Method for producing dendrobine through co-culture of endophytic fungi and dendrobium nobile and special strain

Abstract

The invention belongs to the technical field of liquid fermentation of endophytic fungi, and particularly relates to a method for producing dendrobine by co-culture of endophytic fungi and dendrobium nobile and a special strain. According to the method, fusarium endophytic fungi which are preserved in the China General Microbiological Culture Collection Center (CGMCC) and have the preservation number of CGMCC NO.3.27824 are adopted, dendrobium nobile stems or dry powder is added into an optimized liquid culture medium to serve as a homologous stimulant for co-culture, and the yield of dendrobine is remarkably increased. Experiments show that by means of the method, the yield of dendrobine can be increased by dozens of times or above and up to about 99 times, the process is stable, cost is low, and large-scale production is easy to achieve. The invention provides a new way for large-scale green production of dendrobine, and reduces the dependence on wild dendrobium resources.

Description

A method and specific strain for producing dendrobine by co-culturing endophytic fungi with Dendrobium nobile Technical Field

[0001] This invention belongs to the field of liquid fermentation technology of endophytic fungi, and specifically relates to a method and a special strain for producing dendrobine by co-culturing endophytic fungi with Dendrobium nobile. Background Technology

[0002] Dendrobine is a sesquiterpene alkaloid with antiviral, antitumor, antioxidant, hepatoprotective, blood sugar regulating, and neuroprotective effects. Currently, Dendrobium is the main source of dendrobine, but due to its low content in Dendrobium and the difficulty in direct extraction from Dendrobium, it can no longer meet the huge demand of the medical market. Therefore, it is crucial to explore other methods for producing dendrobine.

[0003] Endophytic fungi are filamentous fungi that parasitize plant tissues. Through long-term co-evolution with their host plants, they can produce metabolites that are the same as or similar to those of their hosts. Endophytic fungi play a crucial role in the synthesis of secondary metabolites from medicinal plants. Moreover, compared to plants, endophytic fungi have advantages such as faster growth, easier cultivation, and easier genetic manipulation. Therefore, utilizing endophytic fungi to synthesize secondary metabolites from medicinal plants, especially dendrobine, a secondary metabolite with significant medicinal value, is of great significance in meeting market demand for dendrobine. Summary of the Invention

[0004] The present invention aims to provide a liquid fermentation culture method for dendrobine based on endophytic fungi, so as to solve the problems of low yield and difficult acquisition method of extracting dendrobine directly from dendrobium in the prior art.

[0005] In a first aspect, the present invention provides an endophytic fungus belonging to the genus Fusarium HX2, classified as Fusarium sp., and deposited on May 21, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC NO. 3.27824.

[0006] Secondly, the present invention provides a co-culture composition for producing dendrobine, comprising the strain of the endophytic fungus as described in claim 1, and Dendrobium nobile stem strips or Dendrobium nobile powder.

[0007] Thirdly, the present invention also provides a method for producing dendrobine based on the strain of the endophytic fungus, comprising the following steps: S1, activating the strain of the endophytic fungus in PDA solid medium to obtain activated fungal blocks; S2, inoculating the activated fungal blocks into PDB liquid optimized medium containing Dendrobium nobile stems or Dendrobium nobile powder for co-culture; S3, after the culture is completed, collecting the mycelium and extracting dendrobine from the mycelium.

[0008] Furthermore, the pH value of the PDA solid culture medium and the PDB liquid culture medium is 5.6±0.2.

[0009] Furthermore, the PDB liquid optimized culture medium contains the following components: 15-25 g / L soluble starch, 10-15 g / L yeast extract, 0.03-0.09 g / L sodium chloride, 0.05-0.15 g / L chloramphenicol, and potato extract as the solvent.

[0010] Furthermore, in step S2, the co-culture conditions are: temperature 22~28℃, dark culture, and time 20~40 days.

[0011] Furthermore, in step S3, the mycelium is dried at a constant temperature of 45°C before the extraction of dendrobine.

