Application of phlebopus portentosus in production of cytochalasin E and fermentation method for increasing yield

Abstract

The invention belongs to the technical field of production of edible and medicinal fungi, particularly discloses an application of phlebopus portentosum in production of cytochalasin E and a fermentation method for increasing the yield, and particularly provides an application of phlebopus portentosum YAFMF008 with the preservation number of CCTCC NO: M 20232694 in production of the cytochalasin E. The invention further discloses a preparation method of the phlebopus portentosum YAFMF008, and application of the phlebopus portentosum YAFMF008 to preparation of the cytochalasin E. The invention further discloses a preparation method of the phlebopus portentosum YAFMF008. Through gene sequence analysis, fermentation culture and component detection, it is found that the phlebopus portentosum YAFMF008 can produce the cytochalasin E, a fermentation culture method is optimized, the optimized culture medium can remarkably improve the yield of the cytochalasin E, and a new foundation is laid for efficient medicinal value development of the phlebopus portentosum YAFMF008. A way is provided for efficient production of the bioactive component cytochalasin E.

Description

Technical Field

[0001] The present invention belongs to the technical field of edible and medicinal mushroom production, and particularly relates to the application of Phlebopus portentosus in the production of cytochalasin E, as well as a fermentation medium and a fermentation method for increasing the content of cytochalasin E in Phlebopus portentosus YAFMF008. Background Art

[0002] Phlebopus portentosus is a fungus belonging to the order Boletales and the family Boletinellaceae. Its fruit body is large and delicious, and is rich in nutritional value.

[0003] Cytochalasin E is an alkaloid produced by fungi and belongs to the cytochalasin family. These compounds are known for their effects on the cytoskeleton, especially microfilaments (actin filaments). Cytochalasin E can prevent the normal progression of cells during mitosis and cell division by interfering with the polymerization of actin. Actin is a key component in the process of cell division, especially playing an important role in the early stage of cell division (such as the division of the cell membrane). Cytochalasin E has been shown to have the effects of inhibiting growth and inducing cell death in certain cancer cells. It interferes with the proliferation and migration of tumor cells by affecting the remodeling of the cytoskeleton. Since cytochalasin E affects the cytoskeleton and reduces actin polymerization, it can inhibit the migration and adhesion ability of cells, which makes it have certain application potential in the research of cell movement, cancer metastasis, etc. Generally speaking, the main mechanism of action of cytochalasin E is to affect the basic functions of cells such as cell morphology, division, and movement by interfering with the dynamic changes of the cytoskeleton. It is widely used in cell biology research, especially having important application value in the research of cell migration, cell division, and oncology.

[0004] The microbial fermentation method is the main production method of cytochalasin E. Different fermentation media and fermentation methods have significant effects on the growth state, nutritional value, and biological activity of fungi. Therefore, studying different microbial production methods, increasing the production sources of cytochalasin E, and reducing production costs are of great significance for industrial production. Summary of the Invention

[0005] The present invention provides a Phlebopus portentosus YAFMF008 for producing cytochalasin E through gene sequence analysis, fermentation culture, and component detection. Further, by optimizing the fermentation medium formula and improving the fermentation conditions, the content of cytochalasin E in Phlebopus portentosus YAFMF008 is increased, and the pharmacological activity of Phlebopus portentosus YAFMF008 is enhanced.

[0006] The technical solution of the present invention is as follows: On the one hand, the present invention provides an application of Phlebopus portentosus YAFMF008 with a preservation number of CCTCC NO: M 20232694 in the production of cytochalasin E.

[0007] On the other hand, the present invention provides an application of the fermentation broth of Phlebopus portentosus YAFMF008 in the production of cytochalasin E. The preservation number of Phlebopus portentosus YAFMF008 is CCTCC NO: M 20232694, and the fermentation broth is obtained by inoculating Phlebopus portentosus YAFMF008 into a medium for fermentation culture.

[0008] On the other hand, the present invention provides a method for producing cytochalasin E. After inoculating Phlebopus portentosus YAFMF008 with a preservation number of CCTCC NO: M 20232694 into a medium for culture, the fermentation broth is collected, separated and purified to obtain cytochalasin E.

[0009] On the other hand, the present invention provides a method for producing cytochalasin E with increased yield. After inoculating Phlebopus portentosus YAFMF008 with a preservation number of CCTCC NO: M 20232694 into a medium containing 7.5 g / L Malt Extract Broth, 5 g / L malt-yeast, and 1 g / L yeast powder for culture, the fermentation product is collected, separated and purified to obtain cytochalasin E. The culture conditions are constant temperature of 28 o °C and dark culture at a rotation speed of 150 rpm.

