CN111172049A - Toxoflavin high-yield strain and application thereof - Google Patents
Toxoflavin high-yield strain and application thereof Download PDFInfo
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- CN111172049A CN111172049A CN201811328269.7A CN201811328269A CN111172049A CN 111172049 A CN111172049 A CN 111172049A CN 201811328269 A CN201811328269 A CN 201811328269A CN 111172049 A CN111172049 A CN 111172049A
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- burkholderia
- toxoflavin
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- SLGRAIAQIAUZAQ-UHFFFAOYSA-N toxoflavin Chemical compound CN1N=CN=C2C1=NC(=O)N(C)C2=O SLGRAIAQIAUZAQ-UHFFFAOYSA-N 0.000 title claims abstract description 189
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 66
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000000855 fermentation Methods 0.000 claims abstract description 38
- 230000004151 fermentation Effects 0.000 claims abstract description 38
- 239000001963 growth medium Substances 0.000 claims abstract description 32
- 239000007787 solid Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 20
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- 241001225321 Aspergillus fumigatus Species 0.000 claims abstract description 14
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- 238000004321 preservation Methods 0.000 claims abstract description 5
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 4
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 21
- 238000000605 extraction Methods 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
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- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 1
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 1
- VQUOCQPUWMVVJV-UHFFFAOYSA-N 6-[amino(methyl)amino]-3-methyl-1h-pyrimidine-2,4-dione Chemical compound CN(N)C1=CC(=O)N(C)C(=O)N1 VQUOCQPUWMVVJV-UHFFFAOYSA-N 0.000 description 1
- 241001135516 Burkholderia gladioli Species 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940023064 escherichia coli Drugs 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
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- 239000000194 fatty acid Substances 0.000 description 1
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- 150000004665 fatty acids Chemical class 0.000 description 1
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
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Abstract
The invention discloses a burkholderia capable of stably producing toxoflavin at high yieldBurkholderiasp.) HDXY-02, belonging to the technical field of applied microorganisms. The strain is preserved in China general microbiological culture Collection center (CGMCC for short) in 2017, 4 months and 20 days, and the preservation number is CGMCC No. 14054. After the liquid culture medium of the strain is fermented or the solid plate is coated and cultured for 48-72 hours, the toxoflavin obtained by extracting dichloromethane or chloroform, separating and purifying has the activity of inhibiting pathogenic bacteria aspergillus fumigatus and resisting tumor Lovo cells. The strain provided by the invention can synthesize toxoflavin through fermentation and has high yield, and compared with the existing chemical synthesis method, the method avoids the pollution of chemical substances to the environment and has potential application prospect.
Description
Technical Field
The invention relates to Burkholderia and application thereof, in particular to Burkholderia sp HDXY-02 capable of efficiently synthesizing toxoflavin, and belongs to the technical field of microorganisms.
Background
Natural products are substances produced by microorganisms, plants, or other organisms, wherein microbial natural products provide a rich source of chemical diversity. Toxoflavin is a small-molecular fatty acid exotoxin synthesized by microorganisms, and is found to be used as an antibacterial agent and an antitumor agent. The molecular formula of toxoflavin is C7H7N5O2The molar mass is 193g/mol, and the antibacterial agent can generate resistance to various pathogenic bacteria, and the strong antibacterial property of the antibacterial agent can be caused by inhibiting the respiratory chain and generating peroxide. The toxoflavin also has strong antitumor activity and can kill cancer cells at a lower concentration. Researches prove that the toxoflavin has good inhibitory activity on lung cancer cells, ovarian cancer cells, gastric cancer cells and other cancer cells. The higher biological activity of toxoflavin in the aspects of antibiosis, tumor resistance and the like indicates that toxoflavin has prospect in the research fields of pharmacology, toxicology and the like. The toxoflavin has high research value in the aspects of apoptosis and the like as a new antibiotic and antitumor drug. In addition, the development of post-structure modified toxoflavin as an antibacterial and antitumor agent drug also has high commercial value.
