A method for increasing the yield of betulinic acid in Phellinus bournei production

Abstract

This invention provides a method for increasing the production of betulinic acid from *Sanghuang* bao's mushroom, relating to the field of medicinal fungal culture technology. The method involves inoculating a liquid strain of *Sanghuang* bao's mushroom into a PGO-added solid culture medium, wherein the PGO solid culture medium formulation is 20g ginseng powder + 15ml water + 2g soybean oil. This invention overcomes the shortcomings of existing technologies by effectively promoting the production of betulinic acid from *Sanghuang* bao's mushroom through optimized culture medium formulation, providing a new direction for the development of *Sanghuang* bao's mushroom culture products with high betulinic acid content.

Description

Technical Field

[0001] This invention relates to the field of medicinal fungi cultivation technology, specifically to a method for increasing the yield of betulinic acid in Phellinus bournei. Background Technology

[0002] Betulinic acid is a pentacyclic triterpenoid natural product derived from various plants, known for its diverse and significant biological activities and good safety profile. Betulinic acid exhibits selective cytotoxicity against specific tumor cells such as melanoma, induces apoptosis, and inhibits eukaryotic topoisomerase I. Antiviral activity: It demonstrates anti-HIV activity, and some of its derivatives have successfully completed phase II clinical trials for anti-HIV purposes. Anti-inflammatory and immunomodulatory effects: In a spinal cord injury model, it enhances autophagy through the AMPK-HDAC5-KLF2 signaling pathway, promoting microglia polarization towards the M2 type, thereby alleviating inflammation. Cardiovascular protection: Birch aldehyde (a derivative of betulinic acid) can regulate the phenotype of vascular smooth muscle cells through the PPARγ / mitochondria / ROS axis, alleviating angiogenesis and showing potential therapeutic value for cardiovascular diseases. Studies have also shown that it possesses antibacterial, antimalarial, and antioxidant activities.

[0003] Betulinic acid is currently mainly extracted from various plants such as birch bark and jujube kernel. For example, the purity of betulinic acid extracted from jujube kernel can reach 86.6%. Recently, researchers have also discovered that fungi of the genus *Philobacter* in the family Polyporaceae can also produce betulinic acid under liquid culture conditions. In addition, researchers have also found that adding edible oil can increase the triterpenoid content in *Ganoderma lucidum* and *Antrodia camphorata*.

[0004] Sanghuang, a traditional edible and medicinal fungus, is rich in secondary metabolites such as polysaccharides, terpenes, and polyphenols (Cai, C., Ma, J., Han, C., Jin, Y., Zhao, G., & He, X. (2019). Extraction and antioxidant activity of total triterpenoids in the mycelium of a medicinal fungus, Sanghuangporus sanghuang. Scientific reports, 9(1), 7418.). Its main pharmacological activities include antitumor and antioxidant effects (LiT, Mei Y, Li J, et al. Comparative Compositions and Activities of Flavonoids from Nine Sanghuang Strains Based on Solid-State Fermentation and In VitroAssays[J]. Fermentation, 2023, 9(3): 308.), anti-inflammatory (Lin WC, Deng JS, Huang SS, et al. Evaluation of antioxidant, anti-inflammatory and anti-proliferative activities of ethanol extracts from different varieties of Sanghuang species[J]. Rsc Advances, 2017, 7(13): 7780-7788.), immunomodulation and antidiabetic (Hou R, Zhou L, Fu Y, et al. Chemical characterization of two fractions from Sanghuangporus sanghuang and evaluation of antidiabetic activity[J].Journal of Functional Foods (Chemical characterization and antidiabetic activity evaluation of two components of Sanghuang[J]. Journal of Functional Foods), 2021, 87: 104825., Hepatoprotective (Jiang WP, Deng JS, Huang SS, et al. Sanghuangporus sanghuang mycelium prevents paracetamol-induced hepatotoxicity through regulating the MAPK / NF-κB, Keap1 / Nrf2 / HO-1, TLR4 / PI3K / Akt, and CaMKKβ / LKB1 / AMPK pathways and suppressing oxidative stress and inflammation[J]. Antioxidants (Sanghuang mycelium prevents paracetamol-induced hepatotoxicity by regulating the MAPK / NF-κB, Keap1 / Nrf2 / HO-1, TLR4 / PI3K / Akt and CaMKKβ / LKB1 / AMPK signaling pathways, thereby inhibiting oxidative stress and inflammatory response[J]. Antioxidants) 2021, 10(6): 897.) and neuroprotection. Meanwhile, *Sanghuang* mycelium showed no mutagenic activity in the bacterial reverse mutagenesis test (Ames test) and did not induce chromosomal aberrations; acute (7-day) oral toxicity studies in ICR mice showed LD50 > 12 g / kg bw; in the mouse micronucleus test, *Sanghuang* mycelium did not alter the abundance of reticulocytes or significantly increase the micronucleus rate of reticulocytes, meaning it did not cause chromosomal damage during the cell cycle. These toxicological studies all indicate that *Sanghuang* has extremely low toxicity, proving its safety for humans.