[0012] Furthermore, in step S3, a methanol solution containing 0.05% formic acid is used to extract dendrobine from the mycelium by reflux and rotary evaporation.

[0013] Furthermore, the Dendrobium mentioned is Dendrobium nobile.

[0014] Furthermore, the Dendrobium nobile stem strips are fresh stem strips or fresh stem strips that have undergone surface sterilization treatment; the Dendrobium nobile powder is obtained by drying and pulverizing the Dendrobium nobile stem strips and then passing them through a No. 3 sieve.

[0015] The beneficial technical effects of this invention are as follows: The liquid fermentation culture method for dendrobine based on endophytic fungi proposed in this invention not only successfully overcomes the limitations and inefficiency of directly extracting dendrobine from dendrobium plants, but also achieves efficient and stable production of dendrobine through a series of meticulously designed steps, which has significant technical innovation and practical application value.

[0016] 1. High-efficiency production of dendrobine: By optimizing the culture medium formula, namely PDA solid medium and PDB + stem strip liquid medium, a more suitable growth environment is provided for endophytic fungi, effectively promoting their growth and metabolic activities, thereby increasing the yield of dendrobine. Using the homologous stimulant, dendrobium stem strips, as an inducing factor further stimulates the potential of endophytic fungi to produce dendrobine, resulting in a significant increase in dendrobine yield.

[0017] 2. Stable production performance: By purifying the strains multiple times, the genetic stability of the endophytic fungi used and the consistency of production performance are ensured, thus guaranteeing the stability and reliability of the production of dendrobine. The liquid fermentation culture system has a high degree of controllability and can precisely regulate culture conditions such as temperature and pH value, further improving the stability and repeatability of dendrobine production.

[0018] 3. Reduced production cost: Compared with directly extracting dendrobine from Dendrobium plants, this method uses endophytic fungi for liquid fermentation culture, which has the advantages of fast growth rate, short culture cycle, easy availability of raw materials and low cost, significantly reducing the production cost. The liquid fermentation culture system is easy to realize industrial production, providing the possibility for the large-scale production of dendrobine.

[0019] 4. Environmentally friendly and sustainable: This method avoids over-reliance on wild Dendrobium resources, contributing to the protection of the ecological environment and biodiversity.

[0020] In summary, the method for liquid fermentation culture of dendrobine based on endophytic fungi proposed in this invention not only achieves the efficient and stable production of dendrobine, but also reduces the production cost, provides a research basis, and meets the requirements of environmentally friendly and sustainable development, having broad application prospects and profound social significance. Brief description of the drawings

[0021] Figure 1. Colony and microscopic morphology of the endophytic fungus DN689 of Dendrobium officinale.

[0022] Figure 2. Dendrobine content diagram of single-factor optimization of the fermentation medium of the endophytic fungus DN689 of Dendrobium officinale.

[0023] Figure 3. Three-dimensional contour and response surface analysis of the synthesis of dendrobine by the endophytic fungus DN689 of Dendrobium officinale.

[0024] Figure 4. Prediction diagram of the optimal culture components for the synthesis of dendrobine by the endophytic fungus DN689 of Dendrobium officinale.

[0025] Figure 5. Dendrobine content diagram of the endophytic fungus DN689 of Dendrobium officinale added with different homologous stimulants. Detailed implementation manners

[0026] The following is a further detailed description through specific implementation manners: Biological material preservation information: The strain information preserved in this application is: Fusarium sp. HX2, its taxonomic name is: Fusarium sp., the Chinese taxonomic name is: Fusarium genus, and the preservation number is CGMCC NO. 3.27824; this strain is preserved in the China General Microbiological Culture Collection Center, and the preservation date is May 21, 2025.

[0027] Basic Case 1: Solid Culture of Endophytic Fungi of Dendrobium nobile Step 1: Preparation of PDA solid culture medium: Take fresh peeled potato pieces and place them in boiling water. Keep boiling for 30 minutes, filter, add 20g of glucose and 12.5g of agar powder, and adjust the volume of the culture medium to 1L with potato extract. The pH value is 5.6±0.2. After dispensing, sterilize (121℃, 30 minutes) for later use.

