instrument used an electrospray ionization source (ESI), with positive and negative ion ionization modes. The positive ion spray voltage was 3.50 kV, and 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 70000, with a scan range of 81 - 1000. HCD was used for secondary fragmentation, and the collision voltage was 30 eV. At the same time, dynamic exclusion was used to remove unnecessary MS / MS information.

[0027] Data analysis: According to the detection results in step (2), 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. Differentially expressed compounds were screened by multivariate statistical analysis, and compounds were identified by comparing with the mass spectrometry data in the database. The peak areas of Emodic acid in different samples were obtained, which were the relative content differences of Emodic acid in different samples. Based on this, the specific results of the content differences of Emodic acid in Talaromyces purpureogenus fermented in different formulated media are shown in Table 1 below and Figure 5 。

[0028] The results are shown in Table 1 below: Table 1

[0029] From Table 1 and Figure 5 it can be seen that compared with Gause liquid medium, PDA liquid medium, and ISP liquid medium, the preferred fermentation medium HD liquid medium of the present invention greatly increases the content of Emodic acid, providing a new and effective way for the production of Emodic acid and the efficient utilization of Talaromyces purpureogenus.

[0030] 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 cause the essence of the corresponding technical solutions to 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 the bioactive substance Emodic acid in Talaromyces purpureogenus, characterized in that, The cultivation method is to liquid-culture the mycelium of Talaromyces purpureogenus and then inoculate it into a liquid medium containing black bean powder for cultivation.

2. The culturing method according to claim 1, characterized in that: The specific formula of the liquid medium containing black bean powder is 2 - 6 g / L of black bean powder + 15 - 25 g / L of glucose, and the rest is water.

3. The culturing method according to claim 1, characterized in that: The conditions for inoculating into the liquid medium containing black bean powder for cultivation are culturing in a constant-temperature shaker at 28 ± 2 °C and 150 ± 50 rpm.

4. The cultivation method according to claim 1, characterized in that: The method for obtaining the mycelium of Talaromyces purpureogenus is to inoculate the Talaromyces purpureogenus strain into an activation medium and activate it in a constant-temperature incubator at 26 ± 2 °C and 60 ± 10% humidity for 4 - 8 d until the mycelium covers the entire medium to obtain the mycelium of Talaromyces purpureogenus.

5. The culturing method according to claim 4, characterized in that, The activation medium is a PDA solid medium, and the formula is: 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 1.1 g / 100 mL of agar.

6. The cultivation method according to claim 4, characterized in that: The method for liquid-culturing the mycelium of Talaromyces purpureogenus is to scrape and crush the mycelium of Talaromyces purpureogenus and then inoculate it into a modified malt extract broth liquid medium and culture it in a constant-temperature shaker at 28 ± 2 °C and 150 ± 50 rpm for 8 - 12 d.

7. The cultivation method according to claim 6, characterized in that, The formula of the modified 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.

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