[0005] The main active components of *Sanghuangporus baumii* include polysaccharides, terpenes, flavonoids, and polyphenols, which have been shown to possess various pharmacological activities, including anticancer, anti-inflammatory, antioxidant, immunomodulatory, hypoglycemic, and hypolipidemic effects. Based on these previous studies, developing *Sanghuangporus baumii* cultures with high betulinic acid content is a significant research direction. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for increasing the production of betulinic acid from Phellinus boulardii. By optimizing the culture medium formulation, the method effectively promotes the production of betulinic acid from Phellinus boulardii, providing a new direction for the production of Phellinus boulardii with high betulinic acid content.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An edible culture of Phellinus boulardii with high betulinic acid content, wherein the method involves inoculating the liquid strain of Phellinus boulardii into PGO solid medium for culture, and the formula of PGO solid medium is 20g ginseng powder + 15ml water + 2g soybean oil.

[0009] Preferably, the culture conditions are dark culture at 28°C.

[0010] Preferably, the method for preparing the liquid bacterial strain includes the following steps:

[0011] (1) Inoculate the Phellinus baozi onto MY solid medium, incubate in the dark at 28℃ for 10 days, and collect the mycelium for later use;

[0012] (2) Mix the mycelium with sterile water and steel balls, and grind it with a grinder to obtain a fungal suspension for later use;

[0013] (3) Inoculate the bacterial suspension into liquid MY medium and culture it on a shaker for 7 days to obtain liquid bacterial culture.

[0014] Preferably, in step (2), the grinding speed of the grinder is 180 rpm and the grinding time is 2 min.

[0015] Preferably, in step (3), the shaking speed is 150 rpm, the temperature is 28°C, and the culture method is dark culture.

[0016] This invention provides an edible culture of Phellinus boulardii with high betulinic acid content, which has the following advantages compared with the prior art:

[0017] This invention is the first to utilize traditional Chinese medicine powder fermentation to cultivate *Sanghuang Bao's*. To better suit product development and meet the requirements for both medicinal and edible use, all culture medium raw materials are commercially available medicinal herbs and soybean oil of existing medicinal and edible grade. The culture contains no additives or artificial chemical components. Comparative studies revealed that ginseng powder effectively promotes the content of betulinic acid in *Sanghuang Bao's* mycelium, and the betulinic acid yield is significantly increased compared to cultures containing *Polygonum multiflorum* powder, *Salvia miltiorrhiza* powder, *Coix lacryma-jobi* powder, and pea powder. Adding soybean oil to the ginseng culture medium significantly increases the biomass and betulinic acid content of *Sanghuang Bao's*. This invention provides a new and effective approach for the development of *Sanghuang Bao's* products and for developing *Sanghuang Bao's* cultures with high betulinic acid content. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of PGO solid culture medium culture in an embodiment of the present invention;

[0019] Figure 2This is a schematic diagram of PG solid culture medium culture in an embodiment of the present invention;

[0020] Figure 3 This is a schematic diagram of PS solid culture medium cultivation in an embodiment of the present invention;

[0021] Figure 4 This is a schematic diagram of PM solid culture medium culture in an embodiment of the present invention;

[0022] Figure 5 This is a schematic diagram of CL solid culture medium culture in an embodiment of the present invention;

[0023] Figure 6 The image shows the HPLC detection spectrum of PGO solid culture medium in an embodiment of the present invention, where the peak indicated by the red arrow is betulinic acid;

[0024] Figure 7 The image shows the HPLC detection spectrum of the PG solid culture medium in this embodiment of the invention, where the peak indicated by the red arrow is betulinic acid;

[0025] Figure 8 The image shows the HPLC detection spectrum of PM solid culture medium in this embodiment of the invention, where the peak indicated by the red arrow is betulinic acid.

[0026] Figure 9 The image shows the HPLC detection spectrum of the CL solid culture medium in this embodiment of the invention, where the peak indicated by the red arrow is betulinic acid;

[0027] Figure 10 The image shows the HPLC detection spectrum of the PS solid culture medium in this embodiment of the invention, where the peak indicated by the red arrow is betulinic acid. Detailed Implementation

[0028] 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.

[0029] The purchase link for the *Phellinus basil* used in the following examples is https: / / www.biobw.org / China-strain / bio-089346.html, and the laboratory number is A67.