[0028] Step 2: Isolation and identification of endophytic bacteria. Collect fresh stems of *Dendrobium nobile*, sterilize the surface, and centrifuge in 50ml centrifuge tubes. Add (20-30 ml) of 75% ethanol for 30 seconds, shaking the tube several times every 10 seconds to ensure thorough sterilization. Rinse three times with sterile water, then rinse 3-5 times with (20-30 ml) of 0.1% mercuric chloride for 1 minute. Cut the sterilized stems into 0.5cm segments using surgical scissors and inoculate them into PDA medium. Incubate in a climate chamber at 25°C in the dark. Once endophytic mycelia are observed to grow, immediately use a sterilized toothpick to pick up the mycelia from the tips and inoculate them into fresh PDA solid medium. Repeat this inoculation process 3-5 times to obtain single strains. The purified strains are then preserved short-term at -4°C using the slant culture method for later use.

[0029] Step 3: Strain screening. The bacterial strains were transferred to a fungal liquid culture medium and cultured at 25 ℃ for 25-35 days. Products were extracted from the bacterial samples and culture medium, analyzed by LC-MS, and compared with dendrobine standards. Strains containing dendrobine in their secondary metabolites were selected as candidate strains for co-culture. Comparison with dendrobine standards revealed three endophytic fungi exhibiting peaks at the same time as dendrobine. Strain DN689, with stable growth and the highest dendrobine yield, was selected (Figure 1). ITS sequencing confirmed that all three Dendrobium nobile endophytic fungi belonged to the Fusarium genus and were used as backup strains for subsequent examples.

[0030] Step 4: Determination of Dendrobine Content Produced by Endophytic Fungi in Dendrobium nobile (1) LC-MS Conditions: ACQUITY UPLC® BEH C18 (2.1×100mm, 1.7 µm), mobile phase: 100% acetonitrile (A) - 0.05% formic acid water (B), flow rate: 1 mL / min. Initial temperature: 30℃, increased to 50℃ at a rate of 10℃ / min, held for 5 min, injection volume: 10 µL.

[0031] (2) Preparation of standard solution: Weigh out a suitable concentration of dendrobine reference standard and prepare a solution with a mass concentration of 20 ng / mL using 100% chromatographic 0.05% formic acid methanol.

[0032] (3) Preparation of test solution: Take 0.1g of the liquid fermentation mycelium of Dendrobium nobile endophytic fungi, dry it, add 10ml of 0.05% formic acid methanol solution, reflux and rotary evaporate, and make up to 2ml. Store for later use.

[0033] (4) Construction of the Dendrobine Standard Curve: Accurately weigh 1 mg of dendrobine standard powder, dissolve it in 0.05% formic acid methanol solution and bring the volume to 2 mL. Dilute with 0.05% formic acid methanol solution to 1, 10, 20, 50, and 100 ng / mL. Analyze using liquid chromatography-mass spectrometry (LC-MS). Plot the dendrobine standard curve based on the peak areas of the LC-MS spectrum.

[0034] Step 5: Calculation of Dendrobine Content Produced by Endophytic Fungi in Dendrobium nobile. Based on the Dendrobine standard curve drawn in Step 3, the Dendrobine content in solid culture of endophytic fungi is calculated.

[0035] Basic Case 2: Optimization of Liquid Culture Medium for Endophytic Fungi of Dendrobium nobile Step 1: Replace the glucose carbon source in the PDB liquid culture medium for endophytic fungi with lactose, fructose, sucrose, and soluble starch, keeping the rest of the formula unchanged. Set up three replicates for each group, inoculate DN689 cells, and conduct fermentation culture. Determine the dendrobine content in the fermentation broth. Screen for the optimal carbon source for the culture medium in which endophytic fungi synthesize dendrobine.