[0010] On the other hand, the present invention provides a method for industrialized large-scale culture of Phlebopus portentosus YAFMF008 with a preservation number of CCTCC NO: M 20232694 for the production of cytochalasin E, including the following steps: S1: Primary strain culture The strain of Phlebopus portentosus YAFMF008 is activated and cultured using a 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, and 1.1 g / 100 mL of agar. S2: Secondary strain culture The first-class strain of Boletus luridus YAFMF008 was inoculated into a modified malt extract broth liquid medium: 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, and 7 g / L of glucose, and cultured. S3: Tertiary strain culture The second-class strain of Boletus luridus YAFMF008 was inoculated into the BDMY liquid medium: 7.5 g / L MaltExtract Broth, 5 g / L malt-yeast, and 1 g / L yeast powder, cultured, the fermentation broth was collected, and cytochalasin E was obtained after separation and purification.

[0011] Furthermore, the culture condition in step S1 was dark culture in a constant temperature incubator at 26 o °C and 60% humidity.

[0012] Furthermore, the culture condition in step S2 was constant temperature dark culture at 28 °C and 150 rpm.

[0013] Furthermore, the culture condition in step S3 was constant temperature at 28 o °C and dark culture at a rotation speed of 150 rpm.

[0014] The present invention has the following beneficial effects: Through gene sequence analysis, fermentation culture, and component detection, the present invention determines that Boletus luridus YAFMF008 can produce cytochalasin E. Furthermore, by comparing the effects of different medium formulations on the content of cytochalasin E in Boletus luridus YAFMF008, it is screened out that the BDMY liquid medium is the optimal medium for Boletus luridus YAFMF008 to produce cytochalasin E. In this way, the content of cytochalasin E in the produced Boletus luridus YAFMF008 is the highest, which is more than 5 times that of the PDA solid medium and the liquid PDA medium. It provides a way for the efficient development of the medicinal value of Boletus luridus YAFMF008 and the efficient production of the bioactive component cytochalasin E. Description of the drawings

[0015] Figure 1 It is a comparison diagram of the cytochalasin biosynthesis gene cluster in Boletus luridus YAFMF008 described in the present invention; Figure 2 It is a result diagram of TLC detecting cytochalasin E in the fermentation product of YAFMF008, where A is the fermentation product and B is the standard cytochalasin E; Figure 3This is the LCMS analysis chart of the present invention; A: Cytochalasin E standard, B: Cytochalasin E in the fermentation product of YAFMF008, and the peak indicated by the red arrow is Cytochalasin E; Figure 4 This is the growth situation chart of Boletus phaeocystidiosus YAFMF008 of the present invention on PDA solid medium. A is the morphology of the fungus on the front of the petri dish, and B is the morphology of the fungus on the bottom of the petri dish; Figure 5 This is the growth situation chart of Boletus phaeocystidiosus YAFMF008 of the present invention in BDMY liquid medium. A is the side view, and B is the bottom view; Figure 6 This is the bar chart of the content difference of Cytochalasin E in Boletus phaeocystidiosus YAFMF008 cultured in different media; 1 is the solid PDA fermentation product, 2 is the BDMY fermentation product, and 2 is the PDAY fermentation product. Detailed implementation mode

[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. For the purchased commodities in the test methods, if the specific conditions are not indicated, they shall be carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0017] The Boletus phaeocystidiosus YAFMF008 provided by the present invention has the preservation name of Boletus phaeocystidiosus YAFMF008 (Phlebopus portentosus YAFMF008), is preserved in the China Center for Type Culture Collection, and the preservation address is Wuhan University; the preservation number: CCTCC NO: M 20232694, and the preservation date: December 27, 2023. This strain has been disclosed in the patent CN202410288908.0, a Boletus phaeocystidiosus YAFMF008, its isolation method, and the mycorrhizal seedling infection method.

[0018] All media in the present invention have been sterilized by high-temperature and high-pressure steam: 121 °C, 20 min.

[0019] Example 1 Determine that the genome of Boletus phaeocystidiosus YAFMF008 contains the biosynthetic gene of Cytochalasin E: To explore the potential secondary metabolites of Boletus obscurata YAFMF008, we sequenced its whole genome. The gene sequence accession number of Boletus obscurata YAFMF008 is JAYXKI000000000, and the web link is https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_037892935.1 / ). Through genome analysis, the synthetic genes related to the biosynthesis of cytochalasin E were found, including PKS-NRPS, trans-ER, α / β-hydrolase and other cytochalasin-related skeleton genes, as well as cytochrome P450, MFS, and O-methyltransferase and other modification genes. Its gene cluster structure is similar to the cytochalasin biosynthesis gene cluster of Aspergillus flavipes( Figure 1 ).

