Poplar phaeohyphomycete SH-8 and application thereof

Abstract

The application discloses a poplar phellinus SH-8 and application, and belongs to the technical field of microorganisms, and the strain has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.42969.The poplar phellinus SH-8 is a newly separated phellinus strain, and the optimized liquid fermentation process can realize the yield of 1500 mg / L of triterpenoid compounds; further adding indole-3-acetic acid as a synergistic exogenous inducer during liquid fermentation, the yield of triterpenoid compounds can be increased to 1668.16 mg / L.At the same time, the liquid fermentation process is simple in operation and easy to control, can greatly reduce the production cost of triterpenoid compounds, and has remarkable economic benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a poplar mulberry fungus SH-8 and its applications. Background Technology

[0002] Phellinus linteus is a rare perennial medicinal fungus, renowned as "forest gold" due to its rich content of polysaccharides, flavonoids, terpenes, and steroids. In traditional medicine, Phellinus linteus is commonly used to treat dysentery, night sweats, and rectal prolapse with bloody stools. Modern pharmacological research has confirmed its broad biological activities, including antitumor, antibacterial, antiviral, anti-inflammatory, immunomodulatory, and antioxidant effects, indicating promising applications. Among these, poplar Phellinus linteus (… Sanghuangporus vaninii Belonging to the genus Phellinus in the family Phellinusaceae, it is a highly regarded edible and medicinal species in the Phellinus family. The triterpenoids contained in its fruiting body and mycelium have significant effects on regulating blood lipids, anti-inflammation, and liver protection, and are regarded as the core active ingredients for medicinal development.

[0003] However, the natural wild *Sanghuang* (a type of medicinal mushroom) has an extremely long growth cycle and is a scarce resource; over-harvesting can damage the ecosystem. While artificial solid-state cultivation can partially alleviate the supply-demand imbalance, it takes several months and results in low levels of active substances, making it difficult to meet industrialization needs. Liquid deep fermentation, due to its short cycle, ease of control, and ability to rapidly obtain large quantities of mycelium, has become the preferred method for efficiently obtaining secondary metabolites from *Sanghuang*. However, existing liquid fermentation technologies still have significant limitations: the commonly used ordinary liquid culture media have a single nutrient composition, which, while maintaining basic mycelial growth, severely limits the synthesis of active substances such as triterpenes; most optimization studies are limited to adjusting only the carbon or nitrogen source, lacking a multi-factor systematic investigation, resulting in low mycelial biomass and unstable yields of target triterpenes. This not only restricts the development of high-value-added *Sanghuang* products but also hinders its promotion and application in the health food and pharmaceutical fields. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a newly isolated poplar mulberry fungus SH-8 with optimized fermentation capacity; the second purpose is to provide a fermentation application for the efficient synthesis of triterpenoids based on this poplar mulberry fungus SH-8.

[0005] Technical solution: The poplar mulberry fungus (as described in this invention) Sanghuangporus vaninii SH-8, with accession number CGMCC No.42969.

[0006] Preservation Notes: The *Poplar Sanghuang* SH-8 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 42969, on August 11, 2026. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0007] The microbial preparation of the present invention contains Populus euphratica SH-8.

[0008] Preferably, the microbial preparation further contains a biologically acceptable carrier; more preferably, the biologically acceptable carrier includes: culture medium, enrichment agent, dispersant, stabilizer, and synergist.

[0009] The application of the poplar mulberry fungus SH-8 or microbial preparation described in this invention in the preparation of triterpenoid compounds.

[0010] Preferably, the application steps include: preparing poplar mulberry SH-8 seed liquid and inoculating it into a fermentation medium for liquid fermentation, and extracting triterpenoid compounds after fermentation.

[0011] Preferably, the volume ratio of the poplar mulberry SH-8 seed liquid to the fermentation medium is (0.08~0.1):1.

[0012] Preferably, the fermentation medium contains 18-28 g / L glucose, 5-7.5 g / L yeast extract, 0.57-1.07 g / L K2HPO4, and has a pH of 6.5-7.5.

[0013] Preferably, during the liquid fermentation, indole-3-acetic acid with a final concentration of 80-120 μmol / L is also added to the fermentation system.

[0014] Preferably, the liquid fermentation time is 10 to 11 days.

[0015] The application of the poplar mulberry fungus SH-8 or microbial preparation described in this invention in the preparation of drugs.