[0030] The purchase link for betulinic acid is: https: / / www.echemsrc.com / goodsdetail?id=4657828;

[0031] The link to purchase ginseng powder is: https: / / item.taobao.com / item.htm?id=649780073337&spm=a1z10.3-c.w4002-8102932567.9.419c4c8fhTb6Be&skuId=4859521775998;

[0032] The link to purchase pea flour is: https: / / item.jd.com / 10140719998843.html;

[0033] The link to purchase He Shou Wu powder is: https: / / item.taobao.com / item.htm?id=649027044572&spm=a1z10.3-c.w4002-8102932567.9.9c974c8fwlBRk8;

[0034] The link to purchase Job's tears powder is: https: / / item.taobao.com / item.htm?id=623530598755&spm=a1z10.3-c.w4002-8102932567.9.13384c8fDk6SjC;

[0035] The link to purchase soybean oil is: https: / / item.jd.com / 100108079609.html. Example 1:

[0036] Cultivation of Phellinus balsamina:

[0037] 1. First, remove the preserved Phellinus balsamina A67 from the 4℃ refrigerator. Inoculate it onto Malt-Yeast (MY) medium (Difco, Lawrence, USA) solid medium. Then, place the inoculated medium in a dark incubator at 28℃ for 10 days, and harvest for later use.

[0038] 2. Take a 1cm*1cm mycelium from the cultured solid culture and place it in a 2ml sterile centrifuge tube. Add 700ul of sterile water and two steel columns. Grind the culture at 180rpm for 2 minutes to obtain a culture suspension. Then, inoculate the culture suspension into 100ml of liquid Malt-Yeast (MY) medium (Difco, Lawrence, USA). Incubate on a shaker for 7 days (150rpm, 28°C, dark) to obtain a liquid culture for later use.

[0039] 3. Prepare different culture media according to the following formulas:

[0040] PGO solid culture medium: 20g ginseng powder + 15ml water + 2g soybean oil.

[0041] PG solid culture medium: 20g ginseng powder + 15ml water.

[0042] PM solid culture medium: 20g of Polygonum multiflorum powder + 15ml of water.

[0043] CL solid culture medium: 20g of Job's tears powder + 15ml of water.

[0044] PS solid culture medium: 20g pea powder + 15ml water.

[0045] After thoroughly mixing the powder, soybean oil, and water, all culture media were sterilized by high-temperature and high-pressure steam (121℃, 20 min) before use.

[0046] 4. Take 2 ml of liquid bacterial suspension and inoculate it into the different solid culture media mentioned above, and incubate in the dark at 28°C for 20 days (e.g., Figure 1-5 As shown in the figure, cultures were obtained in different culture media for subsequent detection.

[0047] Detection:

[0048] 1. Determination of dry matter weight of mycelium

[0049] After culturing *Sanghuang baoyi* in different culture media, the biomass of *Sanghuang baoyi* was first visually observed. The mycelium was then scraped from the culture media, dried, and weighed using an electronic balance to obtain the weight of the dry matter of *Sanghuang baoyi* mycelium in each culture medium. Through actual observation, the mycelium of *Sanghuang baoyi* growing in PGO completely covered the entire tissue culture flask. Figure 1 Comparing the dry matter weights, it was found that the dry matter weight of *Sanghuang baozi* mycelium grown on PGO medium was significantly higher than that on other media, as shown in Table 1 below:

[0050] Table 1

[0051]

[0052] II. Fermentation Product Extraction Process

[0053] 1. Sampling and processing: Accurately weigh 30 mg of fungal hyphae under different culture conditions and place them in a 2 mL centrifuge tube;

[0054] 2. Extraction solution treatment: Add 1 mL of 50% methanol solution pre-cooled to 4℃, and add 2 glass beads. Vortex for 30 seconds.

[0055] 3. Freezing treatment: Immerse the centrifuge tubes in liquid nitrogen for 5 minutes to freeze rapidly.

[0056] 4. Grinding process: After thawing at room temperature, grind the tissue at a frequency of 55 Hz for 60 seconds using a high-throughput tissue homogenizer.

[0057] 5. Repeat the process: Repeat steps 2-4 twice, and then let it stand at -20℃ for 30 minutes after completion.

[0058] 6. Concentration treatment: Centrifuge at 12,000 rpm and 4℃ for 20 minutes, and take 850 μL of supernatant to concentrate under vacuum until completely dry.

[0059] 7. Reconstitution treatment: Add 150 μL of 50% methanol solution containing 5 ppm 2-chlorophenylalanine and vortex for 30 seconds.

[0060] 8. Filtration: Centrifuge at 12,000 rpm and 4℃ for 10 minutes, take the supernatant and filter it through a 0.22 μm filter membrane, and collect the filtrate into the test bottle.

