Endophyte for producing scirpusin b

Abstract

The present application provides a strain of endophyte separated from Radix Paeoniae Venetae and producing paeonol B, and belongs to the field of microorganisms.The endophyte is Fusarium equiseti DSJ12, which is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.40233.The endophyte is separated from the tuber of Radix Paeoniae Venetae by a tissue block method, and can ferment to produce paeonol B.The fermentation period can be greatly shortened, the production efficiency can be improved, and the production cost can be reduced by using the endophyte to ferment to produce paeonol B.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to an endophytic bacterium isolated from *Dioscorea opposita* that produces flavoside B. Background Technology

[0002] Dioscorea Bulbifera L., also known as Huangyaozi or Lingyuzi, is a perennial herbaceous wild vine. Its tubers are oval or pear-shaped, with a purplish-black outer skin and densely covered with fibrous roots. The stems are left-spiraling. Leaves are simple, alternate, broadly cordate, with a broad cordate base and a long tail-like apex, and entire margins. It has unisexual flowers; male inflorescences are drooping spikes, clustered in the leaf axils, with small, densely packed, pale greenish-white flowers; female flowers are closely appressed to the central axis, and the stem produces several small oval balls, resembling yam beans. Dioscorea Bulbifera is bitter and cold in nature, possessing the effects of cooling the blood, reducing internal heat, eliminating goiter, and detoxifying. It is an effective remedy for serious illnesses and is clinically used to treat goiter, hematemesis, epistaxis, esophageal cancer, gastric cancer, lymph node tuberculosis, and breast tumors.

[0003] The plant is rich in chemical components. Its tubers contain furan-demethyl diterpenoids, as well as scotyl terpenoid A, scotyl terpenoid B, and scotyl terpenoid C. Among them, scotyl terpenoid B is a unique component of the plant and is closely related to its pharmacological activity. It has anti-inflammatory, anti-tumor, and antibacterial effects and is distributed throughout the entire plant. It can be used to treat cancer.

[0004] The existing production method for huangduxin B is plant extraction, which involves extraction and separation from the huangduxin plant. This method suffers from low production efficiency and low yield. For example, patent CN201110003843.3 discloses an extraction method for huangduxin B, which includes acetone reflux extraction of huangduxin, concentration of the extract until no acetone odor is present, obtaining an extract; mixing the extract with silica gel, adding petroleum ether for extraction, evaporating the filter residue to dryness, adding chloroform for extraction, evaporating the extract to dryness to obtain the chloroform fraction; mixing the chloroform fraction with silica gel, loading onto a column, eluting with a chloroform-methanol mixture at a volume ratio of 98:2, collecting the eluent in fractions; recovering the solvent from the eluent, dissolving and crystallizing in acetone to obtain crystals, loading the filtrate onto a column again, eluting with a chloroform-methanol mixture at a volume ratio of 99:1, recovering the solvent from the eluent, dissolving and crystallizing in acetone, combining the crystals to obtain huangduxin B. Patent CN200710123281.X discloses an effective component of Dioscorea bulbifera, its preparation method, and its uses. The preparation process includes the following steps: Step 1: Extracting Dioscorea bulbifera with a mixture of ethyl acetate and ethanol as a solvent; Step 2: Extracting the residue with ethanol to obtain an extract; Step 3: Eluent obtained by column chromatography; Step 4: Eluent obtained by gradient elution with preparative liquid chromatography, using water and acetonitrile as the mobile phase, and collecting the eluent for 10.8-13.8 minutes to obtain the effective component.

[0005] The production cycle of preparing flavonoid B by plant extraction is long (it takes 4-5 months to grow flavonoids) and the production efficiency is low (about 1.5g / kg of flavonoids). Therefore, there is an urgent need to provide a preparation method for flavonoid B with a short production cycle and high production efficiency. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides an endophytic bacterium isolated from *Dioscorea opposita* that produces flavoside B. This invention uses a tissue block method to isolate an endophytic bacterium from the tuber of *Dioscorea opposita*. This endophytic bacterium can ferment and produce flavoside B. Using this endophytic bacterium for flavoside B fermentation can significantly shorten the fermentation cycle, improve production efficiency, and reduce production costs.

[0007] The technical solution of the present invention is as follows:

[0008] On one hand, the present invention provides an endophytic bacterium, namely Fusarium equiseti, DSJ12, which was deposited on June 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40233.