[0016] Preferably, the drug has one or more pharmacological activities such as anti-tumor, anti-oxidation, anti-inflammation, immunomodulation, hepatoprotection, anti-fibrosis, and metabolic regulation.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The poplar mulberry SH-8 is a newly isolated strain of the mulberry genus. Combined with the optimized liquid fermentation process, a triterpenoid compound yield of 1500 mg / L can be achieved. Furthermore, by adding indole-3-acetic acid as an exogenous synergist during liquid fermentation, the triterpenoid compound yield can be increased to 1668.16 mg / L. The liquid fermentation process is simple to operate and easy to control. While increasing the total triterpenoid yield of mycelium, it can significantly reduce the production cost of triterpenoid compounds, resulting in significant economic benefits. Attached Figure Description

[0018] Figure 1 A statistical chart showing the triterpenoid yield of *Poplaria sinensis* SH-8 under different carbon source conditions; Figure 2A statistical graph showing the triterpenoid yield of Populus euphratica SH-8 under different nitrogen source conditions; Figure 3 A statistical chart showing the triterpenoid yield of Populus euphratica SH-8 under different inorganic salt conditions; Figure 4 A statistical chart showing the triterpenoid yield of *Poplarina mulberry* SH-8 under different fermentation time conditions; Figure 5 A statistical graph showing the triterpenoid yield of *Poplaria sinensis* SH-8 under different pH conditions; Figure 6 A statistical graph showing the triterpenoid yield of Populus euphratica SH-8 under different inoculation amounts; Figure 7 A statistical chart showing the triterpenoid yield of *Poplarina mulberry* SH-8 under different types and concentrations of synergistic exogenous inducers; Figure 8 A statistical chart showing the triterpenoid yield of *Phyllanthus linteus* SH-8 before and after optimization of the liquid fermentation process. Detailed Implementation

[0019] The technical solution of the present invention will be further described below.

[0020] Example 1: Identification of Poplar Phellinus shampoo SH-8 The strain SH-8 was isolated by the Edible Fungi Innovation Team Laboratory of the Vegetable Research Institute of Jiangsu Academy of Agricultural Sciences.

[0021] Mycelia of strain SH-8 were picked and inoculated onto potato dextrose agar (PDA) medium in a clean bench and incubated at 25°C until colonies covered the plate. Mycelia were collected and mycelial DNA was extracted using the M5 Plant Genomic DNA Kit (Beijing Polymer Biotechnology Co., Ltd., MF070-04).

[0022] Using mycelial DNA as a template, PCR amplification was performed using fungal LSU sequence primers LROR-F: 5'-acccgctgaacttaagc-3' and LR5-R: 5'-tcctgagggaaacttcg-3', via 2×T5 Super PCR Mix (Qingke Biotechnology, DLP101). The reaction conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 20 s, 56℃ annealing and extension for 30 s, 34 cycles; and 72℃ extension for 5 min. The PCR products were detected by 1% agarose gel electrophoresis. PCR products with a specific band at approximately 750 bp, compared with a 2000 bp DNA marker, were sequenced by Qingke Biotechnology Co., Ltd.

[0023] The sequencing results were compared with those from the NCBI database using BLAST, and the results were compared with those from *Poplar mulberry* (…). Sanghuangporus vaninii The strain SH-8 was identified as a *Poplar mulberry* strain because it showed the highest similarity to the strain SH-8.

[0024] Example 2: Plate activation and mycelial growth performance determination of Populus euphratica SH-8 Poplar mulberry SH-8 was inoculated onto 90 mm diameter, 15 mm thick PDA medium and incubated at 25°C in the dark until the colonies covered the entire plate. Using a 7 mm diameter sterile punch, holes were made along the edge of the activated plate colonies, and the mycelial blocks were inoculated into the center of fresh PDA medium. After mycelial germination, the initial growth zone (l0) was marked. When the fastest-growing mycelia were 3 mm from the edge of the medium, the outer edge of the mycelia (L) was marked. The distance between l0 and L was measured with a ruler, and the mycelial growth rate (mm / d) was calculated using the following formula: Mycelial growth rate (mm / d) = (L - l0) / number of incubation days. Table 1. Mycelial growth rate

[0025] The results are shown in Table 1. The mycelial growth rate of Populus euphratica SH-8 was 7.958±0.191 mm / d, indicating excellent mycelial growth activity.