At present, toxoflavin can be synthesized chemically, 2-thiobarbituric acid is taken as a raw material, and the toxoflavin is synthesized through the steps of methylation, chlorination, hydrolysis, nitration, catalytic reduction, reflux and the like; can also be used for synthesizing toxoflavin by prolonging the nitration and cyclization of 3-methyl-6- (1' -methylhydrazino) uracil; in addition, the toxoflavin can be synthesized by using methylurea, malonic acid and acetic anhydride as raw materials through the steps of chlorination, condensation, diazotization and the like. However, the steps for chemically synthesizing toxoflavin are complex, the required organic reagents are more, the number of byproducts is large, the environment is polluted, and the like. It is worth noting that toxoflavin can be synthesized not only by chemical methods but also by biological methods. In natural environment, the microorganisms capable of producing toxoflavin include Pseudomonas cocoanut, Burkholderia gladioli, Burkholderia glumae and the like. The biosynthesis has the advantages of small pollution, mild reaction conditions and the like. But the yield of toxoflavin in microorganisms is low at present, and the yield of toxoflavin still has a great space to be improved. Therefore, the method has important research and development values for further obtaining the high-yield toxoflavin strain by separating the toxoflavin production strain in the environment, and is an important way for solving the limitation of the source of the current toxoflavin raw material.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the prior art, the Burkholderia sp (Burkholderia sp.) with high toxoflavin yield is provided, the strain is stable in heredity, and the content of toxoflavin in fermentation liquor is high.
The Burkholderia sp HDXY-02 strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation unit address is No. 3 of Xilu No.1 of Beijing Korean district, the microbial research institute of Chinese academy of sciences, the preservation date is 4 months and 20 days in 2017, and the registration number of the strain is CGMCC No. 14054.
The invention aims to provide Burkholderia sp (HDXY-02) with high toxoflavin yield and application thereof, the bacterial strain has strong toxoflavin production capacity, the yield can reach 387mg/L after being cultured for 48 hours at 30 ℃, and good application prospects are shown.
The second purpose of the invention is to provide a method for biosynthesizing and extracting toxoflavin. The toxoflavin extracted by the invention is obtained by liquid fermentation or solid culture and then extraction of the Burkholderia sp.
Extraction of toxoflavin after liquid fermentation of Burkholderia sp.hdxy-02: inoculating a proper amount of activated and cultured strains into seed liquid, inoculating the strains into a liquid fermentation culture medium according to a certain proportion for fermentation after overnight culture, centrifuging after the fermentation is finished, taking supernatant of fermentation liquid, adding chloroform or dichloromethane for extraction, collecting an organic phase part, concentrating and volatilizing an organic solvent under reduced pressure, adding a little chloroform or dichloromethane for extracting residues once, drying to obtain a crude extract, and further purifying to obtain a pure toxoflavin.
Extraction of toxoflavin after solid culture of Burkholderia sp.hdxy-02: diluting a proper amount of activated and cultured strains, coating the strains on a KMB solid plate, scraping thalli on the surface of the plate after culture, soaking chloroform or dichloromethane in a solid culture medium for extraction, collecting an organic phase part, concentrating and volatilizing the chloroform or dichloromethane under reduced pressure, adding a little chloroform or dichloromethane to extract residues once, drying to obtain a crude extract, and further purifying to obtain a pure toxoflavin.
Detecting fermentation extract of Burkholderia sp.HDXY-02 by thin layer chromatography, liquid phase, mass spectrum and other methods, and confirming that main extract of fermentation liquor of Burkholderia sp.HDXY-02 is toxoflavin with molecular formula of C7H7N5O2The molecular weight is 193 g/mol.
Specifically, the preparation method of toxoflavin comprises the following steps:
(1) preparation of fermentation broth or culture plate
Fermentation liquor: inoculating Burkholderia sp.HDXY-02 as defined in claim 1 into LB culture medium, culturing overnight at 30 ℃, and obtaining seed liquid after culturing for 12-16 h; inoculating the seed liquid into a KMB fermentation culture medium in an inoculation amount of 1-10%, and performing shake culture at 200 rpm.