[0036] Step 2: Based on the screened carbon source, 0.1% nitrogen source was added to the endophytic fungal liquid culture medium PDB. Two inorganic nitrogen sources, ammonium sulfate and ammonium chloride, and three organic nitrogen sources, beef meal, peptone, and yeast extract, were used for fermentation optimization. Each group was set up in triplicate. DN689 was inoculated and fermented, and the dendrobine content in the fermentation broth was measured. The optimal nitrogen source for the endophytic fungus DN689 to synthesize dendrobine was screened.

[0037] Step 3: Based on the screening of carbon and nitrogen sources, sodium chloride, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and magnesium sulfate were selected to prepare culture media for fermentation optimization. Three replicates were set up for each group, inoculated with DN689, and fermentation was carried out. The dendrobine content in the fermentation broth was measured. The optimal inorganic salts for the culture medium in which endophytic fungus DN689 synthesizes dendrobine were screened.

[0038] Step 4: Response surface methodology optimization. Based on the single-factor optimization results of the culture medium, the contents of A (carbon source), B (nitrogen source), and C (inorganic salts) of the culture medium are further optimized to establish a response surface model and seek the optimal values. The optimal values ​​obtained from the experiment are then validated and compared to verify the accuracy of the model.

[0039] Table 1 Response Surface Experimental Design

[0040] Comparative Example 1: Liquid fermentation culture of endophytic bacteria of Dendrobium nobile Step 1: Preparation of potato liquid culture medium (PDB): Fresh peeled potato pieces were placed in boiling water and kept boiling for 30 min. After filtration, 0.1 g of chloramphenicol, optimized carbon source, nitrogen source and inorganic salts were added. The culture medium was adjusted to 1 L with potato extract, and the pH value was 5.6±0.2. After dispensing, the medium was sterilized (121℃, 30 min) for later use.

[0041] Step 2: Inoculate the activated DN689 strain into fresh PDA solid medium and incubate in the dark at 25°C for 3-5 days. Then, pick 1 cm samples from each culture medium. 2 Mycelia were inoculated into PDB liquid fermentation optimized medium and cultured in the dark at 25°C and 120 rpm for about 25-35 days.

[0042] Step 3: Separate the dried bacterial cells from the fermentation broth, and extract the products by reflux and rotary evaporation using a methanol solution containing 0.05% formic acid. Perform LC-MS analysis and compare the results with the standard of dendrobine.

[0043] Step 4: Determination of dendrobine content in endophytic fungi of Dendrobium nobile is the same as in the basic case.

[0044] Comparative Example 2: Adding Dendrobium officinale powder to Dendrobium nobile endophytic fungal solid culture medium. Step 1: Take Dendrobium nobile stems, wash them with distilled water, absorb the moisture from the plants with filter paper, dry them, crush them with a mortar and pestle or grinder, pass them through a No. 3 sieve, accurately weigh 0.1 g, add them to 100 mL of endophytic fungal solid culture medium PDA, and autoclave at 121 ℃ for 30 min.

[0045] Step 2: Inoculate the activated DN689 strain into PDA medium containing Dendrobium officinale powder, and incubate in the dark at 25℃ for 3-5 days. Then, use a sterile toothpick to pick a 1 cm sample. 2 The bacterial blocks were inoculated in PDB optimized medium and cultured at 25°C for 25-35 days.

[0046] Step 3: Filter the mycelium with sterile filter paper, collect the mycelium, dry the mycelium, and extract the product by reflux and rotary evaporation using a methanol solution of 0.05% formic acid. Perform LC-MS analysis and compare the results with the dendrobine standard. Step 4: Determination of dendrobine content in endophytic fungi of Dendrobium nobile is the same as in the basic case.