[0026] Example 3: Use of Poplar Phellinus linteus SH-8 for the preparation of triterpenoids 1. Preparation of Poplar Sanghuang SH-8 Seed Liquid In a clean bench, mycelia of strain SH-8 were picked and inoculated onto PDA medium (90 mm in diameter and 15 mm in thickness). The medium was incubated at 25°C until the colonies covered the plate. After homogenization with 50 mL of sterile water, a homogenate was obtained. Each bottle of PDB liquid seed medium was inoculated with 10% of the homogenate and cultured at 28°C and 150 r / min on a shaker for 7 days. After the culture was completed, the mycelial balls were broken up using a homogenizer to obtain a uniform and homogeneous poplar mulberry SH-8 seed liquid for later use.

[0027] 2. Screening of components in liquid fermentation culture medium In the screening experiment of liquid fermentation medium components, the fermentation system was as follows: 100 mL of medium was added to a 250 mL Erlenmeyer flask, and 10 mL of 10% volume of Populus euphratica SH-8 seed liquid was inoculated. The fermentation system was shaken at 28℃ and 150 r / min for 7 days. After the fermentation was completed, the mycelial dry weight and total triterpenoid content of the mycelium were measured. Each treatment was repeated 3 times.

[0028] The methods for determining the dry weight of mycelium and the total triterpenoid content of mycelium are as follows: (1) Preparation of mycelial samples: After fermentation, mycelial balls were collected by gauze filtration, washed repeatedly with deionized water until the filtrate was clear, dried in an oven at 60°C until constant weight, weighed and then crushed. (2) Extraction of triterpenoids: Accurately weigh 0.250 g of mycelial powder and place it in a 50 mL volumetric flask. Add 15 mL of anhydrous ethanol and extract by ultrasonication at 180 W in a 60℃ water bath for 40 min. Make up to the mark with anhydrous ethanol and filter to obtain the extract. (3) Establishment of standard curve: Prepare 0.2 mg / mL oleanolic acid standard working solution. Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard working solution respectively, and evaporate the solvent in a boiling water bath. Add 0.4 mL of 5% vanillin-glacial acetic acid and 1.6 mL of perchloric acid in sequence, mix well, heat in a water bath at 70℃ for 15 min, cool to room temperature, add 8 mL of ethyl acetate and shake well. Measure the absorbance at 560 nm. Fit the standard curve equation with the mass of oleanolic acid (μg) as the abscissa (x) and the absorbance as the ordinate (y): y = 4.20936x + 0.0352, with a correlation coefficient R. 2 =0.9905; (4) Sample detection: Take 1 mL of the extract obtained in step 2, dilute it 10 times with anhydrous ethanol, take 1 mL, evaporate the solvent, add 0.4 mL of 5% vanillin-glacial acetic acid and 1.6 mL of perchloric acid in sequence, mix well, heat in a water bath at 70℃ for 15 min, cool to room temperature, add 8 mL of ethyl acetate and shake well, measure the absorbance at 560 nm, substitute into the standard curve equation obtained in step 3 to calculate the mass of triterpenoids, and further calculate the triterpenoid concentration and triterpenoid yield according to the following formula: Triterpenoid concentration (mg / g) = (C×V) t ×D×0.001) / (V0×m), Where C is the mass (μg) of the triterpenoid compounds in the sample measured by the standard curve; V t V0 is the total volume of the extract (mL); D is the dilution factor; m is the mass of the mycelium sample (g). Triterpenoid yield (mg / L) = Mycelial dry weight (g / 100 mL) × Triterpenoid concentration (mg / g) × 10.

[0029] 2.1 Carbon source screening Five groups of culture media with different carbon sources were prepared for liquid fermentation: Group 1 contained 28 g / L glucose, Group 2 contained 40 g / L corn flour, Group 3 contained 100 g / L wheat bran, Group 4 contained 20 g / L corn flour + 10 g / L glucose, and Group 5 contained 25 g / L corn flour + 75 g / L wheat bran. The culture media with different carbon sources also contained 10 g / L peptone, 0.5 g / L KH2PO4, 2 g / L MgSO4, and the remainder was water.

[0030] The results are as follows Figure 1As shown, when the carbon source of the culture medium is glucose at 28 g / L, the yield of triterpenes is significantly higher than that of other carbon source formulations.