[0020] Example 2 Determination of cytochalasin E production by Boletus obscurata YAFMF008: To verify the production of cytochalasin E by Boletus obscurata YAFMF008, we purchased a cytochalasin E standard product (Order NO. A606583) from Sangon Biotech (Shanghai) Co., Ltd. After activating Boletus obscurata YAFMF008, it was inoculated into solid PDA medium and incubated statically in the dark at 28 °C for 25 days. Then it was extracted with ethyl acetate, sonicated for 40 min and then left to stand for 12 h. Then the extract was evaporated to remove the ethyl acetate solution with a rotary evaporator (temperature 50 °C, rotation speed 80 r / min) to obtain the fermentation product. The fermentation product was then detected by TLC. According to the comparison with the standard product, A is the fermentation product and B is the standard product cytochalasin E. The presence of cytochalasin E in the fermentation broth of Boletus obscurata YAFMF008 was determined by the color development and RF value( Figure 2 ). Subsequently, we used an LCMS-IT-TOF system (Shimadzu, Kyoto, Japan) to detect Boletus obscurata YAFMF008. The presence of cytochalasin E in the fermentation product of Boletus obscurata YAFMF008 was determined by comparing the retention time, molecular weight and fragment peaks with the standard product cytochalasin E( Figure 3 ).

[0021] Example 3 Scale-up culture of Boletus obscurata YAFMF008 and screening of fermentation medium: 1. Activation of Boletus obscurata YAFMF008 strain (primary strain culture) The activation preparation of the Boletus reticuloceps Fries strain YAFMF008 is as follows: Disinfect the laminar flow cabinet 15 minutes in advance by turning on the ultraviolet sterilization. Prepare the strain 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 a small piece of the original Boletus reticuloceps Fries strain YAFMF008 and inoculate it onto the PDA solid medium. After making marks (strain number, date), seal the film and place it in a constant temperature incubator at 26 o °C and 60% humidity for dark cultivation for 30 days. Observe and record every 5 days during this period. After the mycelium covers the entire medium, place it in a refrigerator at 4 o °C for storage for later use.

[0022] 2. Liquid culture of Boletus reticuloceps Fries strain YAFMF008 (secondary strain culture) Preparation of the liquid culture medium of Boletus reticuloceps Fries strain YAFMF008: Scrape one-fourth of the mycelium in a 60-mm culture dish of Boletus reticuloceps Fries strain YAFMF008 and put it into a 2-mL sterilized centrifuge tube. Then 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 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 rest is water). Place it in a constant temperature shaking incubator at 28°C and 150 rpm for dark cultivation for 10 days.

[0023] 3. Inoculation of Boletus reticuloceps Fries strain YAFMF008 in different fermentation media for cultivation (optimization of the tertiary strain medium) Absorb the mycelial balls of Boletus reticuloceps Fries strain YAFMF008 in liquid culture and inoculate them onto solid PDA: 200 g / L of boiled potato juice, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 5 g / L of yeast powder, 0.1 g / L of VB1, 16 g / L of agar; liquid PDAY: 200 g / L of boiled potato juice, 1 g / L of potassium dihydrogen phosphate, 0.5 g / L of magnesium sulfate, 5 g / L of yeast powder, 0.1 g / L of VB1; BDMY liquid medium: 7.5 g / L of Difco™ Malt Extract Broth, 5 g / L of malt-yeast, 1 g / L of yeast powder; all media have been sterilized by high-temperature high-pressure steam treatment (121 °C, 20 minutes). The culture conditions are: cultivate in a constant temperature shaking incubator (temperature 28 o °C, rotation speed 150 r / min, dark cultivation time 25 days).

[0024] Example 4 Harvest and extract the crude extract of Boletus luridus YAFMF008 after fermentation culture: After the liquid fermentation of Boletus luridus YAFMF008 is completed, harvest the mycelium. The specific operation steps are as follows: 1. Use a funnel to separate the mycelium from the bacterial liquid. Place the obtained bacterial liquid in a 250 mL Erlenmeyer flask, add ethyl acetate in a 1:1 ratio for extraction. After ultrasonic oscillation for 40 min, let it stand for 12 h. Then pour the extracted bacterial liquid into a separatory 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 separatory funnel into a round-bottom flask, and use a rotary evaporator (temperature 50 o °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 air-dry it.

[0025] 2. Blot the obtained mycelium dry with absorbent paper, transfer it to a sterilized centrifuge tube with forceps, immediately put it into liquid nitrogen for quick freezing, and then store it in a -80 o °C refrigerator.

[0026] 3. At the same time, sample and preserve the solid fermentation mycelium of Boletus luridus YAFMF008. Take 3 replicates for each sample of Boletus luridus YAFMF008 fermented and cultured in different formula media.