[0047] Example 1: Adding Dendrobium stems to the liquid culture medium of Dendrobium nobile endophytic bacteria Step 1: Preparation of potato liquid culture medium (PDB): Take fresh peeled potato pieces and place them in boiling water. Keep boiling for 30 minutes, filter, add 0.1g of chloramphenicol, optimized carbon source, nitrogen source and inorganic salts, and dilute the culture medium to 1L with potato extract. The pH value is 5.6±0.2. After dispensing, sterilize (121℃, 30 minutes) for later use.

[0048] Step 2: Inoculate the activated DN689 strain into a new PDA solid medium and incubate in the dark in a 25°C artificial climate chamber for 3-5 days before use.

[0049] Step 3: Wash the fresh Dendrobium stems with distilled water, absorb the water from the plants with filter paper, and weigh them; cut the plants into small pieces, add 0.1g to 100ml of LPDB liquid culture medium, and use a sterile toothpick to pick up 1cm of the sample. 2 DN689 bacterial blocks were inoculated into PDB optimized medium for co-culture. The culture flasks were cultured at 120 rpm and 25°C in the dark for about 25-35 days.

[0050] Step 4: After culturing for about 25-35 days, the fermentation broth was filtered with sterile filter paper, the mycelium was collected, dried at 45℃, and the product was extracted by reflux and rotary evaporation using a methanol solution containing 0.05% formic acid. The product was analyzed by LC-MS and compared with the dendrobine standard. Step 5: The determination of dendrobine content in endophytic fungi of Dendrobium nobile was the same as in the basic case.

[0051] Example 2: Adding sterile Dendrobium stems to the liquid culture medium of Dendrobium nobile endophytic bacteria. Step 1: Preparation of potato liquid culture medium (PDB): Fresh peeled potato chunks were placed in boiling water and kept boiling for 30 minutes. After filtration, 0.1 g of chloramphenicol, optimized carbon source, nitrogen source, and inorganic salts were added. The culture medium was brought to a final volume of 1 L with potato extract, and the pH value was 5.6 ± 0.2. Fresh Dendrobium stems were washed with distilled water, and the water on the plants was absorbed with filter paper. They were weighed. The plants were cut into small pieces, and 0.1 g of each piece was added to 100 ml of PDB liquid culture medium. The mixture was sterilized (121℃, 30 min) for later use.

[0052] Step 2: Inoculate the activated DN689 strain into a new PDA solid medium and incubate in the dark in a 25°C artificial climate chamber for 3-5 days before use.

[0053] Step 3: Use a sterile toothpick to pick up 1 cm 2 DN689 mycelial blocks were inoculated into PDB optimized medium containing sterile Dendrobium nobile stems for co-culture. The culture flasks were cultured at 120 rpm and 25°C in the dark for about 25-35 days.

[0054] Step 4: After culturing for about 25-35 days, the fermentation broth was filtered with sterile filter paper, the mycelium was collected, dried at 45℃, and the product was extracted by reflux and rotary evaporation using a methanol solution containing 0.05% formic acid. The product was analyzed by LC-MS and compared with the dendrobine standard. Step 5: The determination of dendrobine content in endophytic fungi of Dendrobium nobile was the same as in the basic case.

[0055] Example 3: Adding Dendrobium nobile dry powder to Dendrobium nobile endophytic fungal liquid culture medium Step 1: Take Dendrobium nobile stem strips, wash them with distilled water, absorb the moisture from the plants with filter paper, dry them, crush them with a mortar and pestle or grinder, pass them through a No. 3 sieve, accurately weigh 0.1 g, add them to 100 mL of endophytic fungal liquid culture medium PDB, and autoclave at 121 ℃ for 30 min.

[0056] Step 2: Inoculate the activated DN689 strain into a new PDA solid medium and incubate in the dark in a 25°C artificial climate chamber for 3-5 days before use.

[0057] Step 3: Use a sterile toothpick to pick up 1 cm 2 DN689 mycelial blocks were inoculated into PDB optimized medium supplemented with Dendrobium officinale powder for co-culture. The culture flasks were cultured at 120 rpm and 25℃ in the dark for about 25-35 days.

