Method for promoting phlebopus portentosus to produce dehydrotumulin acid

Abstract

The invention provides a method for promoting phlebopus portentosus to produce dehydrotumulic acid, and relates to the technical field of medicinal fungus culture. The method for promoting the phlebopus portentosus to produce the dehydrotumoic acid comprises the step of inoculating a liquid strain of the phlebopus portentosus into a culture medium containing walnut oil residue powder and walnut shell powder for culturing. According to the method, the defects in the prior art are overcome, the capability of the phlebopus portentosus for producing the dehydrotumuloic acid can be effectively improved by optimizing the culture medium, and compared with a common culture medium, a culture medium only added with walnut oil residue powder, a culture medium only added with walnut shell powder and a culture medium added with walnut branch powder, the yield of the phlebopus portentosus is greatly improved. And the yield of the dehydrotumulin acid is obviously improved, and a new direction is provided for the production of the dehydrotumulin acid.

Description

Technical Field

[0001] This invention relates to the field of medicinal fungi culture technology, specifically to a method for promoting the production of dehydrotomonic acid by Boletus thaliana. Background Technology

[0002] Dehydrotumulosic acid (CAS Registry Number 6754-16-1) is a tetracyclic triterpenoid compound and a key active ingredient in Guizhi Fuling capsules. It is a triterpenoid compound isolated from the traditional Chinese medicine Poria cocos. This compound possesses pharmacological activities including antitumor, anti-inflammatory, immunomodulatory, and antiemetic effects. Pharmacologically, it is used to expel dampness, promote diuresis, strengthen the spleen, calm the mind, and regulate the body's immune system. Dehydrotumulosic acid is also a potential glucocorticoid receptor agonist with anti-Coxsackie virus activity.

[0003] *Boletus davidii* is an edible and medicinal mushroom with a delicious flavor and high nutritional value. Belonging to the genus *Boletus* in the family Boletaceae, order Boletales, it is now widely distributed in tropical China, especially in Yunnan, Guangxi, and Hainan (Yang, et al., 2023; Wan, et al., 2021). It is locally known as "black boletus" due to its dark-colored fruiting body. Previous research on *Boletus davidii* has mainly focused on its cultivation and commercial value, and it is the first edible bolete variety that can be artificially cultivated. Previous research on the secondary metabolites of *Boletus davidii* has mainly focused on phenolic substances, alkaloids, and polysaccharides. Subsequent genetic analysis revealed a large number of triterpenoid synthesis-related genes in the Boletus tarda genome, indicating a potential ability to synthesize triterpenoid compounds. However, the content of related components such as dehydrotomolates is low during the actual cultivation of Boletus tarda. Therefore, how to increase the yield of dehydrotomolates during the cultivation of Boletus tarda is a major research direction at present. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for promoting the production of dehydrotomonic acid by Boletus brownii. By optimizing the addition of a combination of walnut oil residue powder and walnut shell powder as exogenous components to the culture medium, the yield of dehydrotomonic acid in Boletus brownii is increased, providing a new direction for the production of dehydrotomonic acid.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A method for promoting the production of dehydrotomonic acid by Boletus thaliana, wherein the method involves inoculating a liquid culture of Boletus thaliana into a culture medium containing walnut oil residue powder and walnut shell powder for cultivation, and the specific culture medium formula is: 7 g / L walnut oil residue powder + 1 g / L walnut shell powder + 2 g / L mannitol + 0.5 g / L ammonium sulfate + 1 g / L magnesium sulfate + 1 g / L dipotassium hydrogen phosphate + water.

[0006] Preferably, the walnut oil residue powder and walnut shell powder are obtained by drying the walnut oil residue and walnut shell and then grinding and pulverizing them to a particle size of 1-30μm.

[0007] Preferably, the specific method for obtaining the liquid culture of *Boletus glomeratus* includes the following steps: (1) Inoculate Boletus glomeratus onto a modified PDAM plate and incubate at 28°C in the dark for 15 days to obtain a solid culture. (2) Cut the above solid strain into small pieces and inoculate them into PMG liquid culture medium. Shake at 150 rpm, incubate in the dark at 28°C for 20 days to obtain Boletus glomeratus liquid strain.

[0008] Preferably, the method for preparing the modified PDAM petri dish in step (1) is as follows: cut 200g of potatoes into granules and put them into 1L of boiling water. After boiling for 6 minutes, filter the potato granules with gauze to obtain potato extract. Add 5g of malt extract broth medium, 20g of glucose, 0.5g of ammonium sulfate, 1g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, and 15g of agar to the potato extract. After steam sterilization at 121℃ for 20 minutes, pour the mixture into a 60cm petri dish and wait for it to solidify before use.