[0027] 4. Detection of cytochalasin E content Detect the cytochalasin E content of the mycelium of Boletus luridus YAFMF008 fermented and cultured in different formula media. 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 H 2 2O (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 supporting 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 a grinder, and oscillate at 55 Hz for 2 min; repeat this step twice; take out the centrifuge tube, centrifuge at 12000 rpm at 4 °C for 10 min, take the supernatant, 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 on-machine detection; (2) Perform LC-MS on-machine detection with the sample to be tested Chromatographic conditions: An ACQUITY UPLC® HSS T3 1.8 µm (2.1×150 mm) chromatographic column was used. The temperature of the auto-sampler was set at 8 °C. The flow rate was 0.25 mL / min, the column temperature was 40 °C, and 2 μL of the sample was injected 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 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).

[0028] 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 70,000, and the scanning range was 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.

[0029] Data analysis: According to the 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. Based on the retention time, molecular mass, and secondary fragmentation spectra, comparison was made with the data of cytochalasin E in the database to determine that the fermentation extract of Boletus obscuratus YAFMF008 contained cytochalasin E. Thus, the corresponding peaks in the HPCs data and the peak areas of cytochalasin E in different samples were found, which were the relative content differences of cytochalasin E in different samples. Based on this, the content differences of cytochalasin E in Boletus obscuratus YAFMF008 fermented and cultured in different formulated media were determined. The results are shown in Table 1.

[0030] Table 1 Content of cytochalasin E in Boletus obscuratus YAFMF008 in different samples

[0031] As can be seen from Table 1, the preferred fermentation medium BDMY of the present invention greatly increases the content of cytochalasin E compared with the PDA solid medium and the PDAY medium, and obtains a preferred fermentation medium for the production of cytochalasin E from the liquid fermentation mycelium of Boletus obscurellus YAFMF008, which can be used for large-scale industrial production and directly used in fermenters. The strain has stable performance, high yield and strong adaptability. The present invention provides a new and effective way for the production of cytochalasin E and the efficient utilization of Boletus obscurellus YAFMF008.

[0032] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A dark brown tricholoma boletus with a deposit number of CCTCC NO: M 20232694 ( Phlebopus portentosus )Application of YAFMF008 in the production of cytochalasin E.

2. Application of fermentation liquid of dark brown boletus YAFMF008 in producing cytochalasin E, characterized in that: The dark brown Dictyostelium Phlebopus portentosus ) The deposit number of YAFMF008 is CCTCC NO: M20232694, and the fermentation liquid is obtained by inoculating dark brown boletus YAFMF008 into a culture medium and fermenting it.

3. A method for producing cytochalasin E, characterized in that: The dark brown tricholoma boletus ( Phlebopus portentosus ) After YAFMF008 was inoculated in the culture medium and cultured, the fermentation broth was collected, separated and purified to obtain cytochalasin E.

4. A method for increasing the yield of cytochalasin E, characterized in that: The dark brown tricholoma boletus ( Phlebopus portentosus ) YAFMF008 was inoculated in a medium containing 7.5 g / L Malt Extract Broth, 5 g / L malt-yeast, and 1 g / L yeast powder, and the fermentation product was collected, separated, and purified to obtain cytochalasin E. The culture conditions were constant temperature 28 o C, dark culture at 150 rpm.

5. A factory-scale culture of dark brown tricholoma boletus with a preservation number of CCTCC NO: M 20232694 ( Phlebopus portentosus ) YAFMF008 A method for producing cytochalasin E, characterized in that: The following steps are involved: S1: Primary bacterial culture The dark brown phorbolus YAFMF008 strain was activated and cultured using PDA solid medium: 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; S2: Secondary bacterial culture The first-level strain of dark brown boletus YAFMF008 was inoculated into a modified malt extract broth liquid medium: animal tissue pepsin digest 5g / L, yeast extract 5g / L, malt extract 5g / L, maltose 3g / L and glucose 7g / L for cultivation; S3: Tertiary bacterial culture The secondary strain of Dictyostelium edulis YAFMF008 was inoculated into BDMY liquid culture medium: 7.5 g / L Malt Extract Broth, 5 g / L malt-yeast and 1 g / L yeast powder for cultivation, and the fermentation liquid was collected, and cytochalasin E was obtained after separation and purification.

6. The method according to claim 4, characterized in that The culture conditions in step S1 are 26 o C, cultured in the dark in a constant temperature incubator with 60% humidity.

7. The method according to claim 4, characterized in that The culture conditions in step S2 are constant temperature dark culture at 28° C. and 150 rpm.

8. The method according to claim 4, characterized in that The culture condition in step S3 is a constant temperature of 28 o C, cultured in the dark at 150 rpm.

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