[0058] Step 4: After culturing for approximately 25-35 days, the fermentation broth was filtered through sterile filter paper to collect the mycelium. The mycelium was then cleaned with sterile water to remove any residual Dendrobium powder. After drying at a constant temperature of 45℃, the product was extracted by reflux and rotary evaporation using a methanol solution containing 0.05% formic acid. The product was analyzed by LC-MS and compared with a Dendrobine standard. Step 5: The determination of Dendrobine content in endophytic fungi of Dendrobium nobile was performed in the same manner as in the basic case.

[0059] The experimental results showed that the content of metabolites accumulated by strain DN689 in different carbon source media varied. The soluble starch group had the highest accumulation of dendrobine, which was significantly higher than other carbon source groups (P<0.01), followed by the glucose group, while the fructose group had the lowest accumulation. Therefore, the optimal carbon source was soluble starch (Figure 2A).

[0060] The analysis of metabolites in culture media with different nitrogen sources revealed variations in the accumulation of metabolites during growth in liquid media with different nitrogen sources. The accumulation of metabolites was significantly different between yeast extract and other nitrogen source groups (P<0.05), followed by the peptone group, while the beef extract group showed the lowest accumulation. Therefore, yeast extract is the optimal nitrogen source for the strain's fermentation (Figure 2B).

[0061] The effect of inorganic salts on the accumulation of dendrobine analogues was determined using the same method. As shown in Figure 2C, the difference in the accumulation of dendrobine by different amounts of inorganic salts was less significant (P<0.05) compared to carbon and nitrogen sources. The NaCl group had the highest accumulation of dendrobine, while the FeSO4 group had the lowest accumulation. Therefore, NaCl was selected as the inorganic salt for subsequent response surface optimization.

[0062] The results of the single-factor optimization experiment of the liquid fermentation medium for dendrobine were obtained. A (carbon source), B (nitrogen source), and C (inorganic salts) were selected. Based on the response value of dendrobine content (Y), the influence of the three factors on dendrobine production by DN689 was investigated, and a three-factor, three-level conditional response surface optimization was performed. The results are shown in Table 2. The binary multiple regression equation of dendrobine content Y on the encoded variables A, B, and C was obtained as follows: Y = 139.62 + 12.45*A + 22.59*B - 1.43*C + 12.37*AB + 15.50*AC + 17.65*BC - 29.49*A² - 14.71*B² - 34.38*C² Table 2: Response Surface Experimental Design and Results

[0063] The regression analysis of the equations is shown in Table 3. Analysis of the p-value test shows that the regression model established with dendrobine yield as the response value is significant (P = 0.0014 < 0.05). The lack-of-fit term is not significant (P = 0.3443 > 0.05), indicating a good fit between the experimental data and the model. The model signal-to-noise ratio (Adeq precision) = 9.9265 > 4, and the model correlation coefficient "R²" is 0.9443, indicating high model fit and reliability, and that it can well reflect the relationship between dendrobine yield and starch, yeast powder, and NaCl.

[0064] Table 3 Regression coefficients of the Box-Behnken design

[0065] Abbreviations: s indicates statistical significance, ns indicates no statistical significance, df indicates degrees of freedom, and AP indicates sufficient precision.

[0066] Response surface analysis of the regression model reveals the impact of various factors and their interactions on the response value, which can be visually reflected in response surface plots and contour plots, as shown in Figure 3. Each response surface represents the interaction between two independent variables.

[0067] As shown in Figure 3A, when NaCl is at a moderate concentration (0.06 g / L), soluble starch concentration is less than 22.06 g / L, and yeast extract concentration is less than 14.97 g / L, the dendrobine content increases with increasing concentrations of both factors. In Figure 3B, when yeast extract is at a moderate concentration (12.5 g / L), soluble starch concentration is less than 21.064 g / L, and NaCl concentration is less than 0.061 g / L, the dendrobine content increases with increasing concentrations of both factors. When soluble starch is at a moderate concentration (20 g / L), yeast extract concentration is less than 14.66 g / L, and NaCl concentration is less than 0.065 g / L, the dendrobine content increases with increasing concentrations of both factors.