Culturing a flat plate: burkholderia sp.HDXY-02 according to claim 1 was inoculated into a seed medium (0.5% beef extract; 1% peptone; 0.5% NaCl; 2% agar; distilled water; pH7.0) by shaking culture for 24 hours, diluted appropriately, and spread uniformly on the surface of KMB solid medium.
(2) Preliminary purification of toxoflavin
Centrifuging the fermentation liquor obtained in the step (1), removing thalli, collecting supernatant, performing isometric extraction on the supernatant by using dichloromethane or chloroform as a solvent, collecting an organic phase, and drying to obtain a crude extract of toxoflavin; or scraping off thallus on the surface of the plate, cutting up the solid culture medium, soaking the solid culture medium in dichloromethane or chloroform as a solvent, collecting the organic phase, and drying to obtain the crude extract of toxoflavin.
(3) Secondary purification of toxoflavin
And (3) performing gradient elution on the crude extract obtained in the last step by adopting column chromatography, separating and purifying, and further recrystallizing twice by using n-butyl alcohol to obtain purified toxoflavin.
In the method of the present invention, it is preferable that the KMB medium used in the step (1) for culturing Burkholderia sp.HDXY-02 has the following composition: 20.0g of peptone, 1.5g of dipotassium phosphate, 0.75g of magnesium sulfate and 15mL of glycerol, wherein the pH value is 5.5-8.5, and the volume is up to 1000 mL. Agar was added to the solid medium to a final concentration of 1.5%.
In the method of the present invention, preferably, the culture temperature in step (1) is 30-35 ℃, and the culture is performed for 48-72 hours.
The biological activity detection result shows that: hdxy-02 can inhibit the growth of various pathogenic bacteria (escherichia coli, aspergillus fumigatus and candida albicans); meanwhile, the composition can inhibit Lovo cells of human colon cancer in a proper concentration range.
The method has the advantages that the toxoflavin is stably produced by the strain after multiple passages, and the capacity is not weakened. The Burkholderia sp.HDXY-02 provided by the invention can produce toxoflavin with high yield, and has potential industrial application prospect.
Drawings
FIG. 1 thin layer chromatography analysis of toxoflavin produced by Burkholderia sp.HDXY-02
FIG. 2 Mass Spectrometry (MS) analysis of toxoflavin produced by Burkholderia sp.HDXY-02
FIG. 3 shows the bacteriostatic test of toxoflavin produced by Burkholderia sp.HDXY-02
FIG. 4 inhibition of spore germination and spore formation of Aspergillus fumigatus by Burkholderia sp.HDXY-02 (A) and (B)
FIG. 5: effect of toxoflavin produced by Burkholderia sp.HDXY-02 on viability of cancer Lovo cells
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way.
Example one extraction of toxoflavin by solid culture of Burkholderia sp.HDXY-02
1. Strain activation
The preserved Burkholderia sp.HDXY-02 strain is picked, streaked on a seed solid culture medium (0.5% beef extract, 1% peptone, 0.5% NaCl, 2% agar, distilled water, pH7.0), and cultured at a constant temperature of 30-35 ℃ for 48-72 h.
2. Solid plate culture
Burkholderia sp.HDXY-02 single colonies are picked and inoculated in a seed culture medium (0.5% beef extract, 1% peptone, 0.5% NaCl, 2% agar, distilled water and pH7.0), after shaking culture for 24 hours, diluted properly, coated on a KMB culture medium (20.0 g of peptone, 1.5g of dipotassium hydrogen phosphate, 0.75g of magnesium sulfate, 15mL of glycerol, pH 5.5-8.5, constant volume to 1000mL, 1.5% agar), and kept stand and cultured for 48-72 hours at a constant temperature of 30-35 ℃.