[0009] Preferably, the preparation method of PMG liquid culture medium in step (2) is as follows: cut 200g of potatoes into granules and put them into 1L of boiling water. After boiling for 6 minutes, filter the potato granules with gauze to obtain potato extract. Add 20g of mannitol, 0.5g of ammonium sulfate, 1g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, and 0.1g of vitamin B1 to the potato extract. Mix thoroughly and dispense into 250ml Erlenmeyer flasks. Sterilize by steaming at 121℃ for 20 minutes before use.

[0010] Preferably, the culture method is to culture in the dark at 28°C for a period of time on a shaker at 150 rpm, then add valproic acid to the culture medium and continue the culture.

[0011] This invention provides a method for promoting the production of dehydrotomonic acid by Boletus thaliana, which has the following advantages compared with the prior art: This invention inoculates a liquid strain of Boletus thunbergii into a culture medium containing walnut oil residue powder and walnut shell powder, which effectively enhances the production of dehydrothomolenic acid by Boletus thunbergii. Compared with ordinary culture medium, culture medium with only walnut oil residue powder, culture medium with only walnut shell powder, and culture medium with walnut branch powder, the yield of dehydrothomolenic acid is significantly increased, providing a new direction for the production of dehydrothomolenic acid. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the liquid culture of Boletus glomeratus YAFMF008 in an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the cultivation of Boletus thunbergii liquid culture medium in an embodiment of the present invention. Figure 3 This is a schematic diagram illustrating the cultivation of Boletus thunbergii liquid culture medium using MNHZ liquid medium in an embodiment of the present invention. Figure 4 This is a schematic diagram illustrating the cultivation of Boletus thunbergii liquid culture medium in an embodiment of the present invention. Figure 5 This is a schematic diagram illustrating the cultivation of Boletus thunbergii liquid culture medium using MMN liquid culture medium in an embodiment of the present invention; Figure 6 This is a schematic diagram illustrating the cultivation of Boletus thunbergii liquid culture medium in an embodiment of the present invention. Figure 7 For the HPLC detection of MNHYK culture in this invention, the peak indicated by the red arrow is dehydrothomonic acid; Figure 8 For the HPLC detection of MNHZ culture in this invention, the peak indicated by the red arrow is dehydrotomolic acid; Figure 9 For the HPLC detection of MNHK culture in this invention, the peak indicated by the red arrow is dehydrothomonic acid; Figure 10 For HPLC detection of MMN cultures in this invention, the peak indicated by the red arrow is dehydrothomonic acid; Figure 11 For the HPLC detection of MNHY culture in this invention, the peak indicated by the red arrow is dehydrotomonic acid. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of 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 only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] The *Boletus glomeratus* used in the following examples is *Boletus glomeratus* YAFMF008, accession number CCTCC NO: M 20232694, which has been disclosed in a prior patent application (Wang Yi, Li Jiang, Zhang Yadong, A *Boletus glomeratus* YAFMF008 and its isolation method and mycorrhizal seedling infection method; Authorization date: May 17, 2024, China, ZL202410288908.0, Application date: March 14, 2024). Example:

[0015] 1. Preparation of liquid culture of Boletus globosum: First, the *Boletus edulis* YAFMF008, stored at 4°C, was removed from the refrigerator. It was inoculated onto a modified PDAM plate and incubated in the dark at 28°C for 15 days to obtain a solid culture.

[0016] Improved PDAM petri dish preparation method: Cut 200g of potatoes into small pieces and place them in 1L of boiling water. Boil for 6 minutes, then filter through cheesecloth to remove the potato pieces and obtain a potato extract. Add 5g of malt-gold powder broth medium, 20g of glucose, 0.5g of ammonium sulfate, 1g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, and 15g of agar to the potato extract. After high-temperature and high-pressure steam sterilization (121℃, 20 min), pour into 60cm petri dishes and allow to solidify before use.

[0017] Liquid culture preparation The *Boletus edulis* YAFMF008 strain, cultured with modified PDAM for 15 days, was cut into 2cm pieces using a sterilized scalpel. 2 Small pieces were inoculated into PMG liquid medium and cultured in the dark at 28°C for 20 days using a shaker (150 rpm). This yielded a liquid culture of *Boletus glomeratus*. Figure 1 ).

[0018] The method for preparing PMG liquid culture medium is as follows: Cut 200g of potatoes into small pieces and put them into 1L of boiling water. Boil for 6 minutes, then filter with gauze to remove the potato pieces and obtain potato extract. Add 20g of mannitol, 0.5g of ammonium sulfate, 1g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, and 0.1g of vitamin B1 to the potato extract. Mix thoroughly and dispense into 250ml Erlenmeyer flasks. After high-temperature and high-pressure steam sterilization (121℃, 20 min), it is ready for use.