[0009] On the other hand, the present invention provides a fermentation product of the above-mentioned endophytic bacteria.

[0010] Specifically, the preparation method of the fermentation product is as follows: collect the fermentation broth after fermentation culture, evaporate it to dryness under reduced pressure, and the product is obtained.

[0011] More specifically, the temperature for the reduced pressure evaporation is 40-60°C, preferably 50°C.

[0012] In another aspect, the present invention provides the application of the above-mentioned endophytic bacteria in the preparation of flavonoid B.

[0013] In another aspect, the present invention provides an extract of the aforementioned endophytic bacteria.

[0014] Specifically, the extract includes flavonoid B.

[0015] In another aspect, the present invention provides a method for culturing the above-mentioned endophytic bacteria, wherein the culturing method is fermentation culture.

[0016] The fermentation culture method is as follows:

[0017] (1) Activation of strains: Inoculate the preserved strains onto the culture medium and culture them step by step to screen out the strains with vigorous activity;

[0018] (2) Fermentation culture: The strains selected in step (1) are inoculated into the culture medium for culture to obtain fermentation broth.

[0019] Specifically, the activation of the strain in step (1) can be achieved by streak plating or coating.

[0020] Specifically, the culture medium for activating the strain in step (1) is PDA solid culture medium.

[0021] Specifically, the cultivation conditions described in step (2) are a cultivation temperature of 21-35℃, preferably 25-32℃, and more preferably 28℃; and a cultivation time of 5-9 days, preferably 7 days.

[0022] Specifically, the culture medium mentioned in step (2) is PDA culture medium, which includes: 180-220 g / L of potato and 15-25 g / L of glucose.

[0023] In another aspect, the present invention provides a method for preparing the above-mentioned endophytic bacterial extract, the method comprising the following steps:

[0024] (1) Filter the fermentation broth to remove the mycelium, and put it into a rotary evaporator to evaporate to dryness under reduced pressure;

[0025] (2) Dissolve in methanol, use ultrasound to aid dissolution for 50-70 minutes, filter to remove precipitate, and obtain fermentation extract.

[0026] In another aspect, the present invention provides a daily chemical product, packaging material, additive, pharmaceutical preparation and medical device comprising the above-mentioned endophytes, endophyte fermentation products or endophyte extracts.

[0027] In another aspect, the present invention provides the application of the above-mentioned endophytic bacteria, endophytic fermentation products or endophytic extracts in the preparation of daily chemical products, packaging materials, additives, pharmaceutical preparations and medical devices with anti-inflammatory, anti-tumor or antibacterial functions.

[0028] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0029] (1) The present invention isolated and screened a strain of Fusarium equiseti DSJ12, which was deposited on June 27, 2022 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40233.

[0030] (2) The Fusarium equisetifolium isolated and screened by the present invention can ferment to produce flavonoid B. Using the Fusarium equisetifolium to ferment and produce flavonoid B can greatly shorten the fermentation cycle, improve production efficiency and reduce production costs.

[0031] Preservation Instructions

[0032] Classification and nomenclature: Fusarium equisetifolium

[0033] Latin name: Fusarium equiseti

[0034] Biomaterials used for ginseng: DSJ12

[0035] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0036] Collection institution abbreviation: CGMCC

[0037] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0038] Date of preservation: June 27, 2022

[0039] CGMCC Registration Number: 40233 Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the morphology of Fusarium equisetifolium.

[0041] Figure 2 This is the liquid chromatogram of the standard of flavonoid B.

[0042] Figure 3 This is a liquid chromatogram of the fermentation product. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0044] Example 1: Preparation of Culture Medium

[0045] 1. PDA solid culture medium: 200g peeled potatoes, boiled in 500mL pure water, filtered, the supernatant was taken and 20g glucose and 20g agar were added, pure water was added to make up to 1L, and sterilized at 121℃, 0.1MPa for 20min.

[0046] 2. PDA liquid culture medium: 200g of peeled potatoes, boiled in 500mL of pure water for 1h, filtered, the supernatant was taken and 20g of glucose was added, pure water was added to make up to 1L, and sterilized at 121℃, 0.1MPa for 20min.