3. Extraction of toxoflavin
Scraping off lawn on the surface of the solid culture medium, chopping the solid culture medium rich in toxoflavin, adding 1/2 volume of chloroform or dichloromethane, shaking for 5-10 min, standing for 20-40 min, and removing the organic phase part. The same procedure was repeated 3 times by soaking the solid medium with chloroform or dichloromethane extraction. And combining the organic phases, and volatilizing the organic solvent in a rotary evaporator at 40-45 ℃. And adding a little chloroform or dichloromethane for extraction once, and drying in a drying oven at 40-45 ℃ to obtain a fermented crude extract.
Example two production of toxoflavin by liquid fermentation of Burkholderia sp.HDXY-02
1. Strain activation
The preserved Burkholderia sp.HDXY-02 strain is picked, streaked on a seed solid culture medium (0.5% beef extract, 1% peptone, 0.5% NaCl, 2% agar, distilled water, pH7.0), and cultured at a constant temperature of 30-35 ℃ for 48-72 h.
2. Liquid fermentation process
The Burkholderia sp.HDXY-02 strain on a seed culture medium is diluted and uniformly mixed by using normal saline, 1-10mL of overnight culture is absorbed and inoculated into a 250mL triangular flask filled with 100mL of KMB liquid culture medium (20.0 g of peptone, 1.5g of dipotassium hydrogen phosphate, 0.75g of magnesium sulfate, 15mL of glycerol, pH 5.5-8.5, constant volume to 1000mL), and the mixture is cultured for 48-72 hours at 30-35 ℃ at 150-200 rpm under continuous shaking.
3. Extraction of toxoflavin
And after the shaking culture is finished, centrifuging the fermentation liquor for 4-5 min at the temperature of 4-20 ℃ at 4000-6000 g, sucking the supernatant, adding 1/2 volume of chloroform or dichloromethane into the supernatant, shaking for 5-10 min, standing for 20-40 min, and removing the organic phase part. The same procedure was repeated 3 times by extracting the aqueous portion once with chloroform or dichloromethane. And combining the organic phases, and volatilizing the organic solvent in a rotary evaporator at 40-45 ℃. And adding a little chloroform or dichloromethane for extraction once, and drying in a drying oven at 40-45 ℃ to obtain a fermented crude extract.
EXAMPLE III toxoflavin detection
And (3) secondary purification of toxoflavin: separating and purifying the crude extract by silica gel column chromatography, wherein the gradient elution process comprises the following steps: (dichloromethane: methanol) 90:10 → 80:20 → 60:40 → 50:50 → 30:70 → 0:100 (v/v). And further recrystallizing twice through n-butanol to obtain purified toxoflavin.
The toxoflavin extracted from the strain is compared with a toxoflavin standard.
Thin layer chromatography: the fermentation broth extract of the strain and the standard toxoflavin were dissolved in the same volume of methanol, spotted on one end of a thin-layer silica gel G plate, and developed in a developing solvent (chloroform: methanol: water) (70:25:5, V/V), respectively (see FIG. 1).
Mass spectrum detection: the fermentation liquor extract produced by Burkholderia sp.HDXY-02 liquid fermentation has the same ion peak of 194.0714 m/z as that of the standard toxoflavin. The results show that the fermentation liquor extract for producing toxoflavin by liquid fermentation of Burkholderia sp.HDXY-02 is toxoflavin, and the specific molecular formula and the molecular weight are shown in figure 2.
EXAMPLE four passage verification of Burkholderia sp.HDXY-02 stability of toxoflavin production
Firstly, the Burkholderia sp.HDXY-02 preserved in a refrigerator is streaked on an LB solid culture medium, then after 24 hours of culture, the colonies are removed from the first solid culture medium and then are transplanted onto the second solid culture medium, and so on, ten generations of solid plate colonies are cultured. Respectively inoculating the above materials into seed culture medium, culturing overnight to obtain seed liquid, inoculating into fermentation culture medium, performing shake flask fermentation at 30 deg.C and 200rpm, and continuously shaking for 48 hr. The content of flavins in the fermentation supernatant was determined and the results are shown in Table 1.
TABLE 1 Burkholderia sp.HDXY-02 toxoflavin production passage stability test
As can be seen from the results in Table 1, after multiple passages, the production of toxoflavin by Burkholderia sp.HDXY-02 is stable, and the yield of toxoflavin is not reduced after 10 passages.