[0068] As shown in Figure 4, the optimal culture medium composition ratio is 20.82 g / L soluble starch, 11.12 g / L yeast extract, and 0.05 g / L sodium chloride. Experiments yielded a dendrobine content of 324.67 ± 91.7 ng / g, exceeding the predicted level of 122.048 ng / g, thus validating the prediction. Subsequent fermentation condition optimization experiments were conducted based on this composition ratio.

[0069] The dendrobine content in the control group and the treatment group was calculated. The results showed that the dendrobine content in the control group was as shown in Table 4 and Figure 5.

[0070] Table 4. Dendrobine content in endophytic fungi under different culture conditions

[0071] The results showed that the dendrobine content in the liquid culture medium supplemented with Dendrobium stems was 14157.4667.9±3025.03 ng / g, 750.6±203.62 ng / g, and 32334.2±2550.14 ng / g, respectively, with yields increasing by approximately 44 times, 2 times, and 99 times (Figure 2). There was no significant difference in dendrobine content among the endophytic fungi in the solid culture medium supplemented with Dendrobium powder compared to the control group.

[0072] In this invention, by changing the culture conditions of endophytic fungi and adding Dendrobium officinale powder and Dendrobium officinale stem strips to the endophytic fungi liquid fermentation medium, it was found that adding Dendrobium officinale stem strips and Dendrobium officinale powder to the endophytic fungi liquid fermentation medium can significantly increase the content of dendrobine, providing a reference for the molecular mechanism of subsequent dendrobine synthesis, and has practical application value and research significance.

Claims

1. An endophytic fungus, characterized by: The endophytic fungus belongs to the genus Fusarium, and its taxonomic name is Fusarium sp. It was deposited on May 21, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 3.27824.

2. A co-culture composition for producing dendrobine, characterized in that, The strain comprising the endophytic fungus of claim 1, and Dendrobium nobile stem strips or Dendrobium nobile powder.

3. A method for producing dendrobine based on the strain of endophytic fungus described in claim 1, characterized in that, Includes the following steps: S1. The strain of endophytic fungus described in claim 1 is activated and cultured in PDA solid medium to obtain activated fungal blocks; S2. The activated fungal blocks are inoculated into PDB liquid optimized medium containing Dendrobium nobile stems or Dendrobium nobile powder for co-culture; S3. After the culture is completed, the mycelium is collected and dendrobine is extracted from the mycelium.

4. The method for producing dendrobine according to claim 3, characterized in that: The pH value of the PDA solid culture medium and the PDB liquid culture medium is 5.6±0.

2.

5. The method for producing dendrobine according to claim 3, characterized in that: The PDB liquid optimized culture medium contains the following components: 15-25 g / L soluble starch, 10-15 g / L yeast extract, 0.03-0.09 g / L sodium chloride, 0.05-0.15 g / L chloramphenicol, and potato extract as the solvent.

6. The method for producing dendrobine according to claim 3, characterized in that: In step S2, the co-culture conditions are: temperature 22~28℃, dark culture, and time 20~40 days.

7. The method for producing dendrobine according to claim 3, characterized in that: In step S3, the mycelium is dried at a constant temperature of 45°C before the extraction of dendrobine.

8. The method for producing dendrobine according to claim 3, characterized in that: In step S3, dendrobine in the mycelium is extracted by reflux rotary evaporation using a methanol solution containing 0.05% formic acid.

9. The method for producing dendrobine according to any one of claims 3 to 8, characterized in that: The Dendrobium used is Dendrobium nobile.

10. The method for producing dendrobine according to any one of claims 3 to 8, characterized in that: The Dendrobium nobile stems are fresh stems or fresh stems that have undergone surface sterilization; the Dendrobium nobile powder is obtained by drying and pulverizing the Dendrobium nobile stems and then passing them through a No. 3 sieve.

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