Application of dark brown tricholoma boletus in producing cytochalasin E and fermentation method for increasing yield

Abstract

The present invention belongs to the technical field of edible and medicinal fungi production, and specifically discloses an application of dark brown dichotoma boletus in producing cytochalasin E and a fermentation method for improving the yield. Specifically, the present invention provides a dark brown dichotoma boletus ( Phlebopus portentousus ) The application of YAFMF008 in the production of cytochalasin E, the present invention discovered that dark brown tricholoma boletus ( Phlebopus portentousus ) YAFMF008 can produce cytochalasin E, and the fermentation culture method has been optimized. The optimized culture medium can significantly increase the yield of cytochalasin E, providing a way to develop the efficient medicinal value of the dark brown boletus YAFMF008 and the efficient production of the bioactive ingredient cytochalasin E.

Description

Technical Field

[0001] The invention belongs to the technical field of edible and medicinal fungi production, and particularly relates to an application of dark brown dichotoma boletus in producing cytochalasin E, and a fermentation medium and a fermentation method for increasing the content of cytochalasin E in dark brown dichotoma boletus YAFMF008. Background Art

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

[0003] Cytochalasin E (Cytochalasin E) is a fungal alkaloid belonging to the cytochalasin family. This class of compounds is known for its effects on the cytoskeleton, particularly microfilaments (actin fibers). Cytochalasin E can disrupt the normal progression of cells during mitosis and cell division by interfering with actin polymerization. Actin is a key component of cell division, playing a crucial role in the early stages of cell division, such as the fission of the cell membrane. Studies have shown that cytochalasin E has growth-inhibiting and cell death-inducing effects in certain cancer cells. By affecting cytoskeletal remodeling, it interferes with tumor cell proliferation and migration. Because cytochalasin E affects the cytoskeleton and reduces actin polymerization, it can inhibit cell migration and adhesion, making it potentially useful in studying cell motility and cancer metastasis. Overall, cytochalasin E's primary mechanism of action is to disrupt the dynamics of the cytoskeleton, affecting fundamental cell functions such as morphology, division, and motility. It is widely used in cell biology research, with particular application value in the study of cell migration, cell division, and oncology.

[0004] Microbial fermentation is the main production method of cytochalasin E. Different fermentation media and fermentation methods have a significant impact 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 to industrial production. Summary of the Invention

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

[0006] The technical solutions of the present invention are as follows:

[0007] In one aspect, the present invention provides a use of Phlebopus portentosus YAFMF008 with a deposit number of CCTCC NO: M 20232694 in producing cytochalasin E.

[0008] On the other hand, the present invention provides a use of a fermentation broth of Phlebopus portentosus YAFMF008 in producing cytochalasin E. The deposit number of the Phlebopus portentosus YAFMF008 is CCTCCNO: M 20232694. The fermentation broth is obtained by inoculating the Phlebopus portentosus YAFMF008 into a culture medium and fermenting the culture medium.

[0009] In another aspect, the present invention provides a method for producing cytochalasin E, comprising inoculating Phlebopus portentosus YAFMF008 with a deposit number of CCTCC NO: M20232694 into a culture medium for cultivation, collecting the fermentation broth, and separating and purifying the cytochalasin E.

[0010] On the other hand, the present invention provides a production method for increasing the yield of cytochalasin E, comprising inoculating Phlebopus portentosus YAFMF008 with a deposit number of CCTCC NO: M 20232694 into a culture medium containing 7.5 g / L Malt Extract Broth, 5 g / L malt-yeast, and 1 g / L yeast powder, culturing the culture medium, collecting the fermentation product, separating, and purifying the culture medium to obtain cytochalasin E. The culture conditions are a constant temperature of 28 o C, cultured in the dark at 150 rpm.

[0011] In another aspect, the present invention provides a method for producing cytochalasin E by factory-scale culture of Phlebopus portentosus YAFMF008, which has a preservation number of CCTCC NO: M 20232694, comprising the following steps:

[0012] S1: Primary bacterial culture

[0013] The dark brown boletus strain YAFMF008 was activated and cultured using PDA solid medium: 0.5 g potato starch / 100 mL, 0.1 g potassium dihydrogen phosphate / 100 mL, 0.05 g magnesium sulfate / 100 mL, 0.5 g yeast powder / 100 mL, 0.01 g VB1 / 100 mL, and 1.1 g agar / 100 mL.