EXAMPLE five antimicrobial Activity of toxoflavin produced by Burkholderia sp.HDXY-02 fermentation
1. Detection of bacteriostatic activity
Test strains (Escherichia coli, Candida albicans, Aspergillus nidulans and Aspergillus fumigatus) were selected and diluted with physiological saline. Preparing a toxoflavin detection solution produced by Burkholderia sp.HDXY-02 liquid fermentation at a certain concentration, and adding the toxoflavin detection solution into a solid culture medium according to the concentration. A dilution gradient 106～108The bacterial liquid of CFU/mL thallus is cultured on YAG, YPDA and LB solid culture medium added with toxoflavin at the temperature of 37 ℃ for 24h, and whether the strain grows or not is observed. The results showed that toxoflavin produced by Burkholderia sp.HDXY-02 liquid fermentation had the ability to inhibit the growth of pathogens (FIG. 3). MIC values for Escherichia coli, Candida albicans, Aspergillus nidulans and Aspergillus fumigatus were 5mg/L, 100mg/L, 200mg/L and 100mg/L, respectively.
2. Effect of toxoflavin on spore germination and sporulation of Aspergillus fumigatus
Collecting Aspergillus fumigatus spores by using freshly cultured Aspergillus fumigatus, and adjusting the spore concentration to 10 with YAG or MM medium7CFU/mL, adding toxoflavin to a final concentration of 25 μ g/mL, adding dropwise onto a pre-sterilized cover glass, culturing at 37 deg.C for a corresponding time, taking out, observing with microscope, and collecting the liquid containing equal amount of spores without toxoflavinThe body medium served as a control. As shown in FIG. 4A, in the case of no toxoflavin, germination starts immediately after 6h of culture of Aspergillus fumigatus spores, but the spores are not germinated after 20h by the treatment with the toxoflavin, indicating that the toxoflavin can significantly inhibit the germination of Aspergillus fumigatus spores. The spore-forming structure is important for growth and propagation of aspergillus fumigatus, the final concentration of toxoflavin is selected to be 50 mug/mL, the toxoflavin is added into a solid YAG or MM culture medium, aspergillus fumigatus spores are inoculated, cultivation is carried out in an inserting mode, cultivation is carried out at 37 ℃ for proper time, and microscopic observation is carried out. Toxoflavin was found to significantly inhibit the formation of a sporulating structure of A.fumigatus (FIG. 4B).
EXAMPLE six cytotoxicity of toxoflavin produced by Burkholderia sp.HDXY-02 liquid fermentation
The cytotoxicity of toxoflavin produced by Burkholderia sp.HDXY-02 liquid fermentation on human colon cancer cells (Lovo) is detected by applying an MTT method.
1. Preparation of cell line culture
Culture medium: DMEM (Gibco, USA) supplemented with 10% inactivated calf serum and penicillin G (100U/mL). The Lovo cells were added to culture flasks containing 10mL of medium at 37 ℃ with 5% CO2The incubator (2) is used for moisture-keeping culture. Toxoflavin was dissolved in water and diluted to serial concentrations 5min before use in DMEM medium.
Test for detecting cell growth inhibition ability of toxoflavin by MTT method
To each experimental well of a 96-well cell culture plate, 100. mu.L (containing toxoflavin at the corresponding concentration) of 104Cell sap of Lovo cells, each concentration was set at five replicates, toxoflavin final concentrations of 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625, with no toxoflavin control wells. 37 ℃ and 5% CO2And (5) carrying out moisture-preserving culture in an incubator with the concentration. Culturing under the same conditions for 24h, adding 20 μ l MTT (5mg/mL) into each well, culturing for 4h, carefully blotting the solution in each well, adding 100 μ l DMSO, shaking at 37 deg.C for 10min, selecting 570nm wavelength, and detecting the absorbance of each well with microplate reader. The data is obtained by adding and subtracting standard deviation from the average value of five wells, calculating the percentage of survival cells, and drawing a survival curve, the result is shown in figure 5, toxoflavin has obvious inhibition effect on Lovo cells, the inhibition rate and the concentration form positive correlation, and IC50The value was 0.43. mu.g/mL (2.226. mu.M).