[0019] 2. Setting up different culture media: MNHYK liquid culture medium: 7 g / L walnut oil residue powder, 1 g / L walnut shell powder, 2 g / L mannitol, 0.5 g / L ammonium sulfate, 1 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, and the remainder is water; MNHZ liquid medium: 7 g / L walnut branch powder, 2 g / L mannitol, 0.5 g / L ammonium sulfate, 1 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, and the remainder is water; MNHK liquid culture medium: 7 g / L walnut shell powder, 2 g / L mannitol, 0.5 g / L ammonium sulfate, 1 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, and the remainder is water.

[0020] MMN liquid culture medium: mannitol 10 g / L, ammonium sulfate 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, the remainder being water; MNHY liquid culture medium: 7 g / L walnut oil residue powder, 2 g / L mannitol, 0.5 g / L ammonium sulfate, 1 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, and the remainder is water.

[0021] All the above culture media were sterilized by high temperature and high pressure steam (121℃, 20 min) before use; Furthermore, the walnut oil residue powder, walnut shell powder, and walnut branch powder are made by drying the walnut oil residue, walnut shell, and walnut branch and then grinding them into ultra-fine powder of 1-30 micrometers using a grinder.

[0022] 3. Inoculation and culture: Using a pipette, 3 ml of liquid culture of *Boletus glomeratus* was inoculated into 100 ml of each of the following liquid media: MNHYK, MNHZ, MNHK, MMN, and MNHY. The media were then incubated in the dark at 28°C for 10 days using a shaker (150 rpm). Afterward, 200 μL of valproic acid was added to each medium, and the culture was incubated for another 15 days before harvesting the mycelium (the culture conditions for each medium are shown in the figure). Figures 2-6 (As shown).

[0023] 4. Extraction of fermentation products: Product extraction was performed on the fermentation mycelia of the above-mentioned culture media: (1) Sample preparation: Weigh 30 mg of fungal mycelium obtained from different culture media and place them in 2 mL centrifuge tubes.

[0024] (2) Initial extraction: Add 1 mL of pre-cooled 50% methanol solution to each centrifuge tube and add 2 glass beads. Then vortex for 30 seconds to fully suspend the sample.

[0025] (3) Freezing treatment: Quickly immerse the centrifuge tubes in liquid nitrogen and freeze for 5 minutes.

[0026] (4) Grinding and breaking: Take out the centrifuge tube, thaw it at room temperature, put it into a high-throughput tissue homogenizer, and grind it at a frequency of 55 Hz for 60 seconds to break the cells.

[0027] (5) Repeated extraction and freezing: Repeat steps (2) (adding methanol and vortexing) and (3) (freezing with liquid nitrogen) twice to fully extract the target product. Then, place all centrifuge tubes in a -20°C environment for 30 minutes.

[0028] (6) Centrifugation and concentration: Centrifuge the centrifuge tube at 4°C and 12,000 rpm for 20 minutes. Carefully aspirate 850 µL of the supernatant and concentrate it under vacuum until completely dry.

[0029] (7) Resolution: Add 150 µL of 50% methanol solution containing 5 ppm 2-chlorophenylalanine to the dried residue and vortex for 30 seconds to fully resolution.

[0030] (8) Purification and sample preparation: Centrifuge the reconstituted solution at 4°C and 12,000 rpm for 10 minutes. Take the supernatant, filter it through a 0.22 μm microporous membrane, and finally transfer the filtrate to the detection bottle for subsequent analysis.

[0031] 5. Detection and identification methods of compounds using liquid chromatography-mass spectrometry High performance liquid chromatography-tandem high resolution mass spectrometry was used to separate, detect and identify the samples.

[0032] (1) Instrument configuration The analysis was performed using a Shimadzu LC-30A ultra-high performance liquid chromatography system coupled with a SCIEX TripleTOF 6600+ high-resolution mass spectrometer.

[0033] (2) Liquid chromatography conditions Chromatographic column: Waters ACQUITY Premier HSS T3 column (100 mm × 2.1 mm, 1.8 µm).

[0034] Mobile phase: Phase A: Ultrapure water (containing 0.1% formic acid) Phase B: Acetonitrile (containing 0.1% formic acid) Flow rate: 0.4 mL / min.

[0035] Injection volume: 4 µL.

[0036] Gradient procedure: 0 – 2 min: The proportion of phase B increases linearly from 5% to 20%. 2 – 5 min: Phase B proportion increases linearly from 15% to 60%. 5 – 7.5 min: The proportion of phase B increases linearly from 55% to 99% and remains thereafter. 7.5 – 7.6 min: Phase B percentage rapidly decreases from 99% to 5%. 7.6 – 10 min: Maintain phase B ratio at 5% to equilibrate the column. Ion mode: The method operates in both positive and negative ion modes, with a consistent elution gradient.