[0014] S2: Secondary bacterial culture

[0015] The first-level strain of Dictyostelium edulis YAFMF008 was inoculated into a modified malt extract broth liquid medium containing 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 for cultivation;

[0016] S3: Tertiary bacterial culture

[0017] 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, and cultured. The fermentation broth was collected, and cytochalasin E was obtained after separation and purification.

[0018] Furthermore, the culture conditions in step S1 are 26 o C, cultured in the dark in a constant temperature incubator with 60% humidity.

[0019] Furthermore, the culture conditions in step S2 are constant temperature dark culture at 28° C. and 150 rpm.

[0020] Furthermore, the culture condition in step S3 is a constant temperature of 28 o C, cultured in the dark at 150 rpm.

[0021] The present invention has the following beneficial effects:

[0022] The present invention confirms that Dictyostelium edulis YAFMF008 can produce cytochalasin E through gene sequence analysis, fermentation culture, and component testing. Furthermore, by comparing the effects of different culture medium formulations on the cytochalasin E content in Dictyostelium edulis YAFMF008, the present invention identifies BDMY liquid culture medium as the optimal culture medium for cytochalasin E production by Dictyostelium edulis YAFMF008. The cytochalasin E content in Dictyostelium edulis YAFMF008 produced in this manner is the highest, exceeding five times that of PDA solid culture medium and liquid PDA culture medium. This provides a method for the efficient medicinal development of Dictyostelium edulis YAFMF008 and the efficient production of the bioactive ingredient cytochalasin E. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is an alignment diagram of the cytochalasin biosynthesis gene cluster in the dark brown boletus YAFMF008 of the present invention;

[0024] Figure 2This is a graph showing the results of TLC detection of cytochalasin E in the YAFMF008 fermentation product according to the present invention, wherein A is the fermentation product and B is the standard cytochalasin E;

[0025] Figure 3 LCMS analysis diagram of the present invention; A: cytochalasin E standard, B: cytochalasin E in YAFMF008 fermentation product, the peak indicated by the red arrow is cytochalasin E;

[0026] Figure 4 This is a diagram showing the growth of the dark brown boletus YAFMF008 described in the present invention in a PDA solid culture medium, A shows the bacterial morphology on the front of the culture dish, and B shows the bacterial morphology on the bottom of the culture dish;

[0027] Figure 5 This is a diagram showing the growth of the dark brown boletus YAFMF008 described in the present invention in BDMY liquid culture medium, with A showing the side view and B showing the bottom view;

[0028] Figure 6 1 is a bar graph showing the difference in the content of cytochalasin E in the dark brown boletus YAFMF008 cultured in different culture media; 1 is the solid PDA fermentation product, 2 is the BDMY fermentation product, and 2 is the PDAY fermentation product. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, 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. For purchased commodities in the test method, if no specific conditions are specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they can all be conventional products purchased from the market.

[0030] The dark brown dichotoma boletus YAFMF008 provided by the present invention has a preservation name of dark brown dichotoma boletus YAFMF008 (Phlebopus portentosus YAFMF008), which is preserved in the China Center for Type Culture Collection, and the preservation address is Wuhan University; the preservation number is: CCTCC NO: M 20232694, and the preservation date is: December 27, 2023. The strain has been disclosed by patent CN202410288908.0 A dark brown dichotoma boletus YAFMF008 and its isolation method and mycorrhizal seedling infection method.

[0031] All culture media in the present invention were sterilized by high-temperature and high-pressure steam sterilization: 121°C, 20 min.

[0032] Example 1

[0033] The genome of Dictyostelium edulis YAFMF008 was found to contain cytochalasin E biosynthesis genes.

[0034] To explore the potential secondary metabolites of Dictyostelium fuscae YAFMF008, we sequenced its entire genome (Dictyostelium fuscae YAFMF008 gene sequence accession number: JAYXKI000000000, web link: https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_037892935.1 / ). Genomic analysis revealed that the biosynthesis genes involved in cytochalasin E include cytochalasin-related skeleton genes such as PKS-NRPS, trans-ER, and α / β-hydrolase, as well as modification genes such as cytochrome P450, MFS, and O-methyltransferase. The gene cluster structure is similar to the cytochalasin biosynthesis gene cluster of Aspergillus flavus ( Figure 1 ).