[0031] 2.2 Nitrogen Source Screening Yeast extract powder (yeast powder) was purchased from Thermo Fisher Scientific Inc., catalog number LP0021B.

[0032] Five groups of culture media with different nitrogen sources were prepared for liquid fermentation: Group 1 contained 5 g / L peptone, Group 2 contained 10 g / L peptone, Group 3 contained 15 g / L peptone, Group 4 contained 5 g / L yeast extract, and Group 5 contained 2.6 g / L yeast extract + 2.4 g / L peptone. The culture media with different nitrogen sources also contained 25 g / L corn flour + 75 g / L wheat bran, 0.5 g / L KH2PO4, 2 g / L MgSO4, and the remainder was water.

[0033] The results are as follows Figure 2 As shown, when the nitrogen source in the culture medium is 5 g / L yeast extract, the triterpenoid yield is significantly higher than that of other nitrogen source formulations.

[0034] 2.3 Inorganic Salt Screening Five groups of culture media with different nitrogen sources were prepared for liquid fermentation: Group 1: K2HPO4 1.07 g / L, Group 2: KH2PO4 0.5 g / L, Group 3: KH2PO4 0.5 g / L + MgSO4 2 g / L, Group 4: KH2PO4 0.5 g / L + MgSO4 1.32 g / L, and Group 5: K2HPO4 0.5 g / L + MgSO4 2 g / L. The culture media with different nitrogen sources also contained: corn flour 25 g / L + wheat bran 75 g / L, peptone 10 g / L, and the remainder was water.

[0035] The results are as follows Figure 3 As shown, when the inorganic salt content of the culture medium is 1.07 g / L K2HPO4, the triterpene yield is significantly higher than that of other inorganic salt formulations.

[0036] 2.4 Orthogonal Experiment of Culture Medium Components Based on the results of the single-factor experiments, an orthogonal experiment was designed to prepare different combinations of culture media and carry out liquid fermentation. The factor level table is shown in Table 2.

[0037] Table 2. Factor Level Table for Orthogonal Experiment

[0038] The results are shown in Table 3. When the culture medium contains 28 g / L glucose, 5 g / L yeast extract, and 1.07 g / L K2HPO4 (the remainder being water), the yield of triterpenes obtained by liquid fermentation is higher. This formulation was selected for further testing.

[0039] Table 3 Results of the orthogonal experiment

[0040] 3. Optimization of liquid fermentation environmental parameters The fermentation system consisted of 100 mL of the optimal culture medium obtained from the aforementioned orthogonal experiment added to a 250 mL Erlenmeyer flask, and inoculated with Populus euphratica SH-8 seed culture. The fermentation system was shaken at 150 r / min. After fermentation, the mycelial dry weight and total triterpenoid content were measured. Each treatment was replicated three times.

[0041] 3.1 Fermentation Time Screening Inoculate with 10 mL of Populus euphratica SH-8 seed culture at 10% of the culture medium volume, without adjusting the initial pH, and ferment at 28℃ for 6, 7, 8, 9, 10 or 11 days with shaking.

[0042] The results are as follows Figure 4 As shown, the triterpenoid yield was highest when the fermentation time was 10 days.

[0043] 3.2 Initial pH Screening Inoculate with 10 mL of Populus euphratica SH-8 seed culture at 10% culture medium volume, adjust the initial pH to 5, 6, 7, 8 or 9 using 1 mol / L HCl and 1 mol / L NaOH solutions, and ferment at 28℃ with shaking for 10 days.

[0044] The results are as follows Figure 5 As shown, the triterpenoid yield was highest when the culture medium pH was 7.

[0045] 3.3 Screening of Inoculation Volume Inoculate with 6%, 8%, 10%, 12% or 14% of the culture medium volume of Populus euphratica SH-8 seed liquid (6, 8, 10, 12 or 14 mL), adjust the initial pH to 7, and ferment at 28℃ with shaking for 10 days.

[0046] The results are as follows Figure 6 As shown, the triterpenoid yield was highest when the inoculum volume of Populus euphratica SH-8 seed liquid was 10% of the culture medium volume.

[0047] 4. Screening of exogenous synergistic inducers Accurately weigh 0.3504 g indole-3-acetic acid, 0.276 g salicylic acid, and 0.448 g methyl jasmonate, dissolve them thoroughly in a small amount of anhydrous ethanol, and transfer them to 100 mL volumetric flasks. Make up to 100 mL for each flask to prepare 0.02 mol / L stock solutions of indole-3-acetic acid, salicylic acid, and methyl jasmonate. Filter the solutions through a 0.22 μm sterile membrane for sterilization before use.