Experimental data show that toxoflavin produced by Burkholderia sp.HDXY-02 liquid fermentation also has the obvious effect of inhibiting Lovo tumor cells.
Claims (5)
1. Burkholderia capable of stably producing toxoflavin at high yieldBurkholderiasp.) HDXY-02, which is preserved in China general microbiological culture Collection center (CGMCC for short) in 2017 in 20 months 4, and the preservation number is CGMCC number 14054.
2. A method of using the Burkholderia bacterium (B) of claim 1Burkholderiasp.) fermentation culture method for producing toxoflavin by HDXY-02, which is characterized in that Burkholderia with the preservation number of CGMCC No 14054 (Burkholderia plantarii: (CGMCC) number)Burkholderiasp.) HDXY-02 for liquid fermentation culture or solid plate culture:
(1) liquid fermentation: activated Burkholderia bacterium (b), (c), (d), (Burkholderiasp.) HDXY-02 is inoculated in a seed culture medium for overnight culture, and then is inoculated in a KMB fermentation culture medium with the inoculation amount of 1-10%, the culture condition is 30-35 ℃, 150-200 rpm, continuous shaking culture is carried out for 48-72 h, and the pH value of the culture medium is 5.5-8.5;
(2) solid plate culture: activated Burkholderia bacterium (b), (c), (d), (Burkholderiasp.) HDXY-02 is inoculated in a seed culture medium, after shaking culture is carried out for 24 hours, the mixture is diluted properly and coated on a KMB solid culture medium under the culture condition of 30-35 ℃ and is kept still for 48-72 hours at constant temperature.
3. Burkholderia (Burkholderia) according to claim 1Burkholderiasp.) method for extracting flavin from HDXY-02 fermentation broth, which is characterized by comprising the following steps: after the shaking culture is finished, centrifuging the fermentation liquor for 4-5 min at 4-20 ℃ at 4000-6000 g, sucking supernatant, adding 1/2 volume of chloroform or dichloromethane into the supernatant, shaking for 5-10 min, standing for 20-40 min, and removing an organic phase part; extracting the aqueous phase with chloroform or dichloromethane once, repeating for 3 times, and mixingAnd volatilizing the organic solvent in a rotary evaporator at 40-45 ℃, adding a little chloroform or dichloromethane for extraction once, and drying in a drying oven at 40-45 ℃ to obtain a fermented crude extract.
4. Burkholderia (Burkholderia) according to claim 1Burkholderiasp.) method for extracting flavin from HDXY-02 solid medium, characterized in that it is carried out by the following steps: scraping off lawn on the surface of the solid culture medium, chopping the solid culture medium rich in toxoflavin, adding 1/2 volume of chloroform or dichloromethane, shaking for 5-10 min, standing for 20-40 min, and removing an organic phase part; extracting and soaking the solid culture medium with chloroform or dichloromethane by the same method, repeating for 3 times, combining organic phases, volatilizing the organic solvent in a rotary evaporator at 40-45 ℃, adding a little chloroform or dichloromethane for extraction once, and drying in a drying oven at 40-45 ℃ to obtain a fermented crude extract.
5. Burkholderia (Burkholderia) according to claim 1Burkholderiasp.) the purification method of the crude toxoflavin extracted from the HDXY-02 fermentation liquor or solid culture is characterized by comprising the following steps: the crude toxoflavin extract is separated and purified by silica gel column chromatography, and the gradient elution process comprises the following steps: (dichloromethane: methanol) 90:10 → 80:20 → 60:40 → 50:50 → 30:70 → 0:100 (v/v); and further recrystallizing twice by n-butanol to obtain purified toxoflavin which can inhibit the growth of various pathogenic bacteria (including aspergillus fumigatus); can also inhibit Lovo cell, Ic of human colon cancer50It was 0.43. mu.g/mL (2.226. mu.M).
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