[0035] Example 2

[0036] Determination of Cytochalasin E Production by Dictyostelium edulis YAFMF008:

[0037] To verify that the dark brown Dictyostelium boletus YAFMF008 produces cytochalasin E, we purchased a cytochalasin E standard (Order NO. A606583) from Shanghai Shenggong Technology Co., Ltd. We activated the dark brown Dictyostelium boletus YAFMF008 and inoculated it into solid PDA culture medium. The culture was then incubated at 28°C in the dark for 25 days. The culture was then extracted with ethyl acetate, ultrasonically shaken for 40 minutes, and allowed to stand for 12 hours. The extract was then removed from the ethyl acetate solution using a rotary evaporator (temperature 50°C, speed 80 r / min) to obtain the fermentation product. The fermentation product was then detected by TLC. Based on the comparison with the standard, A is the fermentation product and B is the standard cytochalasin E. The color and RF value were used to determine that the fermentation broth of the dark brown Dictyostelium boletus YAFMF008 contained cytochalasin E ( Figure 2 Subsequently, we used LCMS-IT-TOF system (Shimadzu, Kyoto, Japan) to detect the dark brown boletus YAFMF008 and confirmed that the dark brown boletus YAFMF008 fermentation product contained cytochalasin E ( Figure 3 ).

[0038] Example 3

[0039] Expansion culture and fermentation medium screening of dark brown boletus YAFMF008:

[0040] 1. Activation of the dark brown boletus YAFMF008 strain (primary strain culture)

[0041] To activate the Dictyostelium edulis YAFMF008 strain, disinfect the clean bench and perform UV sterilization 15 minutes in advance. Prepare the strain 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. After cooling, pick a small piece of the original Dictyostelium edulis YAFMF008 strain and inoculate it into the PDA solid culture medium. Label it (strain number, date), seal it with film, and place it in a 26-hour refrigeration oven. o C. Culture in a constant temperature incubator with 60% humidity for 30 days in the dark. Observe and record every 5 days. When the mycelium covers the entire culture medium, place it in a 4 o C Store in refrigerator for future use.

[0042] 2. Liquid culture of dark brown boletus YAFMF008 (secondary strain culture)

[0043] Preparation of liquid culture of Dictyostelium boletum YAFMF008: scrape one-quarter of the mycelium from a 60 mm culture dish of Dictyostelium boletus YAFMF008 and place it in a 2 mL sterile centrifuge tube. Add 1 mL of sterile water and crush it for 2 min. Then, take 500 µL of the crushed sample liquid and inoculate it into 250 mL conical flasks 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 the flask in a constant temperature shaker at 28°C and 150 rpm and culture in the dark for 10 days.

[0044] 3. Cultivation of dark brown boletus YAFMF008 in different fermentation media (optimization of tertiary culture medium)

[0045] Liquid culture pellets of Dictyostelium edulis YAFMF008 were inoculated onto solid PDA (200 g / L boiled potato juice, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 5 g / L yeast powder, 0.1 g / L VB1, and 16 g / L agar); liquid PDAY (200 g / L boiled potato juice, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 5 g / L yeast powder, and 0.1 g / L VB1); and BDMY (7.5 g / L Difco™ Malt Extract Broth, 5 g / L malt-yeast, and 1 g / L yeast powder). All media were sterilized with high-temperature and high-pressure steam (121°C for 20 min) and cultured in a constant-temperature shaking incubator (28 o C, rotation speed 150 r / min, dark culture time 25 d).

[0046] Example 4

[0047] Harvest, extract, ferment and culture the crude extract of Dictyostelium edulis YAFMF008:

[0048] After the liquid fermentation of dark brown boletus YAFMF008 is completed, the mycelium is harvested. The specific steps are as follows:

[0049] 1. Use a funnel to separate the mycelium from the bacterial solution. The obtained bacterial solution is placed in a 250 mL Erlenmeyer flask and extracted with 1:1 ethyl acetate. Ultrasonic vibration is performed for 40 minutes and then allowed to stand for 12 hours. The extracted bacterial solution is then poured into a separatory funnel for full extraction. During this period, the solution is shaken up and down (3 to 5 times). After standing for 3 hours, the upper layer of the extract in the separatory funnel is poured into a round-bottom flask and evaporated using a rotary evaporator (temperature 50 o C, speed 80 r / min) to obtain a crude extract, rinse the crude extract with acetone, transfer it to a 2 mL weighing centrifuge tube, and dry it.

[0050] 2. The obtained mycelium was dried with absorbent paper, transferred to a sterilized centrifuge tube with tweezers, immediately frozen in liquid nitrogen, and stored at -80 o C refrigerator.