[0048] The fermentation system consisted of: 100 mL of the optimal culture medium obtained from the aforementioned orthogonal experiment was added to a 250 mL Erlenmeyer flask; 10 mL of Populus euphratica SH-8 seed culture was inoculated into the flask; the initial pH was adjusted to 7; and the aforementioned 0.02 mol / L indole-3-acetic acid, salicylic acid, or methyl jasmonate stock solution was added to bring the final concentrations of indole-3-acetic acid, salicylic acid, and methyl jasmonate in the fermentation system to 100 μmol / L, 200 μmol / L, 300 μmol / L, or 400 μmol / L, respectively, with three replicates for each group. Simultaneously, to eliminate the interference of the solvent on the experimental results, a control without an inducer (Mock) and a control with 2 mL of anhydrous ethanol added to the fermentation system (CK) were set up.

[0049] All the above fermentation systems were fermented at 28℃ and 150 r / min for 10 days. After fermentation, the dry weight of mycelium and the total triterpenoid content of mycelium were measured. Each treatment was repeated 3 times.

[0050] The results are as follows Figure 7 As shown, the highest triterpene yield was achieved when the inducing agent was 100 μmol / L indole-3-acetic acid.

[0051] 5. Preparation of triterpenoids from *Poplarina mulberry* SH-8 using a fully optimized process Fermentation was carried out using a complete optimized process. The fermentation system was as follows: 100 mL of the optimal culture medium obtained from the aforementioned orthogonal experiment (containing 28 g / L glucose, 5 g / L yeast extract, and 1.07 g / L K2HPO4) was added to a 250 mL Erlenmeyer flask, and 10 mL of Populus euphratica SH-8 seed culture (10% of the culture medium volume) was inoculated. The initial pH was adjusted to 7, and indole-3-acetic acid (100 μmol / L) was added to the fermentation system. Fermentation was carried out at 28℃ and 150 r / min for 10 days with shaking. At the same time, 100 mL of ordinary potato broth (PDB) culture medium was added to a 250 mL Erlenmeyer flask, and 10 mL of Populus euphratica SH-8 seed culture (10% of the culture medium volume) was inoculated. Fermentation was carried out at 28℃ and 150 r / min for 7 days with shaking, serving as a control before optimization.

[0052] After fermentation, the dry weight of mycelium and the total triterpenoid content of mycelium were measured, with each treatment repeated three times.

[0053] The results are as follows Figure 8 As shown, triterpenoids were prepared by using Populus euphratica SH-8. The yield of triterpenoids based on the fully optimized process reached 1668.16 mg / L, which is about 56.08 times the yield of triterpenoids before optimization.

Claims

1. A type of poplar mulberry fungus ( Sanghuangporus vaninii SH-8, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 42969.

2. A microbial preparation, characterized in that, It contains Poplar Phellinus SH-8.

3. The use of the poplar mulberry fungus SH-8 as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of triterpenoid compounds.

4. The application according to claim 3, characterized in that, The application steps include: preparing poplar mulberry SH-8 seed liquid and inoculating it into a fermentation medium for liquid fermentation, and extracting triterpenoid compounds after fermentation.

5. The application according to claim 4, characterized in that, The volume ratio of the poplar mulberry shrub SH-8 seed liquid to the fermentation medium is (0.08~0.1):

1.

6. The application according to claim 4, characterized in that, The fermentation medium contains 18-28 g / L glucose, 5-7.5 g / L yeast extract, 0.57-1.07 g / L K2HPO4, and has a pH of 6.5-7.

5.

7. The application according to claim 4, characterized in that, During the liquid fermentation, indole-3-acetic acid with a final concentration of 80-120 μmol / L is also added to the fermentation system.

8. The application according to claim 4, characterized in that, The liquid fermentation process takes 10 to 11 days.

9. The use of the poplar mulberry fungus SH-8 as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of a drug.

10. The application according to claim 9, characterized in that, The drug is a drug with one or more of the following pharmacological activities: anti-tumor, anti-oxidation, anti-inflammation, immunomodulation, hepatoprotection, anti-fibrosis, and metabolic regulation.

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