[0061] III. Compound Detection Methods

[0062] A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) system was used, wherein:

[0063] Chromatography system: LC-30A ultra-high performance liquid chromatography system (Shimadzu, Japan);

[0064] Mass spectrometry system: TripleTOF 6600+ high-resolution mass spectrometer (SCIEX, USA);

[0065] 1. Chromatographic conditions:

[0066] (1) Chromatographic column: Waters ACQUITY Premier HSS T3 (1.8 μm, 2.1×100 mm);

[0067] (2) Mobile phase:

[0068] Phase A: Ultrapure water (containing 0.1% formic acid)

[0069] Phase B: Acetonitrile (containing 0.1% formic acid);

[0070] (3) Flow rate: 0.4 mL / min;

[0071] (4) Gradient procedure:

[0072] 0 min: 5% B

[0073] 2 min: 30% B

[0074] 5 min: 70% B

[0075] 6-7.5 min: 95% B

[0076] 7.6-10 min: 5% B;

[0077] (5) Injection volume: 4 μL;

[0078] (6) Analysis mode: positive / negative ion mode (the same gradient is used in negative ion mode);

[0079] 2. Mass spectrometry conditions

[0080] (1) Acquisition mode: Information Dependency Acquisition (IDA);

[0081] (2) Ion source parameters:

[0082] Ion source gas 1 / 2: 50 psi

[0083] Curtain air: 25 psi

[0084] Temperature: 550°C

[0085] Declustering voltage: ±60 V (positive / negative mode)

[0086] Ion spray voltage: ±5000 / 4000 V (positive / negative mode);

[0087] (3) Primary mass spectrometry parameters:

[0088] Quality range: 50-1000 Da

[0089] Cumulative time: 200 ms

[0090] Dynamic background removal: Enabled;

[0091] (4) Secondary mass spectrometry parameters:

[0092] Quality range: 25-1000 Da

[0093] Cumulative time: 40 ms

[0094] Collision energy: ±30 V (positive / negative mode)

[0095] Collision energy diffusion: 15

[0096] Intensity threshold: 100 cps

[0097] Maximum number of candidate ions: 18 / cycle.

[0098] IV. Compound Identification Methods

[0099] 1. Based on the PSNGM metabolomics database integration platform of Pasenno, including: self-built standard database, mzCloud mass spectrometry database, LIPID MAPS lipid database, HMDB human metabolomics database, MoNA metabolite and chemical entity database, NIST 2020 MSMS standard mass spectrometry library, and AI predicted MSMS spectrum library.

[0100] 2. Main identification parameters: Level 1 mass spectrometry tolerance: 0.01 Da; Level 2 mass spectrometry tolerance: 0.05 Da; Smoothing level: 3; Minimum peak height: 10,000; Minimum peak width: 5; Mass slice width: 0.05; Identification score threshold: 70 points; All data analysis was completed using the Analyst TF 1.7.1 software platform.

[0101] By comparing with database data, the compound with a retention time of 2.913 minutes was identified as betulinic acid.

[0102] V. Identification of betulinic acid content in fermentation products:

[0103] The betulinic acid in each fermentation product was detected using the method described above, and the specific peak chromatograms are shown below. Figure 6-10 As shown in Table 2 below, the differences in betulinic acid content among various fermentation products were compared by calculating the relative ratios of their peak areas.

[0104] Table 2

[0105]

[0106] As can be seen from the table above, PGO solid fermentation medium significantly increases the content of betulinic acid compared to other culture methods, providing a new and effective approach for the development and utilization of Phellinus balsamina products and the development of Phellinus balsamina cultures with high betulinic acid content.

[0107] 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 increasing the yield of betulinic acid in the production of Phellinus bournei, characterized in that, The method involves inoculating the liquid strain of Phellinus boulardii into a PGO-added solid medium for culture, wherein the PGO solid medium is formulated as 20g ginseng powder + 15ml water + 2g soybean oil; and the culture conditions are dark culture at 28℃.

2. The method according to claim 1, characterized in that: The method for preparing the liquid bacterial strain includes the following steps: (1) Inoculate the Phellinus baozi onto MY solid medium, incubate in the dark at 28℃ for 10 days, and collect the mycelium for later use; (2) Mix the mycelium with sterile water and steel balls, and grind it with a grinder to obtain a fungal suspension for later use; (3) Inoculate the bacterial suspension into liquid MY medium and culture it on a shaker for 7 days to obtain liquid bacterial culture.

3. The method according to claim 2, characterized in that: In step (2), the grinding speed of the grinder is 180 rpm and the grinding time is 2 min.

4. The method according to claim 2, characterized in that: In step (3), the shaking speed of the shaker is 150 rpm, the temperature is 28℃, and the culture method is dark culture.

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