[0047] Example 1. Isolation and purification of strains

[0048] 1. Separation Method - Tissue Block Method

[0049] Clean the surface of the *Dioscorea opposita* fruit and vine stems with tap water, pat dry with sterile paper towels, rinse with 75% alcohol for 4-5 minutes, rinse 3-4 times with sterile water, pat dry again with sterile paper towels, rinse with 10% sodium hypochlorite for 2-3 minutes, and rinse 4-5 times with sterile water. Under sterile conditions, cut the *Dioscorea opposita* fruit and vine stems into 5mm × 5mm pieces and place them on PDA agar plates for incubation at 28℃. After 3-4 days of incubation, promptly pick mycelia or colonies with different morphologies and transfer them to fresh PDA agar plates. Purify 3-4 times to ensure that the obtained colonies are pure cultures. Transfer the purified culture to the appropriate slant agar plates and incubate for 7 days before storing in a refrigerator.

[0050] Blank control: Apply sterile water from the final rinse to a new PDA plate as a blank control to check if surface disinfection was thorough. If cultures are formed, it indicates incomplete surface disinfection, and the separation results are invalid. The experiment is restarted.

[0051] After separation, endophytic bacteria were obtained, and the morphology of the endophytic bacteria was as follows: Figure 1 As shown.

[0052] 2. Identification of Endophytic Bacteria

[0053] The bacterial strain was sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification.

[0054] ITS1 (5'-TCCGTAGGTGAACCTGCGC-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used as primers for amplification. The amplified sequence was compared with the nucleic acid sequence in the NCBI database using BLAST analysis. The comparison results showed that the strain was identified as Fusarium equiseti.

[0055] Example 2. Microbial fermentation

[0056] 120 mL of PDA liquid culture medium was placed into a 250 mL Erlenmeyer flask. The bacterial cells were picked up with an inoculation needle and inoculated into the sterilized culture medium. Fermentation was carried out at 28 °C and 120 r / min for 7 days.

[0057] Example 3. Preparation and content determination of fermentation extract

[0058] 1. Extract preparation

[0059] The fermentation broth after filtering out the mycelium was placed in a rotary evaporator and evaporated to dryness under reduced pressure at 50°C. 10 mL of methanol (chromatographic grade) was added to dissolve the broth, and the mixture was sonicated for 60 min to aid dissolution. The insoluble matter was dissolved in 10 mL of deionized water to obtain the fermentation extract.

[0060] 2. Determination of extract content

[0061] (1) Reagents: Huangdusu B standard (Shanghai Yuanye Biotechnology Co., Ltd.), acetonitrile (chromatographic grade).

[0062] (2) Experimental instruments

[0063] High performance liquid chromatograph: Agilent 1260, chromatographic column: C18 column (250mm×4.60mm, 5μm), analytical balance, ultrasonic instrument.

[0064] (3) Preparation of standard solution of flavonoid B

[0065] Accurately weigh 5.36 mg of flavonoid B and place it in a 10 mL volumetric flask. Dissolve it in methanol (analytical grade) and use ultrasonication to assist dissolution while heating to 35 °C for 120 min. Make up to volume with methanol.

[0066] (4) Liquid chromatography conditions

[0067] Mobile phase: acetonitrile (chromatographic grade), detection wavelength: 220 nm, flow rate: 1.0 mL / min -1 Column temperature: 25℃, injection volume: 10μL.

[0068] (5) The fermentation product obtained by vacuum evaporation and concentration and the standard sample (21.2 μg / mL) were determined under the above chromatographic conditions. After comparison with the peak area of ​​the standard and conversion, the concentration of the fermentation broth was calculated to be 17.24 μg / mL. The liquid chromatogram of the flavonoid B standard is shown below. Figure 2 The HPLC chromatogram of the fermentation products is shown below. Figure 3 .

[0069] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An endophytic bacterium, characterized in that, The endophytic bacteria is Fusarium equiseti DSJ12, which was deposited on June 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 40233.

2. The application of the endophytic bacteria according to claim 1 in the preparation of flavonoid B.

3. A method for culturing endophytic bacteria as described in claim 1, characterized in that, The cultivation method is fermentation culture, which includes the following steps: (1) Activation of strains: Inoculate the preserved strains onto the culture medium and culture them step by step to screen out the strains with vigorous activity; (2) Fermentation culture: The strains selected in step (1) are inoculated into the culture medium for culture to obtain fermentation broth.

4. The cultivation method according to claim 3, characterized in that, The culture conditions described in step (2) are a culture temperature of 21-35℃ and a culture time of 5-9 days.

5. The cultivation method according to claim 3, characterized in that, The culture medium mentioned in step (2) is PDA culture medium, which includes: 180-220 g / L of potato and 15-25 g / L of glucose.

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