[0037] (3) Mass spectrometry conditions Data acquisition was performed in information-dependent acquisition mode, controlled by Analyst TF 1.7.1 software. Ion source parameters (ESI) were: Ion source gas 1 and gas 2: both 50 psi, curtain gas: 25 psi, ion source temperature: 550 ℃, de-clustering voltage: 60 V in positive mode / -60 V in negative mode. Ion spray voltage: 5000 V in positive mode / -4000 V in negative mode Level 1 mass spectrometry scan parameters: Scan range: 50 – 1000 Da, cumulative time: 200 ms, dynamic background subtraction enabled. Secondary mass spectrometry scanning parameters: Scan range: 25 – 1000 Da, cumulative time: 40 ms Collision energy: Positive mode 30 V / Negative mode -30 V (collision energy spread is 15 V) In each cycle, up to 18 candidate parent ions with an intensity greater than 100 cps are selected for fragmentation, and isotopic ions in the 4 Da range are excluded.

[0038] (4) Compound identification methods Raw data were imported into the PGNLM database by Pasenno for compound identification. This database integrates multiple authoritative metabolite databases (including mzCloud, HMDB, LIPID MAPS, MoNA, NIST 2020 MSMS, and AI-predicted spectral libraries). Key database search parameters were set as follows: Level 1 mass spectrometry precision: 0.01 Da, Level 2 mass spectrometry precision: 0.05 Da, identification score cutoff: 70 points. By comparing with database data, the compound with a retention time of 3.914 minutes was identified as dehydrotomonic acid.

[0039] Relevant HPLC detections of various liquid culture media cultures, such as Figure 7-11 As shown in the figure, the peaks indicated by the red arrows in each figure are dehydrotumoic acid.

[0040] After determining the peak pattern of dehydrotemmolic acid, the relative ratios of its peak areas were calculated to compare the differences in dehydrotemmolic acid content in the mycelium of *Boletus glomeratus* YAFMF008 after cultivation in different culture media. The results are shown in the table below:

[0041] As can be seen from the table above, the MNHYK liquid medium significantly increases the content of dehydrotumoic acid compared to other culture methods, providing a new and effective approach for the large-scale production of dehydrotumoic acid.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for promoting the production of dehydrotomonic acid by Boletus brownii, characterized in that, The method involves inoculating a liquid culture of Boletus thunbergii into a culture medium containing walnut oil residue powder and walnut shell powder for cultivation. The specific culture medium formula is as follows: 7 g / L walnut oil residue powder + 1 g / L walnut shell powder + 2 g / L mannitol + 0.5 g / L ammonium sulfate + 1 g / L magnesium sulfate + 1 g / L dipotassium hydrogen phosphate + water.

2. The method according to claim 1, characterized in that: The walnut oil residue powder and walnut shell powder are obtained by drying walnut oil residue and walnut shells and then grinding and pulverizing them to a particle size of 1-30μm.

3. The method according to claim 1, characterized in that, The specific method for obtaining the liquid culture of the *Boletus edulis* includes the following steps: (1) Inoculate Boletus glomeratus onto a modified PDAM plate and incubate at 28°C in the dark for 15 days to obtain a solid culture. (2) Cut the above solid culture into small pieces and inoculate them into PMG liquid culture medium. Shake at 150 rpm, incubate in the dark at 28°C for 20 days to obtain Boletus glomeratus liquid culture.

4. The method according to claim 3, characterized in that: The method for preparing the modified PDAM petri dish in step (1) is as follows: Cut 200g of potatoes into small pieces and put them into 1L of boiling water. After boiling for 6 minutes, filter the potato pieces with gauze to obtain potato extract. Add 5g of malt extract broth medium, 20g of glucose, 0.5g of ammonium sulfate, 1g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, and 15g of agar to the potato extract. After steam sterilization at 121℃ for 20 minutes, pour the mixture into a 60cm petri dish and wait for it to solidify before use.

5. The method according to claim 3, characterized in that: The preparation method of PMG liquid culture medium in step (2) is as follows: cut 200g of potatoes into granules and put them into 1L of boiling water. After boiling for 6 minutes, filter the potato granules with gauze to obtain potato extract. Add 20g of mannitol, 0.5g of ammonium sulfate, 1g of magnesium sulfate, 1g of dipotassium hydrogen phosphate, and 0.1g of vitamin B1 to the potato extract. Mix thoroughly and dispense into 250ml Erlenmeyer flasks. Sterilize by steaming at 121℃ for 20 minutes before use.

6. The method according to claim 1, characterized in that: The culture method involves shaking at 150 rpm and incubating in the dark at 28°C for a period of time, followed by the addition of valproic acid to the culture medium and continued culturing.

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