[0051] 3. At the same time, samples of the solid fermentation mycelium of Dictyostelium edulis YAFMF008 were collected and preserved. Three replicates were taken for each sample of Dictyostelium edulis YAFMF008 fermented in different culture media.

[0052] 4. Cytochalasin E content detection

[0053] The cytochalasin E content of the mycelium of Dictyostelium edulis YAFMF008 fermented in different culture media was detected. The specific steps are as follows:

[0054] (1) Metabolite extraction

[0055] Weigh 60 mg of 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 and immerse it in liquid nitrogen for rapid freezing for 5 min. Remove the centrifuge tube and freeze and thaw it at room temperature. Place the centrifuge tube again in the 2 mL adapter, install it in the grinder, and oscillate at 55 Hz for 2 min. Repeat this step twice; remove the centrifuge tube, centrifuge at 12,000 rpm at 4°C for 10 min, collect the supernatant, and concentrate it to dryness by centrifugation; dissolve the sample in 300 µL of 50% 2-chlorophenylalanine (4 ppm) methanol-water solution (1:1, 4°C), filter it through a 0.22 µm membrane, and perform LC-MS analysis.

[0056] (2) Perform LC-MS testing on the sample to be tested

[0057] 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 phases 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).

[0058] 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, and full scans were performed at a resolution of 70,000 over a scan range of 81 to 1000. HCD was used for secondary fragmentation at a collision voltage of 30 eV, and dynamic exclusion was used to remove unnecessary MS / MS information.

[0059] Data Analysis: Based on the test results, metabolomics data analysis was performed on the samples tested. The specific steps are as follows: Data preprocessing, including format conversion, peak identification, filtering, alignment, and normalization, was performed. Chromatograms and quality control were used to verify the data. Retention time, molecular mass, and secondary fragmentation spectrum were compared with the cytochalasin E data in the database to confirm the presence of cytochalasin E in the fermentation extract of Dictyostelium edulis YAFMF008. The corresponding peaks in the HPCs data and the peak areas of cytochalasin E in different samples were identified, indicating the relative content differences of cytochalasin E in different samples. This was used to determine the differences in cytochalasin E content in Dictyostelium edulis YAFMF008 fermented in different culture media formulations. The results are shown in Table 1.

[0060] Table 1 The content of cytochalasin E in different samples of dark brown boletus YAFMF008

[0061]

[0062] As can be seen from Table 1, the preferred fermentation medium BDMY of the present invention greatly increases the content of cytochalasin E compared to PDA solid medium and PDAY medium, thereby obtaining a preferred fermentation medium for producing cytochalasin E from liquid fermentation mycelium of Dictyostelium boletum YAFMF008. The medium can be used for large-scale industrial production and can be directly used in fermentation tank production. The strain has stable performance, high yield, and strong adaptability. The present invention provides a new and effective approach for the production of cytochalasin E and the efficient utilization of Dictyostelium boletus YAFMF008.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for increasing the yield of cytochalasin E, characterized in that: The following steps are involved: S1: Primary bacterial culture The dark brown boletus YAFMF008 strain was activated and cultured using PDA solid medium: potato starch 0.5g / 100mL, potassium dihydrogen phosphate 0.1g / 100mL, magnesium sulfate 0.05g / 100mL, yeast powder 0.5g / 100mL, VB10.01g / 100mL and agar 1.1g / 100mL; the culture conditions were 26 o C, dark culture in a constant temperature incubator at 60% humidity, the deposit number of the dark brown boletus YAFMF008 is CCTCC NO: M 20232694; S2: Secondary bacterial culture The first-level strain of Dictyostelium edulis YAFMF008 was inoculated into a modified malt extract broth liquid culture medium containing 5 g / L animal tissue pepsin digest, 5 g / L yeast extract, 5 g / L malt extract, 3 g / L maltose, and 7 g / L glucose for culturing; in step S2, the culturing conditions were constant temperature dark culturing at 28° C. and 150 rpm; S3: Tertiary bacterial culture The secondary strain of Dictyostelium edulis YAFMF008 was inoculated into BDMY liquid medium containing 7.5 g / L Malt Extract Broth, 5 g / L malt-yeast and 1 g / L yeast powder, and cultured. The fermentation liquid was collected and separated and purified to obtain cytochalasin E. The culture conditions were constant temperature 28 o C, cultured in the dark at 150 rpm.

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